具有改善的特性的β-葡萄糖苷酶I变体的制作方法

相关申请的交叉引用本专利申请要求2009年11月20日提交的美国临时申请序列号61/263,240的权益，该申请据此全文以引用方式并入。关于联邦政府资助的研究与开发所作出的发明的权利的声明本发明是在由美国能源部授予的附带条件奖No:De-Fc36-08go18078的政府支持下进行。政府具有本发明的某些权利。
技术领域：
本发明总体涉及酶，具体地讲为β-葡萄糖苷酶变体。还描述了编码β-葡萄糖苷酶变体的核酸、包含β-葡萄糖苷酶变体的组合物、使用β-葡萄糖苷酶变体的方法和鉴定另外有用的β-葡萄糖苷酶变体的方法。
背景技术：
：纤维素和半纤维素是通过光合作用产生的最丰富的植物材料。它们能被许多会产生能够将高分子底物水解成单体糖的胞外酶的微生物(例如，细菌、酵母和真菌)降解并且用作能源(Aro等人，J.Biol.Chem.,276:24309-24314,2001)。因为非可再生资源方法的局限性，纤维素成为主要可再生能源的潜能是巨大的(Krishna等人，BioresourceTech.,77:193-196,2001)。通过生物过程对纤维素的有效利用是克服食品、饲料和燃料短缺的一种途径(Ohmiya等人，Biotechnol.Gen.EngineerRev.,14:365-414,1997)。纤维素酶是水解纤维素(β-1,4-葡聚糖或βD-糖苷键)导致形成葡萄糖、纤维二糖、纤维低聚糖等的酶。传统上将纤维素酶分成三个主要类型：内切葡聚糖酶(EC3.2.1.4)(“EG”)、外切葡聚糖酶或纤维二糖水解酶(EC3.2.1.91)(“CBH”)和β-葡萄糖苷酶([β]-D-葡萄糖苷酶；EC3.2.1.21)(“BG”)(Knowles等人，TIBTECH5:255-261,1987；和Schulein,MethodsEnzymol.,160:234-243,1988)。内切葡聚糖酶主要作用在纤维素纤维的非晶部分，而纤维二糖水解酶还能够降解结晶纤维素(Nevalainen和Penttila，Mycota,303-319,1995)。因此，纤维二糖水解酶在纤维素酶体系中的存在是结晶纤维素有效溶解所必需的(Suurnakki等人，Cellulose,7:189-209,2000)。β-葡萄糖苷酶起到从纤维二糖、纤维低聚糖和其他葡萄糖苷释放D-葡萄糖单位的作用(Freer,J.Biol.Chem.,268:9337-9342,1993)。已知纤维素酶由大量细菌、酵母和真菌产生。某些真菌会产生能够降解结晶形式的纤维素的整个纤维素酶体系，使得纤维素酶容易通过发酵大量产生。丝状真菌发挥特别的作用，因为许多酵母例如酿酒酵母(Saccharomycescerevisiae)缺乏水解纤维素的能力(参见，例如，Wood等人，MethodsinEnzymology,160:87-116,1988)。CBH、EG和BG的真菌纤维素酶分类可进一步扩展以包括每个分类内的多个组分。例如，已从包括里氏木霉(Trichodermareesei)(也称为红褐肉座菌(Hypocreajecorina))的多个真菌来源分离了多种CBH、EG和BG，里氏木霉含有两种CBH的已知基因，即，CBHI(“CBH1”)和CBHII(“CBH2”)、至少8种EG的已知基因，即，EGI、EGII、EGIII、EGIV、EGV、EGVI、EGVII和EGVIII以及至少5种BG的已知基因，即，BG1、BG2、BG3、BG4、BG5和BG7(Foreman等人(2003),J.Biol.Chem.278(34):31988-31997)。EGIV、EGVI和EGVIII也具有木葡聚糖酶活性。为了将结晶纤维素有效地转化成葡萄糖，需要包含来自CBH、EG和BG每个分类的组分的整个纤维素酶体系，而分离的组分在水解结晶纤维素方面不太有效(Filho等人，Can.J.Microbiol.,42:1-5,1996)。内切-1,4-β-葡聚糖酶(EG)和外切纤维二糖水解酶(CBH)催化纤维素水解为纤维低聚糖(纤维二糖作为主要产物)，而β-葡萄糖苷酶(BGL)将低聚糖转化为葡萄糖。已观察到来自不同分类的纤维素酶组分之间的协同关系。具体地讲，EG型纤维素酶和CBH型纤维素酶协同地相互作用以有效地降解纤维素。虽然先前已经描述了β-葡萄糖苷酶组合物，但仍然需要新型的改善的β-葡萄糖苷酶组合物。改进的β-葡萄糖苷酶组合物例如在糖化生物质方面有用。尤其值得关注的是在表达、活性和稳定性之一者或多者中表现出所需的水平的β-葡萄糖苷酶。技术实现要素：本发明提供具有β-葡萄糖苷酶活性的多肽。本发明部分基于β-葡萄糖苷酶变体。还描述了编码β-葡萄糖苷酶变体的核酸、包含β-葡萄糖苷酶变体的组合物、使用β-葡萄糖苷酶变体的方法和鉴定另外有用的β-葡萄糖苷酶变体的方法。在本发明的一个方面，β-葡萄糖苷酶变体是红褐肉座菌(Hypocreajecorina)β-葡萄糖苷酶1(BGL1)变体。因此，在一个方面，本发明提供具有至少两种相比野生型BGL1改善的活性的β-葡萄糖苷酶1(BGL1)变体，所述至少两种改善的活性选自：与野生型BGL1相比，(a)预处理玉米秸秆(PCS)水解活性、(b)纤维二糖酶活性、(c)蛋白质表达(HPLC)、(d)通过在氨预处理玉米芯(CC)存在下纤维二糖酶活性或通过CC水解活性量度的β-葡萄糖苷酶活性、(e)热稳定性、(f)磷酸溶胀纤维素(PASC)水解活性和(g)在葡萄糖存在下增加的水解活性，其中BGL1变体是如表4-8、3-2、4-2和4-3所示的任何变体。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(b)和(d)活性，其中所述BGL1变体是L266A,I567E,S283F,S283P,T258E,T258I,T258K,T258Q,P536T,P536W,I532Y,Y530T,P607D,Q406M,Q406S,V602T,G300M,A630S,A630T,T180H,T180M,A450M,I444E,I444F,I444N,I444W,I444Y,V500Q,A633I,S482P,A667V,A485L,A485W,Y678R,V603G,L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,Y530S,Q684N,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,F260W,Y530F,Q406D,G605C,N263T,P607I,A450P,T242H,A630Y,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,L293F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(b)和(e)活性，其中所述BGL1变体是P607H,T011E,T011Y,N146E,I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,P536Q,N369E,N369W,N369Y,N146A,N146Q,P607K,N369T,A655N,I671K,F260T,P607S,F260D,F260G,F260Q,P607G,N400S,P607F,P607I,A450P,T242H,T568E,A630Y,A655D,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(b)和(f)活性，其中所述BGL1变体是N261C,T258C,F392Q,S624E,P607C,P604M,A377Q,N461A,N461F,N461P,T436A,T436C,T436F,T436I,T436M,T436Q,T436Y,Q220C,A655L,T646H,Y678F,A468I,D177M,P661E,L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,P536Q,N369E,N369W,N369Y,T436E,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260W,Y530F,N461V,I671C,K206A,A450P,T242H,E170F,S507G,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,L293F,A633C,S312C或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(a)和(b)活性，其中所述BGL1变体是I567Q,A565F,A565K,A565Q,A565V,F556E,F260I,P607E,G605R,G300C,A377C,A377D,S308C,N146H,N146S,A655C,A655G,P176L,T209I,L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,A565C,A655N,I671K,F260T,P607S,Y639V,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,P607F,Q406D,G605C,N263T,N461VI671C,K206A,T568E,E170F,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(b)和(c)活性，其中所述BGL1变体是I567K,I567R,A565E,A565S,A565Y,F392Y,Q406H,Q406T,P604C,N038F,T568A,N461G,Y639L,Y639M,T243A,T243C,Q245H,Q245M,Q245T,T646A,T646C,I671F,I671L,I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,A565C,Y639G,Y530F,N461V,I671C,K206A,T568E,A630Y,A655D,S507G,F260E,T568K，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(a)和(d)活性，其中所述BGL1变体是I567S,G606E,G606H,G606N,G606S,L293A,S308R,I444C,M201D,R542N,L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,N566F,L293M,Q220P,S692L,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,Q406D,G605C,N263T,S308E,A338D,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(e)和(g)活性，其中所述BGL1变体是L266F,I567Y,A270R,S384C,A630W,E128R,N146M,N146V,N146W,L181F,V043C,Y639P,S507F,Q245P,G662C,A630H,V466T,N146A,N146Q,P607K,N369T,S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D,P607F,A630Y,S308E,A655D，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(c)和(e)活性，其中所述BGL1变体是N261E,N261K,N400A,V602K,L293I,N461S,D457A,V043Q,Q303N,K320S,G662D,F260A,S474R,I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,A601D,S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D,D564T,T568E,A655D,A338D,F260E,T568K，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(a)和(f)活性，其中所述BGL1变体是N566L,N566P,N566W,A270K,A270N,F556H,F556K,P604N,N461D,N463E,K206G,A468Q,A468Y,N566F,N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,A468T,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,N461VI671C,K206A,E170F,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(a)和(c)活性，其中所述BGL1变体是S283D,A270D,N146Y,F260A,S474R,A565C,K206S,D564T,N461VI671C,K206A,T568E,A338D,F260E,T568K，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(a)和(e)活性，其中所述BGL1变体是F556G,F260S,P604E,P604V,N146D,Y639T,T221C,N473S,N583R,R645G,G662Y,F260A,S474R,A655N,I671K,F260T,P607S,S692L,D564T,F260D,F260G,F260Q,P607G,N400S,P607F,T568E,S308E,A338D,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(c)和(d)活性，其中所述BGL1变体是D259S,T243V,Y530F,A338D，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(d)和(e)活性，其中所述BGL1变体是S550Q,P607R,N400Q,V602F,A601G,A601L,L293K,Y575C,Y575R,A450Q,I486C,I486Y,A655S,Q245F,D329A,P536G,P607Q,A655Q,Y575A,Y575K,A630H,V466T,S692L,F260D,F260G,F260Q,P607G,N400S,P607I,A450P,T242H,S308E,A630Y,A338D,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(d)和(f)活性，其中所述BGL1变体是P536F,F392C,S624L,S624R,S624W,I486F,I486W,A667G,A667S,L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,N566F,Y575A,Y575K,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260W,P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,L293F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(c)和(g)活性，其中所述BGL1变体是S384G,S384W,N038E,N038M,N038P,V043H,V043W,Y068E,Y068G,Y068M,L110C,L110G,L110Q,L110W,A655H,N264L,S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D,Y639G,K206S,A655D，或S507G。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(d)和(g)活性，其中所述BGL1变体是G606D,Y068V,L293M,Q220P,A630H,V466T,Y530S,Q684N,F260W,Q406D,G605C,N263T,S308E,A630Y,L293F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(b)和(g)活性，其中所述BGL1变体是A377I,N461Y,N146A,N146Q,P607K,N369T,T436E,Y639G,Y530S,Q684N,Y639V,F260W,P607F,Q406D,G605C,N263T,A630Y,A655D,E170F,S507G,L293F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(c)和(f)活性，其中所述BGL1变体是K206D,A601D,Y530F,N461VI671C,K206A,S507G,F260E，或T568K。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(e)和(f)活性，其中所述BGL1变体是A468G,P536Q,N369E,N369W,N369Y,A601D,Y575A,Y575K,P607I,A450P,T242H,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F。在另一方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的(a)和(g)活性，其中所述BGL1变体是R179V,L293M,Q220P,K206S,A468T,Y639V,P607F,Q406D,G605C,N263T,S308E,E170F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)和(d)中的任何两种或所有三种的活性，其中所述BGL1变体是L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,Q406D,G605C,N263T,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(d)和(f)中的任何两种或所有三种的活性，其中所述BGL1变体是L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260W,Y530F,P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C,N455D，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(c)和(e)中的任何两种或所有三种的活性，其中所述BGL1变体是F260A,S474R,D564T,T568E,A338D,F260E,T568K，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(c)和(e)中的任何两种或所有三种的活性，其中所述BGL1变体是I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,T568E,A655D,F260E,T568K，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(d)和(f)中的任何两种或所有三种的活性，其中所述BGL1变体是N566F,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)和(f)中的任何两种或所有三种的活性，其中所述BGL1变体是N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,N461V,I671C,K206A,E170F,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)和(c)中的任何两种或所有三种的活性，其中所述BGL1变体是A565C,N461V,I671C,K206A,T568E,F260E,T568K，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(d)和(e)中的任何两种或所有三种的活性，其中所述BGL1变体是P536G,P607Q,A655Q,F260D,F260G,F260Q,P607G,N400S,P607I,A450P,T242H,A630Y,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(e)和(f)中的任何两种或所有三种的活性，其中所述BGL1变体是P536Q,N369E,N369W,N369Y,P607I,A450P,T242H,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(c)、(e)和(f)中的任何两种或所有三种的活性，其中所述BGL1变体是A601D,F260E或T568K。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(d)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是L293M,Q220P,Q406D,G605C,N263T,S308E,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(d)、(e)和(f)中的任何两种或所有三种的活性，其中所述BGL1变体是Y575A,Y575K,P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(d)、(e)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是A630H,V466T,S308E,A630Y，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(e)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是N146A,N146Q,P607K,N369T,P607F,A630Y,A655D，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(c)、(e)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D，或A655D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(f)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是T436E,F260W,E170F,S507G,L293F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(c)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是Y639G,P607F,A655D，或S507G。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)和(e)中的任何两种或所有三种的活性，其中所述BGL1变体是A655N,I671K,F260T,P607S,F260D,F260G,F260Q,P607G,N400S,P607F,T568E,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(c)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是K206S或P607F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(d)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是Y530S,Q684N,F260W,Q406D,G605C,N263T,A630Y,L293F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(f)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是A468T,E170F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(d)和(e)中的任何两种或所有三种的活性，其中所述BGL1变体是S692L,F260D,F260G,F260Q,P607G,N400S,S308E,A338D,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)和(g)中的任何两种或所有三种的活性，其中所述BGL1变体是Y639V。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(d)和(f)活性中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C，或S683W。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(d)和(e)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是F260D,F260G,F260Q,P607G，或N400S。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(d)、(f)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是F260W,L293F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(c)、(d)和(f)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是Y530F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(e)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是P607F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(d)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是Q406D,G605C,N263T,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(c)和(f)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是N461V,I671C,K206A,F260E或T568K。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(d)、(e)和(f)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(c)和(e)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是T568E,F260E,T568K，或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(d)、(e)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是S308E。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(d)、(e)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是A630Y或L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(c)、(e)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是A655D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(f)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是E170F,A633C,S312C，或N455D。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(c)、(d)和(e)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是A338D或F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(c)、(f)和(g)中的任何两种、任何三种或所有四种的活性，其中所述BGL1变体是S507G。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(c)、(e)和(f)中的任何两种、任何三种、任何四种或所有五种的活性，其中所述BGL1变体是F260E或T568K。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(d)、(e)和(f)中的任何两种、任何三种、任何四种或所有五种的活性，其中所述BGL1变体是P536C,A630Q,D215S,G372A,G547A,F611A,G662C，或G662F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(c)、(d)和(e)中的任何两种、任何三种、任何四种或所有五种的活性，其中所述BGL1变体是F260L。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(b)、(d)、(e)、(f)和(g)中的任何两种、任何三种、任何四种或所有五种的活性，其中所述BGL1变体是L293F。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，所述BGL1变体具有相比野生型BGL1改善的选自(a)、(b)、(d)、(f)和(g)中的任何两种、任何三种、任何四种或所有五种的活性，其中所述BGL1变体是A633C,S312C，或N455D。本发明还提供具有至少两种相比野生型BGL1改善的活性的BGL1变体，所述至少两种改善的活性选自：(a)预处理玉米秸秆(PCS)水解活性、(b)纤维二糖酶活性、(c)蛋白质表达、(d)通过氨预处理玉米芯(CC)水解活性量度的β-葡萄糖苷酶活性、(e)热稳定性、(f)磷酸溶胀纤维素(PASC)水解活性和(g)在葡萄糖存在下的水解活性，其中BGL1变体包含两个或更多个置换，所述置换选自表5-1列出的那些置换。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(a)和(c)并且置换是：L167W|D225Q,T242S|S312Y,D178K|A338K|S474D|G662L,K345E|N369T|G372A|K428N|P661L|S683W,D177M|D225Q|D564V|Q684G，和D178N|N264K|A338D|S474R|G662K,D177M|D564T|Q626F|Q684A,K428N|S683W,K345E|K428N|S683W,Q226Y|G372A|V603G|T666C,L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662D,L167W|D225Q|D564V|Q626F|Q684N,D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,K345E|N369E|G372A|P661E,N369T|P661L|S683W,R265M|K560S,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(b)和(f)并且置换是：L167W|D177M|D225Q|Q626F|Q684G,L167W|D177M|D564V|Q684G,D215S|S312Y,E170F|S312Y|N369Y,L167W|D225Q|Q626F|Q684R,L167W|D564T|Q626F,P176L|Q226W|Q316T|K320S|V522Y|G662C,R363E|V522Y|G662F,Q316T|K320S|V522Y|G662F,Q226W|K320Y|V522Y,Q316T|K320S|V522Y，和Q226W|K320S|R363E|V522Y|G662F,L167W|D177M|D225Q|D564V,D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,P176L|K320S|V522Y|G662C,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,E170F|Q226Y|N369Y|G372A|P661F，和L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|G372A|S683W,N369E|S683W,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(e)和(g)并且置换是：L167W|D225Q|Q626F|Q684D,L167W|D225Q|Q684N,L167W|D225Q|D564T|,Q626F|Q684C,Q626F|Q684D,N264M|R265P|N369I|D370W,R179V|N238F|D370W,R179V|N238F|K656R,R179V|N264M|D370W,R179V|N238F|R265M,R179V|R265P|D370W|K656R,R179V|N238W|N264M|R265M|N369I,R179V|N369I|D370W|K656R,R179V|N264M|R265P|K656R,R179V|R265M|N369I,R179V|N264M|R265M|D370W|K656R,R179V|N264M|R265M|N369I,R179V|N238W|N264M,N238W|N264M|R265M|D370W,R179V|N238W|R265P|D370W,R179V|N238W|N264M|D370W|K656R,N264M|R265P,R265P|D370W(任选地也包含G662F),R179V|N264M|R265P|N369I|D370W,R265M|N369I,R179V|R265M|D370W,N238W|N264M|R265P,R179V|N238W|N264M|R265P,N264M|N369I,N238F|R265M|N369I,N263C|K345E|N369E|G372A|K428N|P661E|S683W,N263C|K345E|N369T|G372A|K428N|P661E|S683W,N263C|K345E|N369E|G372A,N263C|P661L|S683W,N263C|K345E|N369T|G372A|K428N,K345E|G372A|K428N|P661E,E170F|Q226Y|N369Y|G372A,Q226Y|T242S|G372A|P661F,Q216E|T282I|S312D|S692K,Q216I|T282K|S312K|A622K,P176L|Q316T|G662C,Q226W|Q316T|V522Y|G662F,P176L|Q316T,A347Y|R542N,N238F|N264M|R265M|N369I,L167W|D225Q|D564V|Q626F|Q684N,E170F|V603G,L167W|D177M|D564T|Q684N,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(d)和(f)并且置换是：L167W|D177M|D564V|Q684R,L167W|D225Q|D564V,D177M|D225Q|D564T|Q626F|Q684N,L167W|Q626F,D225Q|D564V|Q626F|Q684R,D177M|D225Q|D564V|Q684R,Q226W|K320Y,P176L|V522Y,R363E|G662C,L167W|D225Q|Q626F|Q684R,L167W|D564T|Q626F,P176L|Q226W|Q316T|K320S|V522Y|G662C,R363E|V522Y|G662F,Q316T|K320S|V522Y|G662F,Q226W|K320Y|V522Y,Q316T|K320S|V522Y,Q226W|K320S|R363E|V522Y|G662F,L167W|D177M|D564T|Q626F|Q684N,L167W|Q626F|Q684D,L167W|D177M|D564T|Q684R,L167W|D177M|D225Q|Q684D,R179V|R265P|N369I,Q316T|K320Y|R363E|V522Y|G662F,L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,P176L|K320S|V522Y|G662C,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(b)和(e)并且置换是：K345E|N369E|K428N|P661L,Q316T|K320Y|V522Y,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,P176L|K320S|V522Y|G662C,K345E|N369E|P661L,L167W|D177M|D564T|Q684N,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(d)和(e)并且置换是：N263C|K345E|N369E|P661L,N238F|N264M|R265M|N369I,P176L|K320S|V522Y|G662C,K345E|N369E|P661L,E170F|V603G,L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(b)和(g)并且置换是：E170F|T242S|N369Y|G372A|V603G|T666C,E170F|Q226Y|N369Y|V603G|T666C,E170F|Q226Y|S312Y,L167W|D177M|D225Q|D564V,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,L167W|D177M|D564T|Q684N,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(d)和(g)并且置换是：D178I|Q303E|A338I,Q316T|K320Y|G662F,L167W|D177M|D564T|Q626F|Q684N,L167W|Q626F|Q684D,L167W|D177M|D564T|Q684R,L167W|D177M|D225Q|Q684D,R179V|R265P|N369I,Q316T|K320Y|R363E|V522Y|G662F,N238F|N264M|R265M|N369I,N238W|R265P|K656R,N264M|R265P,(任选地也包含G662F),N264L|A338I|S474R|G662D,L167W|D177M|Q626F|Q684G，和L167W|D177M|D564V|Q626F|Q684A,E170F|V603G,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(d)和(f)中的任何两种或所有三种并且置换是：L167W|D225Q|Q626F|Q684R,L167W|D564T|Q626F,P176L|Q226W|Q316T|K320S|V522Y|G662C,R363E|V522Y|G662F,Q316T|K320S|V522Y|G662F,Q226W|K320Y|V522Y,Q316T|K320S|V522Y,Q226W|K320S|R363E|V522Y|G662F,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,P176L|K320S|V522Y|G662C,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(d)、(f)和(g)中的任何两种或所有三种并且置换是：L167W|D177M|D564T|Q626F|Q684N,L167W|Q626F|Q684D,L167W|D177M|D564T|Q684R,L167W|D177M|D225Q|Q684D,R179V|R265P|N369I,Q316T|K320Y|R363E|V522Y|G662F,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)和(e)中的任何两种或所有三种并且置换是：K345E|N369T|G372A|K428N|P661L|S683W,L167W|D225Q|D564V|Q626F|Q684N,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369E|P661L,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(f)和(g)中的任何两种或所有三种并且置换是：L167W|D177M|D225Q|D564V,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)和(d)中的任何两种或所有三种并且置换是：D177M|D225Q|D564V|Q684G,D178N|N264K|A338D|S474R|G662K,L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662D,K345E|N369E|G372A|P661E,N369T|P661L|S683W,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,E170F|V603G,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)和(g)中的任何两种或所有三种并且置换是：D177M|D564T|Q626F|Q684A,N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662D,|D225Q|D564V|Q626F|Q684N,R265M|K560S,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(d)、(e)和(g)中的任何两种或所有三种并且置换是：N238F|N264M|R265M|N369I,E170F|V603G,L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)和(c)中的任何两种或所有三种并且置换是：K428N|S683W,K345E|K428N|S683W,Q226Y|G372A|V603G|T666C,D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,K345E|N369E|G372A|P661E,N369T|P661L|S683W,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(d)和(f)中的任何两种、任何三种或所有四种并且置换是：L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(a)、(c)、(d)和(g)并且置换是：N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662DE170F|V603G,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(e)和(g)中的任何两种、任何三种或所有四种并且置换是：L167W|D225Q|D564V|Q626F|Q684N,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)和(f)中的任何两种、任何三种或所有四种并且置换是：D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|G372A|S683W,N369E|S683W,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)和(d)中的任何两种、任何三种或所有四种并且置换是：K345E|N369E|G372A|P661E,N369T|P661L|S683W,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(d)、(f)和(g)中的任何两种、任何三种或所有四种并且置换是：L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(f)和(g)中的任何两种、任何三种或所有四种并且置换是：R265M|K560S,K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)和(e)中的任何两种、任何三种或所有四种并且置换是：N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369E|P661L,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(d)、(e)和(f)中的任何两种、任何三种或所有四种并且置换是：P176L|K320S|V522Y|G662C,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)和(f)中的任何两种、任何三种、任何四种或所有五种并且置换是：K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)和(e)中的任何两种、任何三种、任何四种或所有五种并且置换是：K345E|N369E|P661L,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(d)、(e)和(g)中的任何两种、任何三种、任何四种或所有五种并且置换是：E170F|V603G,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，和P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(d)、(f)和(g)中的任何两种、任何三种、任何四种或所有五种并且置换是：E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(d)、(e)和(g)中的任何两种、任何三种、任何四种或所有五种并且置换是：L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(e)、(f)和(g)中的任何两种、任何三种、任何四种、任何五种或所有六种并且置换是：K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)、(e)和(g)中的任何两种、任何三种、任何四种、任何五种或所有六种并且置换是：G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)、(e)和(f)中的任何两种、任何三种、任何四种、任何五种或所有六种并且置换是：K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)、(e)、(f)和(g)中的任何两种、任何三种、任何四种、任何五种、任何六种或所有七种并且置换是：N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。本发明提供β-葡萄糖苷酶变体，其中所述变体是具有β-葡萄糖苷酶活性并且包含突变的成熟形式，其中当该突变是单个突变时，其不在选自以下的位置：37、61、125、129、132、133、158、159、166、177、236、237、238、240、252、314、444和449，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。在一些实施方案中，突变是置换。在其他实施方案中，突变是缺失或插入。在一些优选的实施方案中，突变不由选自以下的置换组成：W37A,W37G,W37N,W37D,D61N,D61A,R125K,R125A,K158G,H159A,H159S,E166Q,D177E,D236G,D236N,D236E,W237F,W237A,W237L,W237C，和W237P。在一些优选的实施方案中，与参考BGL1相比，所述置换导致产生出表达、活性和稳定性之一者或多者得以改善的β-葡萄糖苷酶变体。此外，本发明提供β-葡萄糖苷酶变体，其中所述变体是具有β-葡萄糖苷酶活性并且包含置换的成熟形式，所述置换发生在选自以下的一个或多个位置：22、24、25、26、27、28、33、35、36、37、50、51、52、61、67、91、92、93、99、100、125、158、159、163、164、165、166、167、168、169、170、176、177、178、179、194、196、199、204、208、209、214、215、216、224、225、226、236、237、238、242、248、249、263、264、265、276、277、278、279、282、284、287、291、301、302、303、306、312、313、316、320、324、328、329、334、335、336、337、338、339、344、345、347、361、363、369、370、371、372、374、375、380、381、382、396、397、398、399、402、409、410、411、420、426、427、428、441、445、446、447、448、449、452、453、454、455、460、467、473、474、475、489、490、492、496、497、498、521、522、534、542、547、548、553、554、555、560、561、563、564、570、571、581、583、586、591、603、611、612、622、626、627、638、642、643、645、649、650、656、660、661、662、663、666、672、673、674、675、680、681、682、683、684、685、692、702和705，其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。在一些实施方案中，所述变体包含发生在选自以下的一个或多个位置的另外的置换：37、61、158、159、166、236、237、238和449，其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。在一些实施方案中，所述另外的置换选自：W037A,D061N,K158G,H159A或S,E166Q,D236G，和W237P。此外，在一些实施方案中，所述发生在一个或多个位置的置换选自：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24和25个位置。在一些实施方案中，所述变体衍自选自以下的亲本β-葡萄糖苷酶：红褐肉座菌BGL1(TrireBGL1)、异常汉逊酵母(Hansenulaanomala)BGL(HananBglu)、厌气性瘤胃真菌(Piromycessp)BGL(PirspBglu)、厌酷球孢子菌(Coccidioidesimmitis)BGL(CocimBglu)、扣囊复膜酵母(Saccharomycopsisfibuligera)BGL2(SacfiBglu2)、扣囊复膜酵母BGL1(SacfiBglu1)、番茄壳针孢菌(Septorialycopersici)BGL(SeplyBglu)、髓囊毕赤酵母(Kuraishiacapsulata)BGL(KurcaBglu)、里氏木霉BGL7(TrireBGL7)、蚕豆单胞锈菌(Uromycesfabae)BGL(UrofaBglu)、土曲霉(Aspergillusterreus)BGL(AspteBglu)、球毛壳菌(Chaetomiumglobosum)BGL(ChaglBglu)、里氏木霉BGL3(TrireBGL3)、巴西青霉(Penicilliumbrasilianum)BGL(PenbrBGL)、黑团孢霉(Periconiasp)BGL(PerspBglu)、PhaeosphaeriaavenariaBGL(PhaavBglu)、烟曲霉(Aspergillusfumigatus)BGL(AspfuBGL)、米曲霉(Aspergillusoryzae)BGL1(AsporBGL1)、棘孢曲霉(Aspergillusaculeatus)BGL1(AspacBGL1)、黑曲霉(Aspergillusniger)BGL(AspniBGL)、埃默森篮状菌(Talaromycesemersonii)BGL(TalemBglu)和嗜热子囊菌(Thermoascusaurentiacus)BGL(TheauBGL)。在一些优选的实施方案中，所述变体衍自这样的亲本β-葡萄糖苷酶，其氨基酸序列与由以下成员组成的组中的某个成员具有至少75％(80％、85％、90％、95％、96％、97％、98％或99％)同一性：SEQIDNO:3、SEQIDNO:4、SEQIDNO:5、SEQIDNO:6、SEQIDNO:7、SEQIDNO:8、SEQIDNO:9、SEQIDNO:10、SEQIDNO:11、SEQIDNO:12、SEQIDNO:13、SEQIDNO:14、SEQIDNO:15、SEQIDNO:16、SEQIDNO:17、SEQIDNO:18、SEQIDNO:19、SEQIDNO:20、SEQIDNO:21、SEQIDNO:22、SEQIDNO:23和SEQIDNO:24。在一些优选的实施方案中，所述变体包含1至59个保守残基，所述残基选自A16、K28、G34、G44、C58、D61、R67、E100、G105、L110、P112、G124、R125、E128、D133、P134、L136、G147、Q149、K158、H159、R169、S173、D178、P188、F189、M201、Y204、N208、K224、F229、G231、D236、W237、G250、D252、M253、M255、P256、R284、D287、R291、K335、N336、L341、P342、G385、P395、E441、D452、V478、L518、Y559、F562、F573、G574、G576、L577和L651，其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。在这些实施方案的一个子集中，所述变体包含E441和D452。在优选的实施方案中，当与参考BGL1相比时，所述置换导致产生出表达、活性和稳定性之一者或多者得以改善的β-葡萄糖苷酶变体。本发明还提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022置换成A、E、F、G、H、I、P、Q、R、S、V、W或Y；N024置换成A、C、D、E、F、G、K、L、M、P、Q、R、S、T、V或Y；L025置换成A、D、F、G、I、K、N、Q、R、S、T、V、W或Y；Q026置换成C、D、E、G、H、I、K、L、P、R、S、T、V、W或Y；D027置换成A、C、E、L、M、Q、S、T或V；K028置换成L、M、N、S或V；S033置换成C、G或T；V035置换成C、E、G、H、K、L、N、P、Q、R、S、T、W或Y；G036置换成C、D、E、F、I、K、N、R、S、W或Y；W037置换成E、F、H、I、M、S、V或Y；S050置换成A、C、F、G、I、K、L、M、N、P、R、T、V或Y；K051置换成A、C、D、E、G、H、I、L、M、N、Q、R、S、T或V，I052置换成A、D、F、K、M、N、P、Q、S、T或V；D061置换成E、G或P；R067置换成A、C、D、E、F、G、I、L、M、N、P、Q、S、T、V、W或Y；R091置换成A、C、D、E、F、G、H、I、K、L、N、Q、S、T、V、W或Y；E092置换成A、C、D、F、H、I、K、L、M、N、Q、R、T、V或Y；R093置换成A、C、D、E、F、G、H、K、L、M、Q、S、T、V或W；E099置换成A、D、F、I、K、M、N、W或Y；E100置换成A、G、I、K、L、M、N、Q、S、T或Y；R125置换成A或D；K158置换成A、C、H或T；H159置换成C、E、G、N、W或Y；N163置换成A、H或S；E164置换成G或S；Q165置换成C、D、F、G、H、I、K、L、M、N、R、S、T、V、W或Y；E166置换成D、F、K、L、N、P、R、S、T或Y；L167置换成A、C、D、E、F、G、M、N、Q、R、S、V、W或Y；N168置换成A、D、E、G、H、Q、R、T或Y；R169置换成A、C、D、E、F、H、K、Q、S或T；E170置换成A、D、F、I、K、L、M、P、V、W或Y；P176置换成A、D、E、F、G、H、K、L、M、Q、R、S、T、V、W或Y；D177置换成A、C、E、F、G、H、K、L、M、N、Q、R、V、W或Y；D178置换成A、C、E、K、N、P、Q、R、S、T、W或Y；R179置换成A、C、G、I、K、M、Q、S、T、V或W；Q194置换成A、C、E、F、G、H、K、L、M、R、T、W或Y；N196置换成E、G、H、L、M、P、Q、R或T；S199置换成A、G、N、T或V；Y204置换成A、E、F、G、H、I、K、M、P、Q、R、S、T、V或W；N208置换成K或R；T209置换成C、D、E、G、H、I、K、L、M、Q、R、S、V、W或Y；E214置换成A、C、D、G、H、K、L、M、N、P、Q、R、S、T、V、W或Y；D215置换成A、C、E、F、G、H、L、M、N、Q、S、V或W；Q216置换成A、C、D、E、F、G、H、I、K、L、M、N、P、R、S、T、W或Y；K224置换成H、R或V；D225置换成A、C、E、F、G、H、I、L、M、Q、S、T、V、W或Y；Q226置换成A、C、D、E、F、H、I、K、L、M、N、R、S、T、V、W或Y；D236置换成A、P、Q、S或T；W237置换成H、I、K、M、R、S、T或Y；N238置换成A、C、D、E、F、G、M、P、S、T或W；T242置换成A、C、E、F、G、H、I、K、L、M、N、Q、R、S、V、W或Y；N248置换成A、C、F、G、L、T、W或Y；S249置换成A、G、I、M或V；N263置换成A、C、D、E、F、G、H、I、K、L、P、Q、R、S、T、V或Y；N264置换成A、C、D、E、G、H、K、L、M、Q、R、S、T、V或Y；R265置换成A、E、F、G、I、K、L、M、N、P、Q、S、T、V或Y；N276置换成A、C、F、K、M或Q；S277置换成A、C、D、E、F、G、H、I、M、N、P、Q、R、W或Y；N278置换成A、C、D、F、G、H、I、L、M、Q、R、S、T、V、W或Y；Q279置换成C、D、E、G、H、I、K、N、S、T、V或Y；T282置换成C、D、G、H、K、L、N、P、R、S或V；R284置换成H、M或N；D287置换成C、E、F、G、H、I、K、L、M、N、S、V、W或Y；Q301置换成A、E、G、K、L、N、R、S、T或V；D302置换成A、C、E、F、G、K、L、M、N、P、S、T、W或Y；Q303置换成A、C、D、E、F、G、H、I、K、L、M、N、P、R、S、T、V、W或Y；Y306置换成A、C、E、F、G、I、K、L、M、N、P、Q、R、S、T、V或W；S312置换成A、C、D、G、I、K、L、M、N、Q、R、T、V、W或Y；R313置换成A、C、D、E、G、K、L、N、S、V或W；Q316置换成A、C、D、E、F、G、H、I、K、L、M、N、P、R、S、T、V、W或Y；K320置换成A、C、E、G、H、L、M、N、P、Q、R、S、T或Y；R324置换成C、D、E、F、H、I、K、L、M、Q、V、W或Y；R328置换成C、E、F、G、I、K、L、M、Q、S、T、V或Y；D329置换成A、E、F、G、H、M、N、Q、S、T或Y；L334置换成A、C、F、M、T、V或W；K335置换成A、D、F、G、H、I、L、M、N、R、S、T、V或W；N336置换成A、C、G、H、L、M、Q、R、S、T、V或Y；D337置换成A、C、E、G、H、K、L、M、N、R、S、T、V、W或Y；A338置换成C、D、E、F、G、H、I、K、L、M、N、P、Q、R、V、W或Y；N339置换成D、E、G、H、I、K、L、P、Q、R、V或Y；K344置换成D、E、F、G、I、L、M、N、P、Q、R、S、T或V；K345置换成A、D、E、F、G、H、N、P、Q、R、S、T、V、W或Y；A347置换成D、F、H、I、K、L、M、P、Q、R、S或Y；H361置换成A、C、D、E、G、K、L、M、N、P、S、T或Y；R363置换成A、C、E、G、K、L、M、N、Q、S、T、V、W或Y；N369置换成A、C、D、E、F、I、L、M、R、S、T、V、W或Y；D370置换成E、F、G、Q、S、W或Y；K371置换成A、D、F、G、H、L、N、Q、R、S、T、V或W；G372置换成A、C、D、E、K、L、M、N、S、T、V、W或Y；D374置换成A、C、F、G、I、L、M、N、Q、R、S、T、V、W或Y；D375置换成A、C、E、H、I、R、V或W；M380置换成E、F、G、I、L、N、Q、S、T、V或Y；G381置换成H；W382置换成F、N或Y；Y396置换成A、C、D、E、F、G、H、I、K、L、M、N、Q、R、S、T、V或W；D397置换成A、C、E、F、H、I、K、L、M、N、P、Q、R、S、T、V或Y；A398置换成C、D、E、F、G、H、I、K、L、M、N、P、Q、R、S、T、V、W或Y；I399置换成A、C、D、E、F、G、L、M、Q、S、T、V、W或Y；R402置换成A、C、E、F、G、I、L、P、Q、S、V、W或Y；Q409置换成C、D、G、H、I或V；V410置换成A、C、F、G、H、I、L、N、R、S、T、W或Y；T411置换成D、E、F、G、H、I、K、L、N、Q、R、S、V或Y；S420置换成A、C、D、G、H、K、N、Q、T、V或Y；R426置换成A、E、F、I、K、L、M、N、P、Q、S、T、W或Y；G427置换成C、D、E、F、H、K、L、M、N、P、Q、R、S、T、V、W或Y；K428置换成A、C、D、E、F、G、H、I、L、M、N、P、Q、R、S、T、V、W或Y；E441置换成A、C、D或G；T445置换成A、C、D、E、F、G、I、K、L、M、N、P、Q、R、S、V或Y；V446置换成A、C、K、Q或R；E447置换成A、K、L、N、S、V、W或Y；G448置换成A、C、D、E、F、H、K、L、M、N、Q、R、S、T、V或Y；N449置换成A、C、E、F、G、H、K、L、M、P、R、T、V或W；D452置换成N；R453置换成A、E、L、M、Q或S；N454置换成A、F、G、K、L、M、R、S、T或V；N455置换成A、C、D、E、F、G、H、I、L、M、S、T、V、W或Y；H460置换成A、C、D、E、F、G、I、K、L、M、N、Q、R、S、W或Y；Q467置换成A、C、D、E、H、K、N、P、S、V、W或Y；N473置换成A、C、E、F、G、H、K、L、M、P、Q、R、S、T、V或W；S474置换成A、C、D、E、F、G、I、K、L、M、N、P、Q、R、T、V或Y；N475置换成I、K、L、M、P、Q、R、S、T、V、W或Y；E489置换成D或N；Q490置换成A、C、E、F、G、H、K、L、P、R、S、T、V、W或Y；L492置换成A、D、F、H、I、M、N、Q、R、T、W或Y；Q496置换成A、G、K、N、P、S、T、V或W；V497置换成A、C、I、M、N或T；K498置换成A、C、E、F、G、H、I、L、M、N、Q、R、S、T、V或Y；D521置换成A、C、E、F、G、H、I、K、L、M、P、R、S、T、V、W或Y；V522置换成A、C、F、G、H、I、K、L、M、N、P、Q、R、S、T、W或Y；K534置换成C、D、E、F、G、H、I、N、Q、R、S、T或V；R542置换成A、C、D、E、F、G、H、I、K、L、M、N、P、Q、S、T、V、W或Y；G547置换成A、C、E、F、K、L、N、P、Q、R、T、V或Y；S548置换成C、E、F、H、I、L、M、N、Q、R、T、V、W或Y；E553置换成D、I、K、N、Q、W或Y；G554置换成A、C、D、F、H、K、L、M、Q、R、S、T、V或W；L555置换成A、C、D、E、F、G、H、I、K、M、N、P、Q、T、V、W或Y；K560置换成A、C、E、G、H、I、L、M、N、P、Q、R、S、T、V、W或Y；H561置换成A、C、D、E、F、G、I、M、N、Q、S、T、V或W；D563置换成A、C、E、F、I、L、M、Q、R、S、T、V、W或Y；D564置换成A、C、E、F、G、K、L、M、N、Q、R、S、T、V或Y；R570置换成A、C、D、E、G、H、I、M、N、Q、S、T或V；Y571置换成H、M、N、R或W；K581置换成A、C、D、E、F、G、H、I、L、M、N、P、R、S、T、V、W或Y；N583置换成A、C、D、E、F、G、H、I、K、L、M、P、R、S、T、V、W或Y；R586置换成D、E、F、G、H、L、N、P、V、W或Y；S591置换成C、D、F、G、H、I、K、M、P、Q或V；V603置换成A、C、D、E、F、G、H、L、M、N、P、Q、R、S、T、W或Y；F611置换成A、C、D、G、I、K、L、M、N、R、S、T、V、W或Y；Q612置换成C、D、F、G、H、I、K、L、M、R、S、V或W；A622置换成D、E、F、G、H、I、K、L、M、N、P、R、S、T、V、W或Y；Q626置换成E、F、G、H、I、L、M、T或V；V627置换成D、K、P、Q、R、S或Y；T638置换成A、D、E、F、G、I、K、L、M、P、Q、R、S、V、W或Y；S642置换成A、C、D、E、F、G、H、I、K、L、M、N、P、Q、R、T、V、W或Y；A643置换成C、E、F、G、H、K、L、M、N、Q、R、S、T、V、W或Y；R645置换成A、D、E、F、G、H、I、K、L、M、P、Q、S、T、V、W或Y；K649置换成A、C、F、I、L、M、N、Q、S、T、W或Y；Q650置换成A、C、D、E、F、G、H、I、K、L、M、N、R、T、V或Y；K656置换成R；T660置换成C、D、E、F、G、H、I、K、M、N、P、Q、R、S、V、W或Y；P661置换成A、C、D、E、F、G、H、I、K、L、M、Q、R、S、T、V或W；G662置换成A、C、D、E、F、H、I、K、L、M、N、Q、R、S、T、W或Y；Q663置换成A、C、D、E、F、G、H、I、K、L、M、N、R、S、V或W；T666置换成A、C、D、E、F、G、H、K、L、N、R、S、V、W或Y；R672置换成C、D、E、F、G、H、I、K、L、M、N、T、V、W或Y；R673置换成A、C、E、F、G、H、I、K、L、M、N、Q、S、T、V或W；R674置换成K、L、M、Q、T、V或Y；D675置换成C、E、H、L、S或Y；D680置换成A、C、E、F、H、I、K、L、M、N、Q、R、S、V、W或Y；T681置换成A、G、H、K、L、M、N、P、Q、R、S、V、W或Y；A682置换成C、E、I、L、M、N、P、S、W或Y；S683置换成A、C、D、E、F、G、I、K、L、M、P、Q、R、V或W；Q684置换成A、C、D、E、F、G、H、I、K、L、M、N、P、R、S或T；K685置换成A、E、F、G、I、L、M、N、Q、R、S、T、V、W或Y；S692置换成C、E、H、I、K、L、M、N、P、Q、T、V或W；R702置换成C、D、F、G、H、I、K、L、M、N、Q、S、T、V或W；和R705置换成C、F、H、I、L、M、P、S、T、V或W，其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体对于以下特性中的至少一种具有大于1的PI：表达(HPLC)、CNPG酶活性、热稳定性、降低的葡萄糖抑制、pH5下的纤维二糖酶活性、pH6下的纤维二糖酶活性、在铵(ammonium)预处理玉米芯存在下的纤维二糖酶活性和酸预处理玉米秸秆的水解。本发明还提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022S,K022W,N024A,N024D,N024E,N024L,N024P,L025W,V035S,V035W,W037E,W037F,W037H,W037S,W037Y,K051A,D061E,D061G,D061P,R067G,R067L,R067M,R067P,R067T,R067V,R067Y,R091I,R091T,R091Y,E092K,E092L,E092T,R125A,R125D,K158A,K158C,H159C,H159E,H159G,H159N,H159W,H159Y,N163A,N163H,N163S,L167W,R169A,R169C,R169D,R169E,R169K,E170A,E170K,E170L,E170P,E170W,E170Y,P176A,P176D,P176G,D177C,D177G,D177K,D177N,D178A,D178E,D178P,D178T,D178W,Q194A,Q194Y,S199A,Y204A,Y204E,Y204G,Y204H,Y204I,Y204K,Y204P,Y204Q,Y204R,Y204S,Y204T,Y204V,Y204W,Q216D,Q216E,Q216N,Q216R,D225C,Q226A,D236A,D236P,D236Q,D236S,D236T,W237H,W237I,W237K,W237M,W237R,W237S,W237T,T242S,N248A,N248C,S249A,N264D,N264E,N264H,N264L,N264R,N264S,N264V,N264Y,R265A,R265G,R265Y,S277A,S277D,N278A,N278D,T282D,T282N,T282R,T282V,Q303A,Q303E,Q303N,Y306A,Y306E,Y306F,Y306L,Y306W,S312A,S312D,S312G,S312I,S312N,S312R,R313D,R313E,Q316A,Q316D,Q316F,K320A,K320H,K320N,K320S,K320Y,K335L,K335S,K335T,A338D,A338E,A338G,A338N,A338R,A347Y,R363A,R363G,R363K,R363M,R363V,D370E,D370Q,K371A,K371H,D374A,Y396A,D397N,I399L,S420A,S420D,G427E,G427S,K428A,E441A,E441C,E441D,E441G,V446A,E447A,E447N,G448A,G448D,G448F,G448M,G448N,G448R,G448S,G448T,G448Y,N454A,N473S,S474D,S474G,S474K,S474N,S474R,S474T,S474V,S474Y,E489D,D521A,K534Q,R542A,R542D,G547E,G547L,G547P,S548E,S548F,S548H,S548L,K560H,N583D,R586D,V603L,V603M,V603Q,V603S,Q612D,Q612G,Q612K,Q612V,A622L,A622W,A622Y,Q626I,Q626L,Q626T,Q626V,T638D,S642D,A643M,K649A，或K649W；Q650D；G662D,G662E,G662L,G662S，或G662T；Q663D，或Q663G；T666A,R673N,R673W,S683K,Q684D,Q684F,Q684H,Q684K,Q684L,Q684M,Q684R,Q684S，和Q684T，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的表达水平(例如，PI大于1)。另外，本发明提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022F,K022G,K022H,K022I,K022P,K022Q,K022R,K022V,K022Y,N024C,N024F,N024G,N024K,N024M,N024Q,N024R,N024S,N024T,N024V,N024Y,L025A,L025D,L025F,L025G,L025I,L025K,L025N,L025Q,L025R,L025S,L025T,L025V,L025Y,Q026C,Q026D,Q026E,Q026G,Q026H,Q026I,Q026K,Q026L,Q026P,Q026R,Q026S,Q026T,Q026V,Q026W,Q026Y,D027A,D027C,D027E,D027L,D027M,D027Q,D027S,D027T,D027V,K028L,K028M,K028S,K028V,S033C,S033G,S033T,V035C,V035E,V035G,V035H,V035K,V035L,V035N,V035P,V035Q,V035R,V035T,V035Y,G036C,G036D,G036E,G036K,G036N,G036R,G036S,W037M,W037V,S050A,S050C,S050F,S050G,S050I,S050K,S050L,S050M,S050N,S050P,S050R,S050T,S050V,S050Y,K051C,K051D,K051E,K051G,K051H,K051I,K051L,K051M,K051N,K051Q,K051R,K051S,K051T,K051V,I052A,I052F,I052M,I052P,I052S,I052T,I052V,R067A,R067C,R067D,R067E,R067F,R067I,R067N,R067Q,R067S,R067W,R091A,R091D,R091E,R091F,R091G,R091H,R091K,R091L,R091N,R091Q,R091S,R091V,R091W,E092A,E092C,E092D,E092F,E092H,E092I,E092M,E092N,E092Q,E092R,E092V,E092Y,R093A,R093C,R093D,R093E,R093F,R093H,R093K,R093L,R093M,R093Q,R093S,R093T,R093V,R093W,E099A,E099D,E099F,E099I,E099K,E099M,E099N,E099W,E099Y,E100A,E100G,E100I,E100K,E100L,E100M,E100N,E100Q,E100S,E100T,E100Y,K158H,K158T,E164G,E164S,Q165C,Q165D,Q165F,Q165G,Q165H,Q165I,Q165K,Q165L,Q165M,Q165N,Q165R,Q165S,Q165T,Q165V,Q165W,Q165Y,E166D,E166K,E166L,E166N,E166P,E166R,E166S,E166T,E166Y,L167A,L167C,L167D,L167E,L167F,L167G,L167M,L167N,L167Q,L167R,L167S,L167V,L167Y,N168A,N168D,N168E,N168G,N168H,N168Q,N168R,N168T,N168Y,R169F,R169H,R169Q,R169S,R169T,E170D,E170F,E170I,E170M,E170V,P176E,P176F,P176H,P176K,P176L,P176M,P176Q,P176R,P176S,P176T,P176V,P176W,P176Y,D177A,D177E,D177F,D177H,D177L,D177M,D177Q,D177R,D177V,D177W,D177Y,D178C,D178K,D178N,D178Q,D178R,D178S,D178Y,R179A,R179C,R179G,R179I,R179K,R179S,R179T,R179V,R179W,Q194C,Q194E,Q194F,Q194G,Q194H,Q194K,Q194L,Q194M,Q194R,Q194T,Q194W,N196E,N196G,N196H,N196L,N196M,N196P,N196Q,N196R,N196T,S199G,S199N,S199T,S199V,Y204F,Y204M,N208K,N208R,T209C,T209D,T209E,T209G,T209H,T209I,T209K,T209L,T209M,T209Q,T209R,T209S,T209V,T209W,T209Y,E214A,E214C,E214D,E214G,E214H,E214K,E214L,E214M,E214N,E214P,E214Q,E214R,E214S,E214T,E214V,E214Y,D215A,D215C,D215E,D215F,D215G,D215H,D215L,D215M,D215N,D215Q,D215S,D215W,Q216A,Q216C,Q216F,Q216G,Q216H,Q216I,Q216K,Q216L,Q216M,Q216P,Q216S,Q216T,Q216W,Q216Y,K224H,K224R,K224V,D225A,D225E,D225F,D225G,D225H,D225I,D225L,D225M,D225Q,D225S,D225T,D225V,D225W,D225Y,Q226C,Q226D,Q226E,Q226F,Q226H,Q226I,Q226K,Q226L,Q226M,Q226N,Q226R,Q226S,Q226T,Q226V,Q226W,Q226Y,W237Y,N238A,N238C,N238D,N238E,N238F,N238G,N238M,N238P,N238S,N238T,N238W,T242A,T242C,T242E,T242F,T242G,T242H,T242I,T242K,T242L,T242M,T242N,T242Q,T242R,T242V,T242W,T242Y,N248F,N248G,N248L,N248T,N248W,N248Y,S249G,S249I,S249M,S249V,N263A,N263C,N263D,N263E,N263F,N263G,N263H,N263I,N263K,N263L,N263P,N263Q,N263R,N263S,N263T,N263V,N263Y,N264A,N264C,N264G,N264K,N264M,N264Q,N264T,R265E,R265F,R265I,R265K,R265L,R265M,R265N,R265P,R265Q,R265S,R265T,R265V,N276A,N276F,N276K,N276M,N276Q,S277C,S277E,S277F,S277G,S277H,S277I,S277M,S277N,S277P,S277Q,S277R,S277Y,N278C,N278F,N278G,N278H,N278I,N278L,N278M,N278Q,N278R,N278S,N278T,N278V,N278W,N278Y,Q279C,Q279D,Q279E,Q279G,Q279H,Q279I,Q279K,Q279N,Q279S,Q279T,Q279V,Q279Y,T282C,T282G,T282K,T282L,T282P,T282S,R284H,R284N,D287C,D287E,D287F,D287G,D287H,D287I,D287K,D287L,D287M,D287N,D287S,D287V,D287W,D287Y,Q301A,Q301E,Q301G,Q301L,Q301N,Q301R,Q301S,Q301T,Q301V,D302A,D302C,D302E,D302F,D302G,D302K,D302L,D302M,D302N,D302P,D302S,D302T,D302W,D302Y,Q303C,Q303D,Q303F,Q303G,Q303H,Q303I,Q303K,Q303L,Q303M,Q303P,Q303R,Q303S,Q303T,Q303V,Q303W,Q303Y,Y306C,Y306G,Y306I,Y306K,Y306M,Y306N,Y306P,Y306Q,Y306R,Y306S,Y306T,Y306V,S312C,S312K,S312L,S312M,S312Q,S312T,S312V,S312W,S312Y,R313A,R313C,R313G,R313K,R313L,R313N,R313S,R313V,R313W,Q316C,Q316E,Q316G,Q316H,Q316I,Q316K,Q316L,Q316M,Q316N,Q316P,Q316R,Q316S,Q316T,Q316V,Q316W,Q316Y,K320C,K320E,K320G,K320L,K320M,K320P,K320Q,K320R,K320T,R324C,R324D,R324E,R324F,R324H,R324I,R324K,R324L,R324M,R324Q,R324V,R324W,R324Y,R328C,R328E,R328G,R328I,R328K,R328L,R328M,R328Q,R328S,R328T,R328V,D329A,D329E,D329F,D329G,D329H,D329M,D329N,D329Q,D329S,D329T,D329Y,L334A,L334C,L334F,L334M,L334T,L334V,L334W,K335A,K335D,K335F,K335G,K335H,K335I,K335M,K335N,K335R,K335V,K335W,N336A,N336C,N336G,N336H,N336L,N336M,N336Q,N336R,N336S,N336T,N336V,N336Y,D337A,D337C,D337E,D337G,D337H,D337K,D337L,D337M,D337N,D337R,D337S,D337T,D337V,D337W,D337Y,A338C,A338F,A338H,A338I,A338K,A338L,A338M,A338P,A338Q,A338V,A338W,A338Y,N339D,N339E,N339G,N339H,N339I,N339K,N339L,N339P,N339Q,N339R,N339V,N339Y,K344D,K344E,K344F,K344G,K344I,K344L,K344M,K344N,K344P,K344Q,K344R,K344S,K344T,K344V,K345A,K345D,K345E,K345F,K345G,K345H,K345N,K345P,K345Q,K345R,K345S,K345T,K345V,K345W,K345Y,A347D,A347F,A347H,A347I,A347K,A347L,A347M,A347P,A347Q,A347R,A347S,H361A,H361C,H361D,H361E,H361G,H361K,H361L,H361M,H361N,H361P,H361S,H361T,H361Y,R363C,R363E,R363L,R363N,R363Q,R363S,R363T,R363W,R363Y,N369A,N369C,N369D,N369E,N369F,N369I,N369L,N369M,N369R,N369S,N369T,N369V,N369W,N369Y,D370F,D370G,D370S,D370W,D370Y,K371D,K371F,K371G,K371L,K371N,K371Q,K371R,K371S,K371T,K371V,K371W,G372A,G372C,G372D,G372E,G372L,G372M,G372N,G372S,G372T,G372V,G372Y,D374C,D374F,D374G,D374L,D374M,D374N,D374Q,D374S,D374T,D374V,D374Y,D375A,D375C,D375E,D375H,D375I,D375R,D375V,D375W,M380E,M380F,M380G,M380I,M380L,M380N,M380Q,M380S,M380T,M380V,M380Y,W382F,W382N,W382Y,Y396C,Y396D,Y396E,Y396F,Y396G,Y396H,Y396I,Y396K,Y396L,Y396M,Y396N,Y396Q,Y396R,Y396S,Y396T,Y396V,Y396W,D397A,D397C,D397E,D397F,D397H,D397I,D397K,D397L,D397M,D397P,D397Q,D397R,D397S,D397T,D397V,D397Y,A398C,A398D,A398E,A398F,A398G,A398H,A398I,A398K,A398L,A398M,A398N,A398P,A398Q,A398R,A398S,A398T,A398V,A398W,A398Y,I399A,I399C,I399D,I399E,I399F,I399G,I399M,I399Q,I399S,I399T,I399V,I399W,I399Y,R402A,R402C,R402E,R402F,R402G,R402I,R402L,R402P,R402Q,R402S,R402V,R402W,R402Y,Q409C,Q409D,Q409G,Q409H,Q409I,Q409V,V410A,V410C,V410F,V410G,V410H,V410I,V410L,V410N,V410S,V410T,V410W,V410Y,T411D,T411E,T411F,T411G,T411H,T411I,T411K,T411L,T411N,T411Q,T411R,T411S,T411V,T411Y,S420C,S420G,S420H,S420K,S420N,S420Q,S420T,S420Y,R426E,R426F,R426I,R426K,R426L,R426M,R426N,R426P,R426Q,R426S,R426T,R426W,R426Y,G427C,G427D,G427F,G427H,G427K,G427L,G427M,G427N,G427P,G427Q,G427R,G427T,G427V,G427W,G427Y,K428C,K428D,K428E,K428F,K428G,K428H,K428I,K428L,K428M,K428N,K428P,K428Q,K428R,K428S,K428T,K428V,K428W,K428Y,T445A,T445C,T445D,T445E,T445F,T445G,T445I,T445K,T445L,T445M,T445N,T445P,T445Q,T445R,T445S,T445V,T445Y,V446C,V446K,V446Q,V446R,E447K,E447L,E447S,E447V,E447W,E447Y,G448C,G448E,G448H,G448K,G448L,G448Q,G448V,N449A,N449C,N449E,N449F,N449G,N449H,N449K,N449L,N449M,N449P,N449R,N449T,N449V,N449W,D452N,R453A,R453L,R453M,N454F,N454G,N454K,N454L,N454M,N454R,N454S,N454T,N454V,N455A,N455C,N455D,N455E,N455F,N455G,N455H,N455I,N455L,N455M,N455S,N455T,N455V,N455W,N455Y,H460A,H460C,H460D,H460E,H460F,H460G,H460I,H460K,H460L,H460M,H460N,H460Q,H460R,H460S,H460W,H460Y,Q467A,Q467C,Q467D,Q467E,Q467H,Q467N,Q467S,Q467V,Q467W,Q467Y,N473A,N473C,N473E,N473F,N473G,N473H,N473K,N473L,N473M,N473P,N473Q,N473R,N473T,N473V,S474A,S474C,S474E,S474F,S474I,S474L,S474M,S474P,S474Q,N475I,N475L,N475M,N475P,N475Q,N475R,N475S,N475T,N475V,N475W,N475Y,E489N,Q490A,Q490C,Q490E,Q490F,Q490G,Q490H,Q490K,Q490L,Q490P,Q490R,Q490S,Q490T,Q490V,Q490W,Q490Y,L492A,L492D,L492F,L492H,L492I,L492M,L492N,L492Q,L492R,L492T,L492W,L492Y,Q496A,Q496G,Q496K,Q496N,Q496P,Q496S,Q496T,Q496V,V497A,V497C,V497I,V497M,V497N,V497T,K498A,K498C,K498E,K498F,K498G,K498H,K498I,K498L,K498M,K498N,K498Q,K498R,K498S,K498T,K498V,K498Y,D521C,D521E,D521F,D521G,D521H,D521I,D521K,D521L,D521M,D521P,D521R,D521S,D521T,D521V,D521W,D521Y,V522A,V522C,V522F,V522G,V522H,V522I,V522K,V522L,V522M,V522N,V522P,V522Q,V522R,V522S,V522T,V522W,V522Y,K534C,K534D,K534E,K534F,K534G,K534H,K534I,K534N,K534R,K534S,K534T,K534V,R542C,R542E,R542F,R542G,R542H,R542I,R542K,R542L,R542M,R542N,R542P,R542Q,R542S,R542T,R542V,R542W,R542Y,G547A,G547C,G547F,G547K,G547N,G547Q,G547R,G547T,G547V,G547Y,S548C,S548I,S548M,S548N,S548Q,S548R,S548T,S548V,S548W,S548Y,E553D,E553I,E553K,E553N,E553Q,E553W,E553Y,G554A,G554C,G554D,G554F,G554H,G554K,G554L,G554M,G554Q,G554R,G554S,G554T,G554V,G554W,L555A,L555C,L555D,L555E,L555F,L555G,L555H,L555I,L555K,L555M,L555N,L555P,L555Q,L555T,L555V,L555W,L555Y,K560A,K560C,K560E,K560G,K560I,K560L,K560M,K560N,K560P,K560Q,K560R,K560S,K560T,K560V,K560W,K560Y,H561A,H561C,H561D,H561E,H561F,H561G,H561I,H561M,H561N,H561Q,H561S,H561T,H561V,H561W,D563A,D563C,D563E,D563F,D563I,D563L,D563M,D563Q,D563R,D563S,D563T,D563V,D563W,D563Y,D564A,D564C,D564E,D564F,D564G,D564K,D564L,D564M,D564N,D564Q,D564R,D564S,D564T,D564V,D564Y,R570A,R570C,R570D,R570E,R570G,R570H,R570I,R570M,R570N,R570Q,R570S,R570T,R570V,Y571H,Y571M,Y571W,K581A,K581C,K581D,K581E,K581F,K581G,K581H,K581I,K581L,K581M,K581N,K581P,K581R,K581S,K581T,K581V,K581W,K581Y,N583A,N583C,N583E,N583F,N583G,N583H,N583I,N583K,N583L,N583M,N583P,N583R,N583S,N583T,N583V,N583W,N583Y,R586E,R586F,R586G,R586L,R586N,R586P,R586V,R586W,R586Y,S591C,S591D,S591F,S591G,S591H,S591I,S591K,S591M,S591P,S591Q,S591V,V603A,V603C,V603D,V603E,V603F,V603G,V603H,V603N,V603P,V603R,V603T,V603W,V603Y,F611A,F611C,F611D,F611G,F611I,F611K,F611L,F611M,F611N,F611R,F611S,F611T,F611V,F611W,F611Y,Q612C,Q612F,Q612H,Q612I,Q612L,Q612M,Q612R,Q612S,Q612W,A622D,A622E,A622F,A622G,A622H,A622I,A622K,A622M,A622N,A622P,A622R,A622S,A622T,A622V,Q626E,Q626F,Q626G,Q626M,V627D,V627K,V627P,V627Q,V627R,V627S,V627Y,T638A,T638E,T638F,T638G,T638I,T638K,T638L,T638M,T638P,T638Q,T638R,T638S,T638V,T638Y,S642A,S642C,S642E,S642F,S642G,S642H,S642I,S642K,S642L,S642M,S642N,S642P,S642Q,S642R,S642T,S642V,S642W,S642Y,A643C,A643E,A643F,A643G,A643H,A643K,A643L,A643N,A643Q,A643R,A643S,A643T,A643V,A643W,A643Y,R645A,R645D,R645E,R645F,R645G,R645H,R645I,R645K,R645L,R645M,R645P,R645Q,R645S,R645T,R645V,R645W,R645Y,K649C,K649F,K649I,K649L,K649M,K649Q,K649S,K649T,K649Y,Q650A,Q650C,Q650E,Q650F,Q650G,Q650H,Q650I,Q650K,Q650L,Q650M,Q650N,Q650R,Q650T,Q650V,Q650Y,K656R,T660C,T660D,T660E,T660F,T660G,T660H,T660I,T660K,T660M,T660N,T660P,T660Q,T660R,T660S,T660V,T660W,T660Y,P661A,P661C,P661D,P661E,P661F,P661G,P661H,P661I,P661K,P661L,P661M,P661Q,P661R,P661S,P661T,P661V,P661W,G662A,G662C,G662F,G662H,G662I,G662K,G662M,G662N,G662Q,G662R,G662W,G662Y,Q663A,Q663C,Q663E,Q663F,Q663H,Q663I,Q663K,Q663L,Q663M,Q663N,Q663R,Q663S,Q663V,Q663W,T666C,T666D,T666E,T666F,T666G,T666H,T666K,T666L,T666N,T666R,T666S,T666V,T666W,T666Y,R672C,R672E,R672F,R672G,R672H,R672I,R672K,R672L,R672M,R672N,R672T,R672V,R672W,R672Y,R673A,R673C,R673E,R673F,R673G,R673H,R673I,R673K,R673L,R673M,R673Q,R673S,R673T,R673V,R674K,R674L,R674M,R674Q,R674V,D675E,D675H,D675S,D675Y,D680A,D680C,D680E,D680F,D680H,D680I,D680K,D680L,D680M,D680N,D680Q,D680R,D680S,D680V,D680W,D680Y,T681A,T681G,T681H,T681K,T681L,T681M,T681N,T681P,T681Q,T681R,T681S,T681V,T681W,T681Y,A682C,A682E,A682I,A682L,A682M,A682N,A682P,A682S,A682W,A682Y,S683A,S683C,S683D,S683E,S683F,S683G,S683I,S683L,S683M,S683P,S683Q,S683R,S683V,S683W,Q684A,Q684C,Q684E,Q684G,Q684I,Q684N,Q684P,K685A,K685E,K685F,K685G,K685I,K685L,K685M,K685N,K685Q,K685R,K685S,K685T,K685V,K685W,K685Y,S692C,S692E,S692H,S692I,S692K,S692L,S692M,S692N,S692P,S692Q,S692T,S692V,S692W,R702C,R702D,R702F,R702G,R702H,R702I,R702K,R702L,R702M,R702N,R702Q,R702S,R702T,R702V,R702W,R705C,R705F,R705H,R705I,R705L,R705M,R705P,R705S,R705T,R705V，和R705W，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有降低的表达水平(PI大于0.1但小于1)。本发明还提供β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022F,K022G,K022P,K022Q,K022S,K022V,K022W,K022Y,N024A,N024C,L025A,L025D,L025F,L025G,L025I,L025K,L025Q,L025R,L025S,L025T,L025V,L025W,L025Y,Q026C,Q026H,Q026I,Q026K,Q026L,Q026P,Q026R,Q026S,Q026T,Q026V,Q026W,D027A,D027C,D027E,D027L,D027M,D027Q,D027S,D027T,D027V,S033C,S033G,V035C,V035E,V035G,V035H,V035K,V035L,V035N,V035P,V035Q,V035R,V035S,V035T,V035Y,G036D,G036E,G036R,G036S,W037V,W037Y,S050A,K051C,K051D,K051E,K051G,K051H,K051M,K051Q,K051R,K051T,K051V,R067A,R067C,R067D,R067F,R067G,R067N,R067P,R067Q,R067S,R067W,R091A,R091D,R091F,R091L,R091Q,R091V,R091W,E092C,E092K,E092L,E092N,E100A,E100G,E100I,E100M,E100S,E100T,E164S,Q165C,Q165F,Q165H,Q165I,Q165L,Q165M,Q165R,Q165S,Q165T,Q165V,Q165W,Q165Y,E166D,E166K,E166L,E166P,E166R,E166T,L167A,L167C,L167D,L167E,L167G,L167Q,L167R,L167S,L167V,L167W,L167Y,N168A,N168D,N168E,N168G,N168Y,P176F,P176G,P176K,P176L,P176R,P176T,P176V,P176W,D177V,D177W,D178A,D178C,D178Q,D178R,R179W,Q194A,Q194K,Q194Y,N196E,Y204F,T209C,T209D,T209E,T209G,T209H,T209I,T209K,T209L,T209M,T209Q,T209R,T209S,T209V,T209W,T209Y,E214A,E214C,E214D,E214G,E214H,E214L,E214M,E214N,E214Q,E214R,E214S,E214T,E214Y,D215E,D215L,D215N,D215Q,D215S,Q216G,Q216I,Q216L,Q216N,Q216S,Q216Y,K224R,K224V,D225V,Q226A,Q226F,Q226L,Q226W,Q226Y,N238A,N238E,N238G,N238M,N238S,N238T,T242A,T242C,T242E,T242F,T242G,T242H,T242I,T242K,T242L,T242M,T242N,T242Q,T242R,T242V,T242W,T242Y,N248A,N248C,N248F,N248T,N248W,N263A,N263C,N263G,N263H,N263S,N263T,N264C,R265E,R265K,R265L,R265N,R265Q,S277W,N278F,Q279C,T282C,D287C,D287E,D287N,D287S,Q301A,Q301K,Q301L,Q301N,Q301R,Q301S,Q301T,Q301V,D302A,D302C,D302W,Q303A,Q303C,Q303E,Q303H,Q303I,Q303K,Q303L,Q303M,Q303N,Q303R,Q303S,Q303T,Q303V,Q303Y,Y306C,Y306G,Y306I,Y306K,Y306L,Y306M,Y306N,Y306P,Y306Q,Y306R,Y306S,Y306T,Y306V,S312C,S312T,S312V,S312W,S312Y,Q316C,Q316P,Q316T,K320C,R328C,R328E,R328G,R328K,R328L,R328M,R328Q,R328S,R328T,R328V,D329A,D329E,D329G,D329H,D329M,D329N,D329Q,D329S,D329T,K335A,K335D,K335F,K335H,K335I,K335L,K335M,K335N,K335R,K335S,K335T,K335V,K335W,D337A,D337C,D337E,D337G,D337H,D337K,D337L,D337M,D337N,D337R,D337S,D337T,D337V,D337Y,A338C,A338F,A338G,A338H,A338I,A338L,A338M,A338N,A338P,A338R,A338V,A338W,K344D,K344E,K344F,K344G,K344I,K344L,K344M,K344N,K344R,K344S,K344T,K344V,K345A,K345E,K345F,K345H,K345P,K345Q,K345R,K345S,K345T,K345V,K345Y,A347D,A347F,A347P,A347Y,H361G,R363C,R363K,R363L,R363Q,R363T,R363W,R363Y,N369C,N369D,N369E,N369F,N369L,N369M,N369S,N369T,N369V,N369W,N369Y,D370E,D370F,D370G,D370S,D370W,D370Y,K371A,K371F,K371G,K371L,K371N,K371Q,K371R,K371S,K371T,K371V,G372A,G372C,G372E,G372L,G372M,G372T,G372V,D374C,D374F,D374G,D374L,D374M,D374N,D374Q,D374S,D374V,D375A,D375C,D375E,D375I,D375V,M380I,M380L,M380Q,M380S,M380T,M380V,Y396A,Y396C,Y396D,Y396E,Y396F,Y396G,Y396I,Y396K,Y396T,D397C,D397E,D397H,D397I,D397K,D397L,D397M,D397N,D397P,D397Q,D397R,D397S,D397T,D397V,D397Y,A398C,A398D,A398E,A398F,A398G,A398H,A398I,A398K,A398L,A398M,A398N,A398P,A398Q,A398R,A398S,A398T,A398V,A398W,A398Y,I399L,I399V,R402A,V410C,T411D,T411E,T411F,T411G,T411H,T411I,T411K,T411L,T411N,T411Q,T411R,T411S,T411V,T411Y,S420C,S420D,S420G,S420K,S420N,S420Q,S420T,S420Y,R426A,R426T,G427C,G427F,G427H,G427L,G427M,G427S,G427Y,K428A,T445A,T445C,T445E,T445F,T445G,T445M,T445V,T445Y,G448A,G448C,,G448D,G448E,G448H,G448T,N449A,N449C,N449E,N449F,N449M,N449P,N449T,N449V,N455C,N455D,N455W,N473S,S474C,S474F,S474I,S474L,S474M,S474N,S474P,S474R,S474T,S474Y,N475I,N475L,N475M,N475P,N475Q,N475R,N475S,N475T,N475V,N475W,N475Y,Q490A,Q490C,Q490E,Q490F,Q490G,Q490H,Q490K,Q490L,Q490R,Q490S,Q490T,Q490V,Q490Y,L492A,L492D,L492H,L492N,V497A,V497T,K498E,K498L,K498M,K498V,D521A,V522A,V522C,V522F,V522H,V522I,V522K,V522L,V522M,V522Q,V522R,V522S,V522T,V522W,V522Y,K534F,K534V,R542C,R542E,R542F,R542G,R542H,R542I,R542K,R542L,R542M,R542N,R542P,R542Q,R542S,R542T,R542V,R542W,R542Y,G547A,G547L,G547P,S548C,S548E,S548F,S548H,S548I,S548L,S548M,S548N,S548Q,S548R,S548T,S548V,S548W,S548Y,G554D,G554L,G554M,G554Q,G554W,L555C,L555I,L555V,H561M,H561N,D563A,D563M,D563Q,D564A,D564C,D564F,D564L,D564M,D564T,D564V,R570A,Y571W,K581A,K581D,K581E,K581F,K581G,K581H,K581I,K581L,K581M,K581N,K581R,K581S,K581T,K581V,K581W,K581Y,N583A,N583C,N583D,N583G,N583R,N583V,R586E,R586F,R586L,R586N,R586P,R586V,R586W,V603A,V603C,V603D,V603E,V603F,V603G,V603H,V603S,V603T,V603W,V603Y,F611A,Q612C,Q612G,Q612S,A622E,A622F,A622G,A622H,A622K,A622L,A622M,A622R,A622S,A622T,A622V,Q626E,Q626F,Q626G,Q626M,Q626T,T638A,T638D,T638E,T638G,T638I,T638K,T638L,T638M,T638Q,T638R,T638S,T638V,T638W,T638Y,S642C,S642E,S642F,S642H,S642I,S642L,S642M,S642N,S642P,S642Q,S642R,S642T,S642V,S642W,S642Y,A643L,A643M,R645G,R645K,K649A,K649C,K649F,K649I,K649L,K649M,K649Q,K649S,K649W,K649Y,T660C,T660D,T660I,T660W,P661C,P661D,P661F,P661I,P661L,P661S,P661V,P661W,G662A,G662C,G662F,G662H,G662T,G662W,G662Y,Q663A,Q663C,Q663D,Q663E,Q663G,Q663I,Q663K,Q663S,Q663W,T666A,T666C,T666N,R672K,R673K,R673N,R673S,R673T,D675E,D675H,D675S,D675Y,D680A,D680C,D680E,D680M,D680Q,D680R,D680V,D680Y,T681A,T681G,T681M,T681S,T681W,S683G,S683V,S683W,Q684A,Q684C,Q684G,Q684N,Q684P,K685A,K685E,K685F,K685G,K685I,K685L,K685M,K685N,K685Q,K685R,K685S,K685T,K685V,K685W,K685Y,S692C,S692E,S692H,S692I,S692K,S692L,S692M,S692N,S692P,S692Q,S692T,S692V,S692W,R702G,R705I，和R705V，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的CNPG酶活性(PI大于1)。另外，本发明提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022S,N024D,N024E,N024F,N024G,N024K,N024L,N024M,N024P,N024T,L025T,Q026D,Q026P,Q026R,Q026W,Q026Y,K028L,K028M,K028N,W037F,W037I,S050C,S050F,S050G,S050K,S050P,S050R,S050T,S050Y,K051A,K051D,K051G,K051H,K051M,K051T,K051V,I052A,I052F,I052N,I052S,R067A,R067C,R067D,R067E,R067F,R067G,R067I,R067M,R067N,R067P,R067S,R067T,R067V,R067W,R067Y,R091A,R091D,R091E,R091F,R091G,R091H,R091I,R091K,R091L,R091N,R091Q,R091S,R091T,R091V,R091W,E092K,E092T,R093A,R093C,R093D,R093E,R093F,R093G,R093H,R093K,R093L,R093M,R093Q,R093S,R093T,R093V,R093W,E099A,E099F,E099I,E099M,E099W,E099Y,E100I,E100K,E100L,E100Y,H159E,H159G,Q165D,Q165I,Q165K,Q165L,Q165R,Q165V,Q165W,Q165Y,E166F,E166K,E166L,E166N,E166R,E166S,E166T,E166Y,R169A,R169C,R169E,R169F,R169H,R169Q,R169S,R169T,E170F,E170I,E170L,E170M,E170V,E170W,E170Y,P176A,P176D,P176R,177A,D177G,D177L,D177M,D177N,D177Q,D177W,D178S,R179A,R179V,Q194A,N196E,N196G,N196M,N196P,S199A,Y204M,N208R,T209K,T209L,T209M,T209R,E214A,E214K,E214P,E214R,E214W,E214Y,D215A,D215C,D215F,D215G,D215H,D215M,D215N,D215Q,D215S,D215V,D215W,Q216A,Q216C,Q216D,Q216E,Q216F,Q216H,Q216I,Q216K,Q216L,Q216M,Q216P,Q216R,Q216T,Q216W,Q216Y,D225A,D225C,D225E,D225F,D225H,D225I,D225L,D225Q,D225T,D225V,D225W,D225Y,Q226A,Q226C,Q226D,Q226E,Q226I,Q226K,Q226W,W237Y,N238A,N238D,N238F,N238G,N238P,N238S,N238W,T242H,T242S,S249M,N263A,N263C,N263D,N263E,N263F,N263H,N263I,N263K,N263L,N263P,N263Q,N263S,N263T,N263V,N264A,N264C,N264D,N264E,N264G,N264H,N264K,N264L,N264M,N264Q,N264R,N264S,N264T,N264V,N264Y,R265A,R265E,R265F,R265G,R265I,R265M,R265P,R265Q,R265S,R265T,R265V,N276A,S277A,S277C,S277D,S277E,S277F,S277G,S277M,S277N,S277Q,S277R,S277W,N278C,N278D,N278F,N278G,,278Q,N278R,N278V,Q279C,Q279D,Q279V,T282D,T282H,T282K,R284N,D287C,D287F,D287H,D287I,D287K,D287L,D287M,D287V,D287W,D287Y,Q301E,Q301L,Q301T,Q301V,Y306C,S312A,S312C,S312D,S312G,S312I,S312K,S312L,S312M,S312N,S312Q,S312R,S312W,S312Y,R313E,R313G,R313L,R313S,R313V,R313W,Q316A,Q316C,Q316D,Q316E,Q316F,Q316G,Q316H,Q316I,Q316K,Q316N,Q316P,Q316R,Q316S,Q316T,Q316V,Q316W,Q316Y,K320E,K320G,K320M,K320N,K320T,R324C,R324D,R328C,R328E,R328I,R328L,R328Q,D329A,D329F,D329Q,D329T,D329Y,L334M,L334V,K335A,K335G,K335S,K335T,K335V,K335W,N336S,N336T,N336Y,D337A,D337C,D337W,A338F,A338P,A338Q,A338V,A338W,A338Y,N339D,N339I,N339L,N339P,N339Q,N339R,N339V,N339Y,K344D,K345A,K345D,K345E,K345G,K345H,K345Q,K345Y,A347D,A347F,A347I,A347K,A347L,A347M,A347P,A347Q,A347R,A347S,A347Y,H361A,H361C,H361E,H361G,H361K,H361L,H361M,H361P,H361S,H361Y,R363A,R363C,R363E,R363G,R363K,R363L,R363N,R363Q,R363S,R363T,R363V,R363W,N369A,N369C,N369D,N369E,N369I,N369L,N369M,N369R,N369T,N369V,N369W,N369Y,D370E,D370F,D370W,D370Y,K371A,K371D,K371F,K371G,K371L,K371S,K371T,K371W,G372A,G372C,G372D,G372N,G372S,D374A,D374I,D374R,D374W,M380E,M380F,M380G,M380L,M380Q,M380T,M380V,M380Y,W382N,W382Y,D397N,A398C,A398D,A398E,A398F,A398G,A398H,A398L,A398N,A398P,A398S,A398T,A398Y,I399L,R402C,R402E,R402G,R402I,R402L,R402P,R402Q,R402S,R402V,R402Y,Q409C,Q409D,Q409G,Q409H,Q409I,Q409V,V410G,V410L,R426A,R426E,R426F,R426I,R426K,R426L,R426M,R426N,R426Q,R426S,R426T,R426Y,G427D,G427E,G427F,G427L,G427N,G427Q,G427S,G427T,G427V,G427W,,428A,K428P,T445A,T445C,T445E,T445F,T445G,T445M,T445P,T445Q,T445S,T445V,T445Y,E447A,E447L,E447W,G448D,G448E,G448F,G448H,G448K,G448L,G448M,G448N,G448Q,G448S,G448T,G448V,G448Y,N449C,N449E,N449G,N449K,N449L,N449R,N449V,N449W,D452N,R453A,R453E,R453L,R453M,R453Q,R453S,N455A,N455C,N455D,N455I,Q467A,Q467C,Q467D,Q467E,Q467H,Q467K,Q467N,Q467S,Q467V,Q467W,Q467Y,N473F,N473P,N473Q,N473R,N473S,N473T,N473V,S474D,S474G,S474K,S474L,S474M,S474N,S474Q,S474R,S474V,N475I,N475K,N475L,N475P,N475T,N475V,N475W,N475Y,E489N,Q490C,Q490G,L492Y,Q496A,Q496G,Q496K,Q496N,Q496P,Q496T,Q496V,Q496W,V497C,V497N,K498A,K498C,K498E,K498F,K498G,K498H,K498I,K498Q,K498R,K498S,K498T,K498Y,D521E,D521F,D521P,D521T,D521V,V522G,V522K,V522N,K534D,K534E,K534G,K534H,K534I,K534N,K534Q,K534S,K534T,K534V,R542A,R542C,R542D,R542F,R542I,R542K,R542P,R542Q,R542S,R542W,R542Y,G547A,G547C,G547E,G547F,G547K,G547L,G547N,G547P,G547Q,G547R,G547T,G547V,G547Y,S548E,S548F,S548I,S548L,S548Q,S548T,S548V,S548W,E553D,E553I,E553K,E553Q,E553W,E553Y,G554A,G554C,G554D,G554F,G554H,G554K,G554L,G554M,G554Q,G554R,G554S,G554T,G554W,K560A,K560C,K560E,K560G,K560H,K560I,K560L,K560M,K560N,K560P,K560Q,K560R,K560S,K560T,K560V,K560Y,H561C,H561D,H561G,H561M,H561N,H561T,H561W,D563A,D563I,D563R,D563S,D563V,D563Y,D564A,D564C,D564E,D564F,D564G,D564N,D564Y,R570E,R570G,R570M,R570N,R570Q,R570T,R570V,Y571H,Y571M,Y571W,K581C,K581D,K581G,K581L,K581N,K581T,N583D,R586D,S591C,S591D,S591F,S591G,S591H,S591I,S591K,S591M,S591P,S591Q,S591V,F611G,F611I,F611L,F611N,F611R,F11S,F611T,F611V,F611Y,Q612C,Q612D,Q612G,Q612H,Q612I,Q612W,A622D,A622E,A622L,V627D,V627K,V627P,V627Q,V627R,V627S,V627Y,T638A,T638D,T638Q,T638R,S642D,S642E,S642F,S642G,S642I,S642L,S642M,S642N,A643E,A643F,A643G,A643H,A643K,A643M,A643N,A643Q,A643S,A643T,A643V,A643W,A643Y,R645A,R645D,R645E,R645F,R645H,R645I,R645L,R645M,R645P,R645Q,R645S,R645T,R645V,R645W,R645Y,K649A,K649Q,K649W,Q650D,K656R,T660C,T660E,T660F,T660G,T660I,T660K,T660M,T660N,T660P,T660Q,T660R,T660S,T660V,T660W,T660Y,P661A,P661C,P661D,P661F,P661G,P661I,G662E,G662F,G662I,G662K,G662L,G662M,G662N,G662Q,G662S,G662W,Q663V,Q663W,T666A,T666C,T666D,T666E,T666F,T666G,T666H,T666L,T666S,T666V,T666Y,R672M,R673F,R673N,R673W,R674K,R674L,R674Q,D675E,D675H,D675S,D675Y,T681G,T681L,A682C,A682E,A682I,A682L,A682M,A682N,A682P,A682S,A682W,A682Y,S683A,S683C,S683D,S683E,S683K,S683L,S683R,S683V,Q684C,Q684D,Q684E,Q684F,Q684G,Q684H,Q684I,Q684K,Q684L,Q684M,Q684N,Q684P,Q684R,Q684T,S692H,S692L,S692N,S692T,S692V,R702C,R702D,R702F,R702G,R702I,R702K,R702L,R702M,R702Q,R702S,R702V,R70W，和R705P，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有降低的葡萄糖抑制(PI大于1)。本发明还提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022F,K022G,K022I,K022P,K022Q,K022S,K022V,K022W,K022Y,N024A,N024C,N024E,N024M,L025I,L025T,Q026H,Q026K,Q026R,Q026S,V035L,V035S,V035T,W037E,W037F,W037H,W037M,W037S,W037Y,S050A,K051A,K051H,K051M,R091Y,E092K,E092L,E092M,L167R,L167W,E170L,P176A,P176G,D178A,D178T,Q194A,Q194K,Q194R,S199A,D215S,Q216E,Q216L,Q216N,D225C,D225G,D225L,D225Q,D225S,D225T,D225V,Q226A,N238A,T242H,T242S,N248A,N248C,N248L,N248T,S249I,N278D,T282C,T282D,T282G,T282N,T282R,T282V,Q303A,Q303D,Q303E,Q303F,Q303G,Q303I,Q303K,Q303L,Q303M,Q303N,Q303R,Q303S,Q303T,Q303V,Q303Y,Y306F,Y306I,Y306L,Y306R,Y306W,S312C,S312N,K320C,K320H,K320N,K320S,K320Y,D329A,K335A,K335L,K335R,K335S,K335T,K335W,A338C,A338D,A338G,A338I,A338N,A338V,A338W,K344S,A347D,A347F,A347Y,R363L,N369C,N369E,N369F,N369I,N369L,N369M,N369R,N369T,N369V,N369W,N369Y,G372A,I399L,R402A,V410L,T411F,T411H,T411L,T411Q,T411Y,S420A,S420D,R426F,R426N,G427E,G427S,K428A,K428S,T445D,T445G,N473S,S474D,S474G,S474I,S474K,S474M,S474N,S474R,S474T,S474V,S474Y,V497A,V497I,V497M,V497T,D521A,D521S,G547A,G547E,G547K,G547L,G547P,G547R,G547V,S548C,S548E,S548F,S548H,S548I,S548L,S548M,S548V,S548W,G554Q,H561N,D563A,D563E,N583D,N583R,R586D,V603A,V603E,V603H,V603M,V603N,V603Q,V603S,F611A,Q612D,Q612G,A622D,A622G,A622H,A622I,A622L,A622N,A622R,A622S,A622W,A622Y,Q626E,Q626L,Q626T,T638D,A643M,R645D,R645G,R645K,K649L,K649M,K649Q,K649W,T660C,T660D,T660E,T660F,T660H,T660I,T660K,T660M,T660Q,T660V,T660W,T660Y,P661C,P661D,P661S,P661V,G662C,G662D,G662E,G662F,G662H,G662K,G662L,G662M,G662N,G662Q,G662R,G662S,G662T,G662W,G662Y,T666A,R673W,S683V,Q684F,Q684H,Q684K,Q684L,Q684M,Q684S,Q684T,K685A,K685L,K685S,S692H,S692K,S692L,S692M,S692T，和R705L，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的热稳定性(PI大于1)。另外，本发明提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022H,K022R,N024F,N024G,N024K,N024Q,N024R,N024S,N024T,N024V,L025A,L025D,L025F,L025G,L025K,L025N,L025Q,L025R,L025S,L025V,L025Y,Q026C,Q026D,Q026E,Q026G,Q026I,Q026L,Q026P,Q026T,Q026V,Q026W,Q026Y,D027A,D027C,D027E,D027L,D027M,D027Q,D027S,D027T,D027V,K028L,K028M,K028S,K028V,S033C,S033G,S033T,V035C,V035E,V035G,V035H,V035K,V035N,V035Q,V035R,G036R,G036S,W037V,S050C,S050F,S050G,S050I,S050K,S050L,S050M,S050N,S050P,S050R,S050T,S050V,S050Y,K051C,K051D,K051E,K051G,K051I,K051L,K051N,K051Q,K051R,K051S,K051T,K051V,I052V,R067Q,R091A,R091D,R091E,R091F,R091G,R091H,R091K,R091L,R091N,R091Q,R091S,R091V,R091W,E092A,E092C,E092D,E092F,E092H,E092I,E092N,E092Q,E092R,E092V,E099A,E099D,E099F,E099I,E099K,E099M,E099N,E099W,E099Y,E100M,E100Q,E100T,L167A,L167C,L167E,L167F,L167G,L167M,L167N,L167Q,L167S,L167V,L167Y,N168A,N168D,N168E,N168G,N168H,N168Q,N168R,N168T,N168Y,E170D,E170F,E170I,E170M,E170V,P176E,P176H,P176L,P176M,P176Q,P176R,P176S,P176T,P176V,P176Y,D177A,D177E,D177F,D177H,D177L,D177M,D177Q,D177R,D177V,D177Y,D178C,D178K,D178N,D178Q,D178R,D178S,R179A,R179C,R179G,R179I,R179K,R179S,R179T,R179V,R179W,Q194C,Q194E,Q194F,Q194G,Q194H,Q194L,Q194M,Q194T,Q194W,N196E,N196G,N196H,N196L,N196M,N196Q,N196R,N196T,S199G,S199T,S199V,Y204F,Y204M,N208K,T209C,T209D,T209E,T209G,T209H,T209I,T209K,T209L,T209M,T209Q,T209R,T209S,T209V,T209W,T209Y,E214D,E214Q,D215A,D215C,D215E,D215F,D215G,D215H,D215L,D215M,D215N,D215Q,D215W,Q216A,Q216C,Q216F,Q216G,Q216H,Q216I,Q216K,Q216M,Q216P,Q216S,Q216T,Q216W,Q216Y,K224R,K224V,D225A,D225E,D225F,D225H,D225I,D225M,D225W,D225Y,Q226C,Q226D,Q226E,Q226F,Q226H,Q226I,Q226K,Q226L,Q226M,Q226N,Q226R,Q226T,Q226V,Q226W,Q226Y,N238C,N238D,N238E,N238F,N238G,N238M,N238S,N238T,N238W,T242C,T242E,T242F,T242G,T242K,T242N,T242Q,T242R,T242W,T242Y,N248F,N248G,N248W,N248Y,S249G,S249M,S249V,N263A,N263C,N263D,N263E,N263F,N263G,N263H,N263I,N263K,N263L,N263P,N263Q,N263R,N263S,N263T,N263V,N263Y,N264C,N264G,N264K,N264M,N264Q,N264T,R265E,R265F,R265I,R265K,R265L,R265M,R265N,R265P,R265Q,R265S,R265T,R265V,N276A,N276F,N276M,N276Q,S277C,S277E,S277F,S277G,S277H,S277I,S277M,S277N,S277P,S277Q,S277R,S277Y,N278C,N278F,N278G,N278I,N278L,N278M,N278Q,N278R,N278S,N278T,N278V,N278W,N278Y,Q279C,Q279D,Q279E,Q279G,Q279H,Q279I,Q279K,Q279N,Q279S,Q279T,Q279V,Q279Y,T282K,T282L,T282P,T282S,D287C,D287E,D287G,D287H,D287I,D287K,D287L,D287M,D287N,D287S,D287V,Q301E,Q301N,D302A,D302C,D302E,D302F,D302G,D302K,D302M,D302N,D302P,D302S,D302T,D302W,D302Y,Q303C,Q303H,Q303P,Q303W,Y306C,Y306G,Y306K,Y306M,Y306N,Y306P,Y306Q,Y306S,Y306T,Y306V,S312K,S312L,S312M,S312Q,S312T,S312V,S312W,S312Y,R313A,R313C,R313G,R313K,R313L,R313N,R313S,R313V,R313W,Q316C,Q316E,Q316G,Q316K,Q316L,Q316M,Q316R,Q316S,Q316T,Q316V,Q316W,Q316Y,K320E,K320G,K320L,K320M,K320P,K320Q,K320R,K320T,R324C,R324D,R324E,R324F,R324H,R324I,R324K,R324L,R324M,R324Q,R324V,R324W,R324Y,R328C,R328E,R328G,R328K,R328L,R328M,R328Q,R328T,R328V,D329E,D329F,D329G,D329H,D329M,D329N,D329Q,D329S,D329T,D329Y,L334A,L334C,L334F,L334M,L334T,L334V,L334W,K335D,K335F,K335G,K335H,K335I,K335M,K335N,K335V,N336A,N336C,N336G,N336H,N336L,N336M,N336Q,N336R,N336S,N336T,N336V,N336Y,D337A,D337C,D337E,D337G,D337H,D337K,D337L,D337M,D337N,D337R,D337S,D337T,D337V,D337W,D337Y,A338F,A338H,A338K,A338L,A338M,A338P,A338Q,A338Y,N339D,N339E,N339G,N339H,N339I,N339K,N339L,N339P,N339Q,N339R,N339V,N339Y,K344D,K344E,K344F,K344G,K344I,K344L,K344M,K344N,K344P,K344Q,K344R,K344T,K344V,K345A,K345D,K345E,K345F,K345G,K345H,K345N,K345P,K345Q,K345R,K345S,K345T,K345V,K345W,K345Y,A347H,A347I,A347K,A347L,A347M,A347P,A347R,A347S,R363C,R363E,R363Q,R363T,N369A,N369D,N369S,G372C,G372N,G372V,D374C,D374N,D374Y,W382N,W382Y,Y396C,Y396D,Y396E,Y396F,Y396G,Y396H,Y396I,Y396K,Y396L,Y396M,Y396N,Y396Q,Y396S,Y396T,Y396V,Y396W,D397A,D397C,D397E,D397P,D397Q,D397R,D397S,D397T,D397V,A398C,A398D,A398E,A398F,A398G,A398H,A398I,A398K,A398L,A398M,A398N,A398P,A398Q,A398R,A398S,A398T,A398V,A398W,A398Y,I399A,I399C,I399D,I399E,I399F,I399G,I399M,I399Q,I399S,I399T,I399V,I399W,I399Y,R402C,R402E,R402F,R402G,R402I,R402L,R402P,R402Q,R402S,R402V,R402W,R402Y,Q409C,Q409D,Q409G,Q409H,Q409I,Q409V,V410A,V410C,V410F,V410G,V410H,V410I,V410N,V410S,V410T,V410W,V410Y,T411D,T411E,T411G,T411I,T411K,T411N,T411R,T411S,T411V,S420C,S420G,S420H,S420K,S420N,S420Q,S420T,S420Y,R426E,R426I,R426K,R426L,R426M,R426P,R426Q,R426S,R426T,R426W,R426Y,G427C,G427D,G427F,G427H,G427K,G427L,G427M,G427N,G427P,G427Q,G427R,G427T,G427V,G427W,G427Y,K428C,K428D,K428E,K428F,K428G,K428H,K428I,K428L,K428M,K428N,K428P,K428Q,K428R,K428T,K428V,K428W,K428Y,T445A,T445C,T445E,T445F,T445I,T445K,T445L,T445M,T445N,T445P,T445Q,T445R,T445S,T445V,T445Y,V446Q,N449C,N454F,N454K,N454L,N454M,N454R,N454S,N454T,N454V,N455A,N455C,N455D,N455E,N455F,N455G,N455H,N455L,N455M,N455S,N455T,N455V,N455W,N455Y,Q467A,Q467C,Q467D,Q467N,Q467S,N473A,N473C,N473E,N473G,N473H,N473K,N473L,N473M,N473P,N473Q,N473R,N473T,N473V,S474A,S474C,S474E,S474F,S474L,S474P,S474Q,N475I,N475L,N475M,N475P,N475Q,N475R,N475S,N475T,N475V,N475W,N475Y,E489N,Q490A,Q490C,Q490E,Q490F,Q490G,Q490H,Q490K,Q490L,Q490P,Q490R,Q490S,Q490T,Q490V,Q490W,Q490Y,L492A,L492D,L492F,L492H,L492I,L492M,L492N,L492Q,L492R,L492T,L492W,L492Y,Q496A,Q496G,Q496N,Q496P,Q496S,Q496T,V497C,V497N,K498A,K498C,K498E,K498F,K498G,K498H,K498I,K498L,K498M,K498N,K498Q,K498R,K498S,K498T,K498V,K498Y,D521C,D521E,D521F,D521G,D521H,D521I,D521K,D521L,D521M,D521P,D521R,D521T,D521V,D521W,V522A,V522C,V522F,V522G,V522H,V522I,V522K,V522L,V522M,V522N,V522P,V522Q,V522R,V522S,V522T,V522W,V522Y,K534C,K534D,K534E,K534F,K534H,K534I,K534N,K534R,K534S,K534T,K534V,R542C,R542E,R542F,R542G,R542H,R542K,R542L,R542M,R542N,R542P,R542Q,R542S,R542T,R542V,R542W,R542Y,G547C,G547F,G547N,G547Q,G547T,G547Y,S548N,S548Q,S548R,S548T,S548Y,E553D,E553K,E553N,E553W,G554A,G554C,G554D,G554F,G554H,G554K,G554L,G554M,G554R,G554S,G554T,G554V,G554W,L555A,L555C,L555D,L555E,L555F,L555G,L555H,L555I,L555K,L555M,L555N,L555P,L555Q,L555T,L555V,L555W,L555Y,K560A,K560C,K560G,K560I,K560L,K560M,K560N,K560P,K560R,K560S,K560T,K560V,K560Y,H561A,H561C,H561D,H561E,H561F,H561G,H561I,H561M,H561Q,H561S,H561T,H561V,H561W,D563C,D563F,D563I,D563L,D563M,D563Q,D563R,D563S,D563T,D563V,D563W,D563Y,D564A,D564C,D564E,D564F,D564G,D564K,D564L,D564M,D564N,D564Q,D564R,D564S,D564T,D564V,D564Y,R570A,R570C,R570D,R570E,R570G,R570H,R570I,R570M,R570Q,R570S,R570T,R570V,Y571H,Y571M,Y571W,K581A,K581C,K581D,K581E,K581F,K581G,K581H,K581I,K581L,K581M,K581N,K581P,K581R,K581S,K581T,K581V,K581W,K581Y,N583A,N583C,N583E,N583F,N583G,N583H,N583I,N583K,N583L,N583M,N583P,N583S,N583T,N583V,N583W,N583Y,R586E,R586F,R586G,R586L,R586N,R586P,R586V,R586W,R586Y,S591C,S591D,S591G,S591H,S591I,S591K,S591M,S591P,S591Q,S591V,V603C,V603D,V603F,V603G,V603P,V603R,V603T,V603W,V603Y,F611C,F611D,F611G,F611I,F611K,F611L,F611M,F611N,F611R,F611S,F611T,F611V,F611W,F611Y,Q612C,Q612F,Q612H,Q612I,Q612L,Q612M,Q612R,Q612S,Q612W,A622E,A622F,A622K,A622M,A622T,A622V,Q626F,Q626G,Q626M,V627K,V627P,V627Q,V627R,V627S,T638A,T638E,T638F,T638G,T638I,T638K,T638L,T638M,T638P,T638Q,T638R,T638S,T638V,T638Y,S642A,S642C,S642E,S642F,S642G,S642H,S642I,S642K,S642L,S642M,S642N,S642P,S642Q,S642R,S642T,S642V,S642W,S642Y,A643C,A643E,A643F,A643G,A643H,A643K,A643L,A643N,A643Q,A643R,A643S,A643T,A643V,A643W,A643Y,R645A,R645E,R645F,R645H,R645I,R645L,R645M,R645P,R645Q,R645S,R645T,R645V,R645W,R645Y,K649C,K649F,K649I,K649S,K649T,K649Y,Q650A,Q650C,Q650E,Q650F,Q650G,Q650H,Q650I,Q650K,Q650L,Q650M,Q650N,Q650R,Q650T,Q650V,Q650Y,K656R,T660G,T660N,T660P,T660R,T660S,P661A,P661E,P661F,P661G,P661H,P661I,P661K,P661L,P661M,P661Q,P661R,P661T,P661W,G662A,G662I,Q663A,Q663C,Q663E,Q663F,Q663H,Q663I,Q663K,Q663L,Q663M,Q663N,Q663R,Q663S,Q663V,Q663W,T666C,T666D,T666E,T666F,T666G,T666H,T666K,T666L,T666N,T666R,T666S,T666V,T666W,T666Y,R672C,R672F,R672G,R672H,R672I,R672K,R672L,R672N,R672T,R672V,R672W,R673A,R673C,R673E,R673G,R673H,R673I,R673K,R673L,R673M,R673Q,R673S,R673T,R673V,R674K,R674L,R674M,R674Q,R674V,D675E,D675H,D675S,D675Y,D680A,D680C,D680E,D680F,D680H,D680I,D680K,D680L,D680M,D680N,D680Q,D680R,D680S,D680V,D680W,D680Y,T681A,T681G,T681H,T681K,T681L,T681M,T681N,T681P,T681Q,T681R,T681S,T681V,T681W,T681Y,A682C,A682E,A682I,A682L,A682M,A682N,A682P,A682S,A682W,A682Y,S683A,S683C,S683D,S683E,S683F,S683G,S683I,S683L,S683M,S683P,S683Q,S683R,S683W,Q684A,Q684C,Q684E,Q684G,Q684I,Q684N,Q684P,K685E,K685F,K685G,K685I,K685M,K685N,K685Q,K685R,K685T,K685V,K685W,K685Y,S692C,S692E,S692I,S692N,S692P,S692Q,S692V,S692W,R702C,R702D,R702F,R702G,R702H,R702I,R702K,R702L,R702M,R702N,R702Q,R702S,R702T,R702V,R702W,R705C,R705F,R705H,R705I,R705M,R705P,R705S,R705T,R705V，和R705W，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有降低的热稳定性(PI大于0.1但小于1)。本发明还提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022F,K022P,K022Q,N024C,N024F,N024Q,N024R,N024Y,L025D,Q026C,Q026E,Q026H,Q026I,Q026K,Q026L,Q026P,Q026R,Q026S,Q026T,Q026V,Q026W,D027A,D027C,D027E,S033C,V035C,V035P,V035T,G036D,G036E,G036K,G036R,G036S,G036Y,S050C,S050L,K051C,K051E,K051G,K051H,K051I,K051M,K051Q,K051T,K051V,I052D,I052T,R091D,R091G,R091K,R091Q,E092A,E092C,E092D,E099Y,E100A,E100G,E100N,E100Q,E100S,E100T,E100Y,K158H,K158T,Q165I,Q165K,Q165M,Q165N,Q165V,E166D,L167C,L167W,N168A,N168D,N168E,N168G,N168Y,E170D,E170F,P176K,P176R,P176W,P176Y,D177E,D177F,D177H,D177L,D177M,D177V,D177W,D177Y,178C,D178Y,R179C,R179M,R179S,Q194C,N196E,N196L,N196Q,N196R,N196T,S199A,T209C,T209G,T209H,T209I,T209L,T209M,T209Q,T209S,T209V,T209Y,E214W,D215C,D215E,D215L,D215N,D215Q,D215S,Q216A,Q216E,Q216G,Q216H,Q216I,Q216L,Q216M,Q216N,Q216S,Q216W,Q216Y,K224H,K224R,K224V,D225G,D225H,D225I,D225L,D225M,D225T,D225V,D225W,D225Y,Q226F,Q226I,Q226L,Q226M,Q226R,Q226V,Q226W,Q226Y,N238A,N238C,N238E,N238G,T242A,T242C,T242E,T242F,T242G,T242H,T242K,T242L,T242M,T242N,T242Q,T242R,T242S,T242V,T242W,T242Y,N248A,N248F,N248G,N248T,N248W,S249M,S249V,N263C,N263D,N263G,N263S,N263T,N264C,N276A,N276C,N276F,S277C,S277F,S277W,S277Y,N278C,N278F,N278G,N278V,Q279C,T282C,D287C,D287S,Q301G,Q301K,Q301L,D302A,D302C,D302E,D302F,D302G,D302K,D302M,D302T,Q303A,Q303C,Q303K,Q303M,Q303P,Y306G,Y306K,Y306M,Y306Q,Y306R,Y306V,S312C,S312D,S312W,S312Y,Q316K,Q316P,Q316R,Q316S,Q316T,Q316Y,K320C,R328S,D329A,L334A,L334V,K335A,K335D,K335H,K335V,K335W,N336A,N336G,D337C,D337K,D337W,A338C,A338W,N339E,N339G,N339H,N339K,N339L,K344D,K344F,K344I,K344L,K344M,K344P,K344S,K344T,K344V,K345A,K345D,K345E,K345F,K345G,K345S,K345V,K345Y,A347S,A347Y,H361A,H361C,H361G,R363C,R363G,R363K,R363Q,R363S,R363W,R363Y,N369C,N369D,N369E,N369F,N369W,N369Y,K371A,K371G,K371L,K371T,G372A,G372K,G372W,D374C,D374L,D374M,D374Q,D374S,D374V,D375C,D375E,D375W,M380N,M380V,W382F,Y396A,Y396C,Y396E,Y396F,Y396K,Y396V,D397C,D397E,D397H,D397I,D397K,D397L,D397M,D397N,D397Q,D397R,D397S,D397T,D397V,D397Y,A398E,A398R,A398V,A398W,I399C,I399Y,R402A,R402E,R402G,R402L,R402Q,R402S,R402W,Q409G,T411D,T411E,T411F,T411G,T411H,T411I,T411K,T411L,T411N,T411Q,T411R,T411S,T411V,S420C,S420G,S420H,S420K,S420N,S420Q,S420T,S420V,S420Y,R426A,G427C,G427D,G427E,G427F,G427H,G427P,G427V,G427Y,K428A,K428N,T445A,T445C,T445E,T445F,T445G,T445M,T445P,T445V,T445Y,V446Q,V446R,E447V,G448C,G448D,G448E,G448F,G448N,N449A,N449C,N449E,N449G,N449K,454F,N455C,N455D,N455S,N455V,N455W,H460E,H460G,H460M,H460Q,H460S,Q467P,Q467S,N473A,N473E,N473L,N473R,N473W,S474A,S474C,S474D,S474G,S474K,S474L,S474N,N475I,N475M,N475S,N475T,N475Y,Q490C,Q490H,Q490L,Q490R,Q490V,Q490W,Q490Y,L492A,L492D,L492F,L492W,L492Y,Q496G,Q496W,V497C,V497M,V497T,K498A,K498C,K498E,K498F,K498G,K498I,K498M,K498T,K498Y,D521A,D521C,D521W,V522A,V522C,V522K,V522L,V522M,V522Q,V522R,V522S,V522W,K534C,K534D,K534E,K534F,K534N,K534R,K534V,R542S,G547A,G547C,S548C,S548E,S548F,S548L,S548M,S548Q,S548T,S548W,G554C,G554D,G554F,G554H,G554M,G554Q,G554W,L555C,L555D,L555E,L555G,L555H,L555K,L555N,L555P,K560A,K560E,K560G,K560P,K560R,K560W,H561G,H561I,H561M,H561N,H561Q,H561S,H561V,H561W,D563A,D563Q,D563S,D563T,D563Y,D564A,D564C,D564F,D564G,D564K,D564L,D564M,D564N,D564Q,D564T,D564V,D564Y,R570A,R570C,R570D,R570E,R570G,R570I,R570Q,R570S,R570T,R570V,Y571H,Y571M,K581A,K581C,K581D,K581E,K581F,K581G,K581H,K581I,K581L,K581M,K581N,K581R,K581S,K581W,K581Y,N583A,N583C,N583D,N583G,N583H,R586N,R586P,R586V,R586W,V603G,V603H,V603Y,F611A,F611C,Q612C,Q612G,Q612S,A622E,Q626E,Q626H,T638A,T638D,T638G,T638W,S642E,S642F,S642G,S642H,642I,S642L,S642Q,S642R,S642T,S642W,S642Y,A643K,A643V,R645A,R645G,R645I,R645K,R645L,R645M,R645W,R645Y,K649C,K649N,K649T,Q650A,Q650C,Q650D,Q650F,Q650G,Q650K,Q650N,Q650R,Q650T,Q650V,Q650Y,T660C,T660D,T660N,T660S,T660W,T660Y,P661A,P661C,P661D,P661E,P661F,P661H,P661I,P661K,P661L,P661M,P661Q,P661R,P661S,P661T,P661V,P661W,G662A,G662C,G662F,G662H,G662I,G662N,Q663E,T666C,T666D,T666N,R672C,R672D,R672G,R672L,R672M,R672N,R672T,R672V,R673G,R673K,R673L,R673N,R673S,R673T,R674T,R674Y,D680C,D680F,D680I,D680M,D680Q,D680V,D680Y,T681A,T681G,T681P,T681Q,T681S,T681V,T681W,S683F,S683V,S683W,Q684C,Q684G,Q684N,K685A,K685E,K685F,K685G,K685I,K685L,K685M,K685Q,K685S,K685T,K685W,K685Y,S692C,S692H,S692I,S692L,S692M,S692V,S692W,R702C,R702D,R702F,R702G,R702H,R702S,R702T,R702V,R705F,R705I,R705L,R705M,R705S,R705T,R705V，和R705W，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的PASC水解活性(PI大于1)。另外，本发明提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022E,K022F,K022G,K022W,N024A,N024C,N024D,N024E,N024F,N024G,N024L,N024P,N024Q,N024S,N024V,N024Y,L025K,L025N,L025T,L025V,L025Y,Q026C,Q026D,Q026E,Q026G,Q026I,Q026K,Q026L,Q026P,Q026R,Q026S,Q026T,Q026V,Q026W,Q026Y,S033C,S033T,V035C,V035E,V035N,G036S,S050C,S050F,S050G,S050I,S050L,S050M,S050N,S050P,S050R,S050T,S050V,K051A,K051C,K051D,K051H,K051I,K051L,K051Q,K051R,K051S,K051T,K051V,R091D,R091E,R091F,R091G,R091H,R091I,R091K,R091L,R091N,R091Q,R091T,,R091V,R091W,R091Y,E092C,E092D,E092F,E092H,E092K,E092L,E092N,E092Q,E092R,E092T,E092V,E092Y,R093K,E099A,E099D,E099F,E099I,E099K,E099M,E099N,E099W,E099Y,L167C,L167D,L167E,L167F,L167G,L167M,L167V,L167W,N168A,N168D,N168E,N168G,N168Q,N168Y,E170D,E170F,P176E,P176F,P176G,P176H,P176L,P176M,P176Q,P176R,P176S,P176T,P176V,P176W,P176Y,D177F,D177K,D177L,D177M,D177N,D177Q,D177R,D177V,D178A,D178C,D178N,D178R,R179A,R179C,R179G,R179I,R179K,R179M,R179Q,R179S,R179T,R179V,Q194A,Q194C,Q194E,Q194F,Q194G,Q194H,Q194K,Q194L,Q194M,Q194R,Q194T,Q194W,Q194Y,N196E,N196H,N196L,N196R,N196T,S199G,S199N,S199T,S199V,T209C,T209D,T209E,T209G,T209H,T209I,T209K,T209L,T209M,T209Q,T209V,T209W,T209Y,E214D,D215C,D215L,D215M,D215S,D215W,Q216A,Q216C,Q216D,Q216E,Q216F,Q216G,Q216H,Q216I,Q216K,Q216N,Q216P,Q216R,Q216S,Q216T,Q216W,Q216Y,K224R,D225A,D225C,D225F,D225G,D225H,D225I,D225L,D225M,D225Q,D225S,D225T,D225V,D225W,D225Y,Q226C,Q226D,Q226E,Q226F,Q226H,Q226I,Q226K,Q226L,Q226M,Q226N,Q226R,Q226T,Q226V,Q226W,Q226Y,N238A,N238C,N238G,N238M,N238S,T242A,T242C,T242E,T242S,N248T,S249A,S249G,N263A,N263C,N263D,N263Q,N263S,N263T,N264C,R265A,R265E,R265I,R265L,R265M,R265Y,N276A,N276C,S277A,S277C,S277D,S277E,S277F,S277G,S277H,S277I,S277M,S277P,S277Q,S277W,S277Y,N278A,N278C,N278D,N278F,N278G,N278I,N278M,N278Q,N278R,N278S,N278T,N278V,N278W,N278Y,Q279C,Q279D,Q279E,Q279G,Q279H,Q279I,Q279K,Q279N,Q279S,Q279T,Q279V,Q279Y,T282K,D287C,D287G,D287H,D287I,D287K,D287M,D287N,D287S,Q301A,Q301E,Q301G,Q301K,Q301R,D302A,D302C,D302E,D302F,D302G,D302K,D302M,D302N,D302P,D302S,D302T,D302W,D302Y,Q303C,Q303D,Q303R,Q303W,Y306M,Y306R,Y306V,S312C,S312D,S312G,S312N,S312Q,S312R,S312T,S312V,S312Y,R313A,R313C,R313D,R313G,R313K,R313N,Q316K,Q316L,Q316M,Q316R,Q316T,Q316Y,K320C,K320G,K320N,K320P,K320S,K320Y,R324C,R324D,R324E,R324F,R324H,R324I,R324K,R324L,R324M,R324Q,R324V,R324W,R324Y,R328C,R328K,R328S,D329A,D329H,D329S,L334A,L334C,L334F,L334M,L334T,L334V,L334W,K335A,K335D,K335H,K335L,K335R,K335S,K335V,K335W,N336A,N336C,N336G,N336H,N336L,N336M,N336Q,N336R,N336T,N336V,N336Y,D337A,A338C,338D,A338E,A338F,A338G,A338H,A338I,A338K,A338L,A338N,A338P,A338Q,A338R,A338V,A338W,A338Y,K344D,K344F,K344L,K344M,K344N,K344P,K344T,K344V,K345A,K345D,K345E,K345F,K345G,K345H,K345N,K345P,K345R,K345S,K345V,K345W,K345Y,A347D,A347F,A347H,A347I,A347K,A347L,A347M,A347P,A347Q,A347R,A347S,A347Y,H361A,H361G,H361N,R363C,R363K,R363M,R363Q,R363S,R363T,R363V,R363W,N369C,N369D,N369S,K371G,G372A,D374C,D374F,D374N,D374S,Y396D,Y396E,Y396F,Y396G,Y396H,Y396K,Y396L,Y396M,Y396N,Y396Q,Y396R,397C,D397E,D397H,D397I,D397K,D397M,D397N,D397P,D397Q,D397R,D397S,D397T,D397V,D397Y,A398C,A398D,A398E,A398F,A398G,A398H,A398I,A398K,A398L,A398M,A398N,A398P,A398Q,A398R,A398S,A398T,A398V,A398W,A398Y,I399A,I399C,I399D,I399E,I399G,I399M,I399Q,I399S,I399T,I399W,I399Y,R402A,R402C,R402G,R402S,Q409D,V410A,V410C,V410I,V410L,V410N,V410R,V410S,V410T,V410W,T411D,T411E,T411G,T411N,T411Q,T411S,T411Y,S420C,R426A,R426E,R426F,R426I,R426K,R426N,R426P,R426Q,R426S,R426W,R426Y,G427C,G427E,G427F,G427H,G427K,G427N,G427Q,G427R,G427S,G427T,G427V,K428C,K428D,K428E,K428F,K428G,K428H,K428I,K428L,K428M,K428N,K428P,K428Q,K428R,K428S,K428T,K428V,K428W,K428Y,T445A,T445C,T445D,T445K,T445M,T445Q,T445S,V446C,G448A,G448C,G448D,G448E,G448F,G448N,G448S,G448T,G448Y,N449A,N449C,N454F,N454G,N454K,N454L,N454M,N454R,N454S,N454T,N454V,N455A,N455D,N455E,N455F,N455G,N455H,N455I,N455L,N455M,N455S,N455T,N455V,N455W,N455Y,Q467A,N473A,N473C,N473E,N473F,N473G,N473H,N473K,N473L,N473M,N473P,N473Q,N473R,N473S,N473T,N473V,N473W,S474A,S474C,S474D,S474F,S474G,S474I,S474K,S474M,S474N,S474Q,S474R,S474T,S474V,N475I,N475K,N475L,N475M,N475P,N475Q,N475R,N475S,N475T,N475V,N475W,N475Y,E489N,Q490P,Q490W,L492A,L492D,L492F,L492I,L492M,L492Q,L492T,L492W,Q496G,Q496S,Q496W,V497A,V497I,V497T,K498A,K498F,K498H,K498I,K498N,K498Q,D521A,D521C,D521E,D521F,D521G,D521H,D521I,D521K,D521L,D521M,D521P,D521R,D521S,D521T,D521V,D521W,D521Y,V522A,522C,V522F,V522G,V522H,V522I,V522K,,V522L,V522M,V522N,V522P,V522Q,V522R,V522S,V522T,V522W,V522Y,K534C,K534D,K534E,K534F,K534G,K534N,K534R,K534V,R542A,R542C,R542D,R542E,R542F,R542G,R542H,R542I,R542K,R542L,R542M,R542N,R542P,R542Q,R542S,R542T,R542V,R542W,R542Y,,G547A,G547L,S548L,G554A,G554C,G554D,G554F,G554H,G554L,G554V,G554W,L555A,L555C,L555D,L555E,L555F,L555G,L555H,L555I,L555K,L555N,L555P,L555Q,L555V,L555W,L555Y,K560H,K560P,K560R,K560W,H561A,H561C,H561D,H561E,H561F,H561G,H561I,H561M,H561N,H561Q,H561S,H561V,H561W,D563A,D563C,D563F,D563I,D563L,D563Q,D563R,D563S,D563T,D563W,D563Y,D564A,D564C,D564F,D564K,D564L,D564R,D564T,D564V,R570A,R570D,R570G,R570H,R570I,R570S,R570V,Y571H,Y571M,K581W,N583A,N583C,N583D,N583E,N583F,N583G,N583H,N583I,N583K,N583L,N583M,N583P,N583R,N583S,N583T,N583W,N583Y,R586E,R586F,R586G,R586H,R586L,R586N,R586P,R586V,R586W,R586Y,S591D,V603A,V603D,V603G,V603H,V603N,V603Q,V603R,V603Y,F611A,F611C,F611D,F611K,F611L,F611M,F611N,F611R,F611S,F611W,Q612C,Q612F,Q612G,Q612H,Q612I,Q612K,Q612L,Q612M,Q612R,Q612S,Q612W,A622H,A622K,Q626H,Q626M,V627P,T638A,T638D,T638E,T638F,T638G,T638I,T638K,T638L,T638M,T638P,T638Q,T638R,T638S,T638V,T638W,T638Y,S642A,S642C,S642E,S642F,S642G,S642H,S642I,S642K,S642L,S642M,S642N,S642P,S642Q,S642R,S642T,S642V,S642W,S642Y,A643C,A643F,A643G,A643H,A643K,A643L,A643M,A643Q,A643R,A643S,A643T,A643V,A643Y,R645A,R645D,R645F,R645G,R645H,R645I,R645K,R645L,R645M,R645P,R645Q,R645T,R645V,R645W,R645Y,K649A,K649C,K649F,K649L,K649N,K649Q,K649S,K649T,K649Y,Q650C,Q650D,Q650E,Q650F,Q650G,Q650H,Q650I,Q650K,Q650L,Q650M,Q650N,Q650R,Q650V,Q650Y,T660C,T660W,T660Y,P661C,P661F,P661H,P661I,P661K,P661L,P661M,P661Q,P661R,P661T,P661V,P661W,G662A,G662C,G662F,G662H,G662I,G662K,G662R,G662Y,Q663A,Q663C,Q663D,Q663E,Q663F,Q663I,Q663L,Q663M,Q663N,Q663R,Q663S,Q663V,Q663W,T666A,T666C,T666H,T666K,T666N,T666R,T666W,R672C,R672D,R672G,R672I,R672K,R672V,R672W,R673A,R673C,R673G,R673H,R673I,R673K,R673L,R673Q,R673T,R673V,R673W,R674L,R674M,R674T,R674Y,D675C,D680A,D680C,D680E,D680F,D680H,D680I,D680K,D680L,D680M,D680N,D680Q,D680R,D680S,D680V,D680W,D680Y,T681A,T681G,T681H,T681K,T681L,T681M,T681N,T681Q,T681R,T681W,S683A,S683C,S683D,S683F,S683G,S683I,S683L,S683P,S683R,S683V,S683W,Q684A,Q684D,K685A,K685F,K685G,K685I,K685L,K685M,K685N,K685Q,K685R,K685S,K685T,K685V,K685W,K685Y,S692E,S692H,S692K,S692L,S692Q,S692T,S692V,S692W,R702C,R702D,R702F,R702G,R702H,R702I,R702K,R702L,R702M,R702N,R702Q,R702S,R702T,R702V,R702W,R705C,R705F,R705H,R705I,R705L,R705M,R705P,R705S,R705T，和R705W，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的PCS水解活性(PI大于1)。本发明还提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022F,K022P,K022Q,N024C,N024P,N024Q,L025A,L025D,Q026C,Q026I,Q026K,Q026L,Q026P,Q026R,Q026S,Q026T,Q026W,S033C,S033G,V035C,V035E,V035Q,V035R,V035S,V035T,V035Y,G036C,G036D,G036E,G036F,G036I,G036K,G036R,G036S,G036W,G036Y,S050C,S050P,K051A,K051C,K051D,K051G,K051H,K051M,K051Q,K051T,K051V,R091D,R091G,R091K,R091N,R091Q,E092A,E092C,E092D,E092F,E092H,E092I,E092L,E092N,E092R,E092V,E099Y,E100A,E100G,E100M,E100T,E100Y,E164G,E164S,Q165V,E166D,L167C,L167G,L167N,L167V,L167W,N168A,N168D,N168E,N168G,N168H,N168Q,N168R,N168T,N168Y,E170F,P176A,P176D,P176F,P176H,P176K,P176L,P176R,P176V,P176W,P176Y,D177E,D177F,D177H,D177L,D177M,D177R,D177W,D177Y,D178C,D178K,D178R,D178Y,R179K,R179M,R179S,R179W,R94A,Q194C,Q194E,Q194F,Q194G,Q194K,Q194L,N196E,N196L,N196Q,N196T,T209C,T209D,T209E,T209G,T209H,T209I,T209L,T209M,T209Q,T209S,T209V,T209Y,E214W,D215C,D215E,D215G,D215L,D215M,D215N,D215Q,D215S,Q216A,Q216C,Q216F,Q216G,Q216I,Q216K,Q216L,Q216M,Q216S,Q216T,Q216W,Q216Y,K224R,K224V,D225F,D225G,D225H,D225I,D225L,D225M,D225T,D225V,D225W,D225Y,Q226A,Q226C,Q226F,Q226I,Q226L,Q226M,Q226N,Q226R,Q226V,Q226W,Q226Y,N238A,N238C,N238E,T242A,T242C,T242E,T242F,T242H,T242K,T242L,T242M,T242Q,T242V,T242W,T242Y,N248G,N248W,N248Y,N263C,N263E,N263F,N263G,N263Q,N263S,N263T,N263V,N263Y,N264C,R265E,R265K,N276C,S277A,S277C,S277F,S277I,S277M,S277P,S277R,S277W,S277Y,N278A,N278C,N278F,N278G,N278H,N278I,N278L,N278M,N278R,N278S,N278T,N278V,N278Y,Q279C,Q279V,Q279Y,T282C,D287C,D287S,Q301G,Q301K,D302A,D302C,D302F,D302G,Q303A,Q303C,Q303D,Q303P,Y306K,Y306Q,Y306R,S312C,S312W,S312Y,Q316C,Q316P,Q316S,Q316T,Q316Y,K320C,K320N,K320S,K320T,K320Y,R324C,R324Y,R328M,R328S,D329A,D329G,D329N,D329S,L334A,L334C,L334F,L334M,L334T,L334V,K335D,K335R,K335V,K335W,N336R,D337T,D337V,A338C,A338D,A338G,A338I,A338V,A338W,N339E,K344D,K344F,K344I,K344L,K344P,K344Q,K344V,K345A,K345D,K345E,K345F,K345G,K345H,K345S,K345T,K345V,K345Y,A347D,A347Y,H361A,H361C,H361E,H361G,H361L,H361M,H361T,R363C,R363E,R363K,R363L,R363M,R363Q,R363W,R363Y,N369C,N369D,N369E,N369F,N369M,N369S,N369T,N369V,N369W,N369Y,K371T,G372A,G372C,G372D,G372K,G372M,G372N,G372V,G372W,G372Y,D374C,D374F,D374G,D374L,D374M,D374Q,D374S,D374V,D375C,D375E,D375V,D375W,M380N,Y396C,Y396G,Y396K,D397A,D397C,D397E,D397F,D397H,D397I,D397K,D397L,D397M,D397N,D397P,D397Q,D397R,D397S,D397T,D397V,D397Y,A398C,A398D,A398E,A398F,A398I,A398K,A398N,A398P,A398Q,A398R,A398S,A398T,A398V,A398W,A398Y,I399A,I399C,I399D,I399E,I399F,I399Q,I399S,I399T,I399V,I399Y,R402A,R402F,R402G,R402L,R402S,R402W,T411D,T411E,T411F,T411G,T411H,T411K,T411L,T411N,T411Q,T411R,T411S,T411V,S420C,S420G,S420N,S420Q,S420T,S420V,S420Y,R426A,R426F,R426N,R426Q,R426T,R426W,G427C,G427F,G427Y,K428A,T445A,T445C,T445E,T445F,T445G,T445I,T445K,T445L,T445M,T445N,T445P,T445Q,T445R,T445S,T445V,T445Y,V446K,V446Q,V446R,G448A,G448C,G448D,G448E,G448F,N449A,N449C,N449G,N449H,N449K,N449P,N454F,N455C,N455D,N455S,N455W,H460A,H460C,H460D,H460E,H460F,H460G,H460I,H460K,H460L,H460M,,H460Q,H460R,H460S,H460W,H460Y,S474C,S474D,S474F,S474G,S474I,S474K,S474L,S474M,S474N,S474P,S474R,S474T,S474V,N475I,N475K,N475L,N475M,N475P,N475Q,N475R,N475S,N475T,N475V,N475W,N475Y,Q490C,Q490L,Q490V,Q490W,Q490Y,L492A,L492D,L492H,L492I,L492N,L492Q,L492T,L492Y,Q496S,Q496W,V497T,K498C,K498E,K498F,K498G,K498I,D521C,D521W,D521Y,V522A,V522C,V522F,V522G,V522K,V522L,V522M,V522N,V522P,V522Q,V522R,V522S,V522T,V522W,V522Y,K534C,K534D,K534E,K534F,K534G,K534V,R542A,R542D,R542I,R542L,R542N,R542T,R542W,G547A,S548C,S548E,S548F,S548L,S548N,S548Q,S548T,S548W,G554A,G554C,G554D,G554F,G554H,G554L,G554M,G554Q,G554W,L555C,L555E,L555G,L555H,L555K,L555M,L555P,L555Q,K560P,H561I,H561M,H561N,H561Q,H561S,H561V,H561W,D563A,D563I,D563L,D563Q,D563R,D563S,D563T,D563V,D563W,D563Y,D564A,D564C,D564F,D564G,D564K,D564L,D564M,D564N,D564R,D564T,D564V,D564Y,R570A,R570C,R570D,R570E,R570I,R570M,R570Q,R570T,R570V,Y571H,Y571M,Y571N,Y571R,K581A,K581C,K581D,K581E,K581F,K581G,K581M,K581W,N583A,N583C,N583D,N583G,N583V,R586D,R586F,R586N,R586P,R586V,R586W,R586Y,V603C,V603E,V603G,V603H,V603Y,F611A,F611C,F611D,F611K,F611M,F611R,F611W,Q612C,Q612D,Q612G,Q612S,A622E,A622H,Q626E,Q626F,Q626H,T638A,T638D,T638G,T638M,T638Q,T638R,T638S,T638V,T638W,T638Y,S642C,S642E,S642F,S642G,S642H,S642I,S642L,S642M,S642P,S642Q,S642T,S642V,S642W,S642Y,A643E,A643F,A643H,A643K,A643L,A643M,A643N,A643T,A643V,A643Y,R645G,R645K,R645M,R645W,R645Y,K649C,K649N,K649S,Q650C,Q650D,Q650T,Q650V,Q650Y,T660C,T660F,T660N,T660S,T660W,T660Y,P661C,P661D,P661E,P661F,P661H,P661I,P661K,P661L,P661M,P661Q,P661R,P661S,P661T,P661V,P661W,G662A,G662C,G662F,G662I,Q663D,Q663E,Q663G,Q663I,T666C,T666N,R672C,R672D,R672E,R672F,R672G,R672H,R672I,R672K,R672L,R672M,R672N,R672T,R672V,R672W,R673A,R673C,R673E,R673G,R673H,R673I,R673K,R673L,R673M,R673N,R673S,R673V,R674T,R674Y,D680A,D680C,D680E,D680F,D680H,D680I,D680M,D680Q,D680R,D680V,D680W,D680Y,T681G,T681H,T681K,T681L,T681M,T681P,T681Q,T681S,T681V,T681W,T681Y,S683C,S683D,S683E,S683F,S683G,S683I,S683M,S683P,S683Q,S683V,S683W,Q684C,Q684G,Q684N,K685A,K685E,K685G,K685I,K685L,K685M,K685N,K685Q,K685S,K685T,K685V,K685W,K685Y,S692C,S692H,S692I,S692L,S692M,S692W,R702C,R702D,R702F,R702G,R702H,R702I,R702K,R702L,R702N,R702Q,R702S,R702T,R702V,R702W,R705I，和R705V，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的pH5下的纤维二糖水解活性(PI大于1)。另外，本发明提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022E,K022F,K022H,K022P,K022Q,K022R,N024C,N024Q,L025A,L025D,L025N,Q026C,Q026H,Q026I,Q026K,Q026L,Q026P,Q026R,Q026S,Q026T,Q026W,D027C,K028S,K028V,S033C,S033G,V035C,V035E,V035G,V035H,V035K,V035L,V035N,V035Q,V035R,V035S,V035T,V035Y,G036C,G036D,G036E,G036F,G036I,G036K,G036N,G036R,G036S,G036W,G036Y,K051C,K051D,K051G,K051H,K051I,K051L,K051M,K051N,K051R,K051T,K051V,I052D,I052K,I052M,I052N,I052P,I052Q,I052T,I052V,R091C,R091Q,R091W,E092C,E092K,E099Y,E100A,E100G,E100I,E100M,E100N,E100Q,E100S,E100T,E100Y,Q165C,Q165G,Q165I,Q165K,Q165M,Q165N,Q165S,Q165V,E166D,L167C,L167V,L167W,L167Y,N168A,N168D,N168E,N168G,N168Y,E170F,E170Y,P176F,P176H,P176K,P176L,P176R,P176V,P176W,P176Y,D177E,D177F,D177H,D177L,D177M,D177Q,D177R,D177V,D177W,D177Y,D178Y,R179M,R179S,R179T,R179W,Q194L,N196L,N196T,N208K,T209C,T209D,T209E,T209G,T209H,T209I,T209K,T209L,T209M,T209Q,T209S,T209V,T209Y,E214A,E214D,E214G,E214R,E214S,E214W,D215E,D215H,D215L,D215N,D215S,D215W,Q216A,Q216D,Q216F,Q216G,Q216H,Q216I,Q216K,Q216L,Q216M,Q216N,Q216S,Q216T,Q216W,Q216Y,K224H,K224R,K224V,D225E,D225F,D225G,D225H,D225I,D225L,D225M,D225T,D225V,D225W,D225Y,Q226A,Q226C,Q226D,Q226F,Q226I,Q226L,Q226W,Q226Y,N238A,N238C,N238E,T242C,T242E,T242H,T242Q,T242W,T242Y,N248G,N248W,N248Y,N263C,N263G,N263S,N263T,N264C,N276A,N276C,N276F,N276M,N276Q,S277C,S277W,S277Y,N278C,N278F,N278V,N278W,Q279C,Q279D,Q279K,Q279V,Q279Y,T282C,T282D,T282P,T282S,R284H,R284M,D287C,D287S,Q301K,D302A,D302C,D302E,D302F,D302G,D302L,D302M,D302N,Q303C,Y306K,Y306M,Y306Q,Y306R,S312C,S312K,S312W,S312Y,Q316C,Q316D,Q316G,Q316H,Q316I,Q316K,Q316P,Q316R,Q316S,Q316T,Q316Y,K320C,K320E,K320H,K320L,K320M,K320P,K320Q,K320R,K320S,K320T,R324C,R324Y,R328C,R328E,R328F,R328G,R328I,R328K,R328L,R328M,R328Q,R328S,R328V,R328Y,D329A,D329M,D329S,D329T,D329Y,L334A,L334T,K335N,K335V,D337A,D337C,D337T,D337V,A338C,A338D,A338F,A338G,A338I,A338P,A338V,A338W,K344D,K344F,K344I,K344V,K345A,K345E,K345F,K345G,K345Q,K345S,K345T,K345V,K345W,K345Y,A347Y,H361C,H361G,H361M,R363C,R363E,R363G,R363K,R363Q,R363S,R363T,R363W,R363Y,N369C,N369D,N369E,N369F,N369W,N369Y,K371T,G372A,G372K,G372M,G372W,G372Y,D374C,D374F,D374G,D374I,D374L,D374M,D374Q,D374S,D374T,D374V,D374Y,D375C,D375E,D375H,D375I,D375R,D375V,D375W,M380I,M380N,M380T,M380V,M380Y,W382F,Y396C,Y396D,Y396E,Y396F,Y396G,Y396H,Y396I,Y396K,Y396L,Y396M,Y396N,Y396Q,Y396R,Y396S,Y396V,Y396W,D397A,D397C,D397E,D397H,D397I,D397K,D397M,D397N,D397P,D397Q,D397R,D397S,D397T,D397V,D397Y,A398K,A398Q,A398R,A398W,I399A,I399C,I399D,I399E,I399G,I399Q,I399S,I399T,I399V,I399Y,R402A,R402E,R402G,R402L,R402Q,R402S,R402W,R402Y,V410C,V410F,V410H,V410I,V410R,V410S,V410W,V410Y,T411D,T411E,T411F,T411G,T411H,T411I,T411N,T411Q,T411R,T411S,T411V,T411Y,S420C,S420G,S420N,S420Q,S420T,S420V,S420Y,R426A,R426L,R426T,R426Y,G427C,G427F,G427P,G427V,K428A,T445A,T445C,T445F,T445G,T445M,T445N,T445P,T445V,T445Y,V446K,V446Q,V446R,E447K,E447L,E447S,E447V,E447Y,G448C,G448Y,N449C,N449H,N449K,N454F,N454V,N455C,N455D,N455S,N455V,N455W,H460A,H460C,H460E,H460F,H460G,H460I,H460K,H460L,H460M,H460N,H460Q,H460R,H460S,H460W,H460Y,N473W,S474A,S474C,S474E,S474F,S474K,S474L,S474N,S474P,S474T,N475I,N475M,N475S,N475T,N475W,N475Y,E489D,E489N,Q490C,Q490V,Q490W,Q490Y,L492A,L492D,L492F,L492H,L492I,L492N,L492R,L492T,L492Y,Q496W,K498A,K498E,K498F,K498M,K498V,D521C,V522A,V522C,V522G,V522K,V522L,V522M,V522Q,V522R,V522S,V522T,V522W,V522Y,K534C,K534D,K534E,K534R,K534V,R542A,R542C,R542D,R542E,R542F,R542G,R542H,R542I,R542L,R542M,R542N,R542Q,R542S,R542T,R542V,R542W,R542Y,G547A,G547C,S548T,E553I,E553Y,G554C,G554D,G554F,G554H,G554Q,G554W,L555D,L555E,L555F,L555G,L555H,L555K,L555M,L555P,L555Q,L555T,L555V,L555W,L555Y,H561A,H561C,H561D,H561G,H561M,H561S,H561W,D563A,D563E,D563L,D563M,D563Q,D563S,D563T,D563V,D563W,D563Y,D564A,D564C,D564F,D564K,D564L,D564N,D564Q,D564R,D564T,D564V,D564Y,R570A,R570C,R570D,R570E,R570M,R570Q,R570S,R570T,R570V,Y571N,K581A,K581C,K581D,K581F,K581G,K581I,K581S,K581V,K581W,K581Y,N583A,N583C,N583D,N583E,N583F,N583G,N583H,N583I,N583K,N583L,N583M,N583P,N583R,N583S,N583T,N583V,N583W,N583Y,R586D,R586F,R586G,R586L,R586N,R586P,R586V,R586W,R586Y,S591D,V603C,V603D,V603F,V603G,V603H,V603M,V603N,V603P,V603Q,V603R,V603S,V603T,V603W,V603Y,F611A,F611C,F611K,F611R,F611V,F611W,F611Y,Q612C,Q612D,Q612G,Q612H,Q612S,A622D,A622G,A622H,A622I,A622K,A622L,A622M,A622P,A622S,A622T,A622V,A622Y,Q626G,Q626H,Q626L,Q626T,Q626V,T638A,T638D,T638G,T638M,T638Q,T638R,T638S,T638V,T638W,T638Y,S642C,S642E,S642F,S642H,S642L,S642P,S642Q,S642T,S642W,S642Y,A643H,A643K,A643L,A643Q,A643T,A643V,A643W,A643Y,R645A,R645D,R645F,R645G,R645I,R645K,R645L,R645M,R645T,R645V,R645W,R645Y,K649T,Q650E,Q650G,Q650H,Q650I,Q650K,Q650L,Q650N,Q650R,Q650T,Q650Y,T660C,T660D,T660N,T660S,T660W,T660Y,P661A,P661C,P661D,P661E,P661F,P661G,P661H,P661I,P661K,P661L,P661M,P661Q,P661R,P661S,P661T,P661V,P661W,G662A,G662C,G662F,G662I,Q663C,Q663D,Q663E,Q663F,Q663G,Q663H,Q663I,T666C,T666N,T666R,R672C,R672D,R672E,R672F,R672G,R672H,R672I,R672K,R672L,R672M,R672N,R672T,R672V,R672W,R672Y,R673E,R673G,R673I,R673L,R673M,R673Q,R673S,R674T,R674Y,D675L,D680A,D680C,D680E,D680F,D680H,D680I,D680L,D680M,D680Q,D680V,D680W,D680Y,T681A,T681G,T681H,T681K,T681L,T681M,T681N,T681P,T681Q,T681S,T681V,T681W,S683E,S683F,S683I,S683L,S683M,S683P,S683V,S683W,Q684A,Q684C,Q684G,K685I,K685L,K685M,K685N,K685S,K685T,K685V,K685W,K685Y,S692C,S692H,S692I,S692M,S692T,S692V,S692W,R702C,R702D,R702G,R702L,R702Q,R702S,R702T,R702V,R702W,R705F,R705H,R705I,R705L,R705S,R705T,R705V，和R705W，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的pH6下的纤维二糖水解活性(PI大于1)。本发明还提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022F,K022P,N024A,N024C,N024Q,L025A,L025D,L025S,Q026C,Q026D,Q026E,Q026K,Q026S,Q026T,Q026W,D027A,D027L,D027M,D027Q,D027S,S033C,V035C,V035E,V035G,G036D,G036E,G036S,G036Y,S050C,S050L,K051C,K051D,K051H,K051T,I052A,I052D,I052N,I052P,I052V,R091D,R091E,R091F,R091N,E092A,E092C,E100A,E100G,E100M,E100N,E100S,E100Y,L167C,L167W,N168Y,P176A,P176F,P176K,P176R,P176S,P176V,P176W,P176Y,D177E,D177F,D177M,D177N,D177V,D177W,D178A,Q194A,Q194C,N196E,N196L,T209C,T209D,T209E,T209G,T209L,T209S,T209V,T209W,T209Y,E214D,E214V,D215C,D215E,D215N,D215S,Q216A,Q216G,Q216H,Q216I,Q216L,Q216S,Q216W,Q216Y,D225E,D225G,D225H,D225I,D225L,D225T,D225W,D225Y,Q226C,Q226D,Q226F,Q226N,Q226R,Q226S,Q226W,Q226Y,N238A,T242C,T242E,T242H,T242L,T242S,T242W,T242Y,N248C,N248W,N263A,N263C,N263D,N263E,N263G,N263H,N263L,N263Q,N263R,N263S,N263T,N264C,N276A,N276C,N276F,N276K,S277C,S277W,N278F,N278W,Q279C,T282C,T282L,T282P,R284M,D287C,D287K,D302A,D302C,D302E,D302F,D302G,Q303C,Y306C,Y306G,Y306M,Y306Q,Y306V,S312C,S312D,S312W,S312Y,Q316C,Q316P,Q316S,Q316T,Q316Y,K320C,K320N,R324C,R328S,D329A,D329S,K335D,N336A,N336C,N336H,D337A,D337C,D337K,D337M,D337T,A338C,A338D,A338E,A338F,A338G,A338K,A338L,A338M,A338P,A338V,A338W,A338Y,K344D,K344F,K344G,K344L,K344Q,K344R,K344V,K345A,K345E,K345F,K345S,K345Y,A347Y,H361D,H361G,R363A,R363C,R363E,R363G,R363K,R363M,R363Q,R363T,R363V,R363W,R363Y,N369C,N369D,N369E,N369T,N369W,K371A,K371L,K371T,G372A,D374C,D374L,D374M,D374S,D375C,M380T,M380V,G381H,W382F,D397C,D397N,D397R,D397T,D397V,D397Y,A398R,A398W,I399L,I399V,R402A,R402I,V410F,V410R,V410S,T411E,T411H,T411N,S420C,S420D,S420G,S420N,S420T,S420V,R426A,G427C,G427E,G427F,G427H,G427L,G427Y,T445A,T445C,T445D,T445E,T445F,T445G,T445I,T445M,T445P,T445V,T445Y,V446A,V446C,G448C,G448F,G448H,N449A,N449C,N454F,N455C,N455D,N455W,H460D,H460F,H460G,H460K,H460Q,S474A,S474C,S474E,S474F,S474I,S474M,S474N,S474T,S474V,S474Y,N475S,Q490C,Q490E,Q490G,Q490H,Q490L,Q490V,Q490W,Q490Y,V497T,K498A,K498E,K498M,D521A,D521S,D521W,D521Y,V522C,V522S,V522W,V522Y,K534C,K534E,K534F,K534N,K534V,R542A,R542C,R542D,R542E,R542F,R542G,R542K,R542L,R542M,R542N,R542Q,R542T,R542V,R542W,G547A,S548C,S548E,S548F,S548W,E553N,G554C,G554F,G554Q,G554W,K560H,H561F,H561G,H561I,H561M,H561N,H561S,H561T,H561V,H561W,D563A,D563S,D564A,D564F,D564S,D564T,K581A,K581C,K581D,K581F,K581G,K581H,K581P,K581S,K581T,K581V,K581W,N583A,N583C,N583D,R586D,R586N,R586P,R586V,R586Y,V603A,V603C,V603D,V603E,V603F,V603G,V603H,V603Y,F611A,Q612C,A622D,A622E,A622F,A622G,A622H,A622M,A622N,A622R,A622S,A622T,A622V,Q626E,Q626F,Q626G,Q626H,Q626M,T638A,T638G,T638W,S642F,S642L,S642W,A643V,R645G,K649A,K649C,K649S,Q650E,Q650G,Q650H,Q650V,Q650Y,T660W,P661A,P661C,P661D,P661F,P661I,P661K,P661L,P661M,P661Q,P661R,P661S,P661T,P661V,P661W,G662A,G662C,G662D,G662F,Q663A,Q663C,Q663D,Q663E,Q663F,Q663G,Q663H,Q663L,Q663N,Q663R,Q663S,Q663V,T666A,T666C,T666N,R672C,R672K,R672T,R673A,R673C,R673G,R673H,R673I,R673K,R673L,R673M,R673S,R673T,R673V,R673W,R674M,R674T,R674V,D680A,D680C,D680M,D680Q,D680V,D680W,D680Y,T681A,T681G,T681K,T681L,T681M,T681P,T681Q,T681S,T681V,T681W,A682M,S683E,S683M,S683V,S683W,Q684A,Q684C,Q684G,Q684N,K685A,K685E,K685I,K685L,K685M,K685S,K685T,K685W,K685Y,S692C,S692H,S692I,S692L,S692M,S692P,S692V,S692W,R702C,R702G,R702S,R705C,R705I,R705T，和R705V，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的在氨预处理玉米芯存在下的纤维二糖水解作用(PI大于1)。另外，本发明提供包含置换的β-葡萄糖苷酶变体，其中所述置换包括由以下置换组成的组中的一种或多种置换：K022A,K022E,K022F,K022G,K022H,K022P,K022Q,K022R,K022S,K022V,K022W,K022Y,N024A,N024C,N024D,N024E,N024F,N024G,N024L,N024P,N024Q,N024R,N024S,N024V,N024Y,L025A,L025D,L025F,L025G,L025I,L025K,L025N,L025Q,L025R,L025S,L025T,L025V,L025W,L025Y,Q026C,Q026D,Q026E,Q026G,Q026H,Q026I,Q026K,Q026L,Q026P,Q026R,Q026S,Q026T,Q026V,Q026W,Q026Y,D027A,D027C,D027E,D027L,D027M,D027Q,D027S,D027T,D027V,K028S,K028V,S033C,S033G,S033T,V035C,V035E,V035G,V035H,V035K,V035L,V035N,V035P,V035Q,V035R,V035S,V035T,V035Y,G036C,G036D,G036E,G036F,G036I,G036K,G036N,G036R,G036S,G036W,G036Y,W037V,W037Y,S050A,S050C,S050F,S050G,S050I,S050L,S050M,S050N,S050P,S050R,S050T,S050V,K051A,K051C,K051D,K051E,K051G,K051H,K051I,K051L,K051M,K051N,K051Q,K051R,K051S,K051T,K051V,I052A,I052D,I052K,I052M,I052N,I052P,I052Q,I052T,I052V,R067A,R067C,R067D,R067F,R067G,R067N,R067P,R067Q,R067S,R067W,R091A,R091C,R091D,R091E,R091F,R091G,R091H,R091I,R091K,R091L,R091N,R091Q,R091T,R091V,R091W,R091Y,E092A,E092C,E092D,E092F,E092H,E092I,E092K,E092L,E092N,E092Q,E092R,E092T,E092V,E092Y,R093K,E099A,E099D,E099F,E099I,E099K,E099M,E099N,E099W,E099Y,E100A,E100G,E100I,E100M,E100N,E100Q,E100S,E100T,E100Y,K158H,K158T,E164G,E164S,Q165C,Q165F,Q165G,Q165H,Q165I,Q165K,Q165L,Q165M,Q165N,Q165R,Q165S,Q165T,Q165V,Q165W,Q165Y,E166D,E166K,E166L,E166P,E166Q,E166R,E166T,L167A,L167C,L167D,L167E,L167F,L167G,L167M,L167N,L167Q,L167R,L167S,L167V,L167W,L167Y,N168A,N168D,N168E,N168G,N168H,N168Q,N168R,N168T,N168Y,E170D,E170F,E170Y,P176A,P176D,P176E,P176F,P176G,P176H,P176K,P176L,P176M,P176Q,P176R,P176S,P176T,P176V,P176W,P176Y,D177E,D177F,D177H,D177K,D177L,D177M,D177N,D177Q,D177R,D177V,D177W,D177Y,D178A,D178C,D178K,D178N,D178Q,D178R,D178Y,R179A,R179C,R179G,R179I,R179K,R179M,R179Q,R179S,R179T,R179V,R179W,Q194A,Q194C,Q194E,Q194F,Q194G,Q194H,Q194K,Q194L,Q194M,Q194R,Q194T,Q194W,Q194Y,N196E,N196H,N196L,N196Q,N196R,N196T,S199A,S199G,S199N,S199T,S199V,Y204F,N208K,T209C,T209D,T209E,T209G,T209H,T209I,T209K,T209L,T209M,T209Q,T209R,T209S,T209V,T209W,T209Y,E214A,E214C,E214D,E214G,E214H,E214L,E214M,E214N,E214Q,E214R,E214S,E214T,E214V,E214W,E214Y,D215C,D215E,D215G,D215H,D215L,D215M,D215N,D215Q,D215S,D215W,Q216A,Q216C,Q216D,Q216E,Q216F,Q216G,Q216H,Q216I,Q216K,Q216L,Q216M,Q216N,Q216P,Q216R,Q216S,Q216T,Q216W,Q216Y,K224H,K224R,K224V,D225A,D225C,D225E,D225F,D225G,D225H,D225I,D225L,D225M,D225Q,D225S,D225T,D225V,D225W,D225Y,Q226A,Q226C,Q226D,Q226E,Q226F,Q226H,Q226I,Q226K,Q226L,Q226M,Q226N,Q226R,Q226S,Q226T,Q226V,Q226W,Q226Y,N238A,N238C,N238E,N238G,N238M,N238S,N238T,T242A,T242C,T242E,T242F,T242G,T242H,T242I,T242K,T242L,T242M,T242N,T242Q,T242R,T242S,T242V,T242W,T242Y,N248A,N248C,N248F,N248G,N248T,N248W,N248Y,S249A,S249G,S249M,S249V,N263A,N263C,N263D,N263E,N263F,N263G,N263H,N263L,N263Q,N263R,N263S,N263T,N263V,N263Y,N264C,R265A,R265E,R265I,R265K,R265L,R265M,R265N,R265Q,R265Y,N276A,N276C,N276F,N276K,N276M,N276Q,S277A,S277C,S277D,S277E,S277F,S277G,S277H,S277I,S277M,S277P,S277Q,S277R,S277W,S277Y,N278A,N278C,N278D,N278F,N278G,N278H,N278I,N278L,N278M,N278Q,N278R,N278S,N278T,N278V,N278W,N278Y,Q279C,Q279D,Q279E,Q279G,Q279H,Q279I,Q279K,Q279N,Q279S,Q279T,Q279V,Q279Y,T282C,T282D,T282K,T282L,T282P,T282S,R284H,R284M,D287C,D287E,D287G,D287H,D287I,D287K,D287M,D287N,D287S,Q301A,Q301E,Q301G,Q301K,Q301L,Q301N,Q301R,Q301S,Q301T,Q301V,D302A,D302C,D302E,D302F,D302G,D302K,D302L,D302M,D302N,D302P,D302S,D302T,D302W,D302Y,Q303A,Q303C,Q303D,Q303E,Q303H,Q303I,Q303K,Q303L,Q303M,Q303N,Q303P,Q303R,Q303S,Q303T,Q303V,Q303W,Q303Y,Y306C,Y306G,Y306I,Y306K,Y306L,Y306M,Y306N,Y306P,Y306Q,Y306R,Y306S,Y306T,Y306V,S312C,S312D,S312G,S312K,S312N,S312Q,S312R,S312T,S312V,S312W,S312Y,R313A,R313C,R313D,R313G,R313K,R313N,Q316C,Q316D,Q316G,Q316H,Q316I,Q316K,Q316L,Q316M,Q316P,Q316R,Q316S,Q316T,Q316Y,K320C,K320E,K320G,K320H,K320L,K320M,K320N,K320P,K320Q,K320R,K320S,K320T,K320Y,R324C,R324D,R324E,R324F,R324H,R324I,R324K,R324L,R324M,R324Q,R324V,R324W,R324Y,R328C,R328E,R328F,R328G,R328I,R328K,R328L,R328M,R328Q,R328S,R328T,R328V,R328Y,D329A,D329E,D329G,D329H,D329M,D329N,D329Q,D329S,D329T,D329Y,L334A,L334C,L334F,L334M,L334T,L334V,L334W,K335A,K335D,K335F,K335H,K335I,K335L,K335M,K335N,K335R,K335S,K335T,K335V,K335W,N336A,N336C,N336G,N336H,N336L,N336M,N336Q,N336R,N336T,N336V,N336Y,D337A,D337C,D337E,D337G,D337H,D337K,D337L,D337M,D337N,D337R,D337S,D337T,D337V,D337W,D337Y,A338C,A338D,A338E,A338F,A338G,A338H,A338I,A338K,A338L,A338M,A338N,A338P,A338Q,A338R,A338V,A338W,A338Y,N339E,N339G,N339H,N339K,N339L,K344D,K344E,K344F,K344G,K344I,K344L,K344M,K344N,K344P,K344Q,K344R,K344S,K344T,K344V,K345A,K345D,K345E,K345F,K345G,K345H,K345N,K345P,K345Q,K345R,K345S,K345T,K345V,K345W,K345Y,A347D,A347F,A347H,A347I,A347K,A347L,A347M,A347P,A347Q,A347R,A347S,A347Y,H361A,H361C,H361D,H361E,H361G,H361L,H361M,H361N,H361T,R363A,R363C,R363E,R363G,R363K,R363L,R363M,R363Q,R363S,R363T,R363V,R363W,R363Y,N369C,N369D,N369E,N369F,N369L,N369M,N369S,N369T,N369V,N369W,N369Y,D370E,D370F,D370G,D370S,D370W,D370Y,K371A,K371F,K371G,K371L,K371N,K371Q,K371R,K371S,K371T,K371V,G372A,G372C,G372D,G372E,G372K,G372L,G372M,G372N,G372T,G372V,G372W,G372Y,D374C,D374F,D374G,D374I,D374L,D374M,D374N,D374Q,D374S,D374T,D374V,D374Y,D375A,D375C,D375E,D375H,D375I,D375R,D375V,D375W,M380I,M380L,M380N,M380Q,M380S,M380T,M380V,M380Y,G381H,W382F,Y396A,Y396C,Y396D,Y396E,Y396F,Y396G,Y396H,Y396I,Y396K,Y396L,Y396M,Y396N,Y396Q,Y396R,Y396S,Y396T,Y396V,Y396W,D397A,D397C,D397E,D397F,D397H,D397I,D397K,D397L,D397M,D397N,D397P,D397Q,D397R,D397S,D397T,D397V,D397Y,A398C,A398D,A398E,A398F,A398G,A398H,A398I,A398K,A398L,A398M,A398N,A398P,A398Q,A398R,A398S,A398T,A398V,A398W,A398Y,I399A,I399C,I399D,I399E,I399F,I399G,I399L,I399M,I399Q,I399S,I399T,I399V,I399W,I399Y,R402A,R402C,R402E,R402F,R402G,R402I,R402L,R402Q,R402S,R402W,R402Y,Q409D,Q409G,V410A,V410C,V410F,V410H,V410I,V410L,V410N,V410R,V410S,V410T,V410W,V410Y,T411D,T411E,T411F,T411G,T411H,T411I,T411K,T411L,T411N,T411Q,T411R,T411S,T411V,T411Y,S420C,S420D,S420G,S420H,S420K,S420N,S420Q,S420T,S420V,S420Y,R426A,R426E,R426F,R426I,R426K,R426L,R426N,R426P,R426Q,R426S,R426T,R426W,R426Y,G427C,G427D,G427E,G427F,G427H,G427K,G427L,G427M,G427N,G427P,G427Q,G427R,G427S,G427T,G427V,G427Y,K428A,K428C,K428D,K428E,K428F,K428G,K428H,K428I,K428L,K428M,K428N,K428P,K428Q,K428R,K428S,K428T,K428V,K428W,K428Y,T445A,T445C,T445D,T445E,T445F,T445G,T445I,T445K,T445L,T445M,T445N,T445P,T445Q,T445R,T445S,T445V,T445Y,V446A,V446C,V446K,V446Q,V446R,E447K,E447L,E447S,E447V,E447Y,G448A,G448C,G448D,G448E,G448F,G448H,G448N,G448S,G448T,G448Y,N449A,N449C,N449E,N449F,N449G,N449H,N449K,N449M,N449P,N449T,N449V,N454F,N454G,N454K,N454L,N454M,N454R,N454S,N454T,N454V,N455A,N455C,N455D,N455E,N455F,N455G,N455H,N455I,N455L,N455M,N455S,N455T,N455V,N455W,N455Y,H460A,H460C,H460D,H460E,H460F,H460G,H460I,H460K,H460L,H460M,H460N,H460Q,H460R,H460S,H460W,H460Y,Q467A,Q467P,Q467S,N473A,N473C,N473E,N473F,N473G,N473H,N473K,N473L,N473M,N473P,N473Q,N473R,N473S,N473T,N473V,N473W,S474A,S474C,S474D,S474E,S474F,S474G,S474I,S474K,S474L,S474M,S474N,S474P,S474Q,S474R,S474T,S474V,S474Y,N475I,N475K,N475L,N475M,N475P,N475Q,N475R,N475S,N475T,N475V,N475W,N475Y,E489D,E489N,Q490A,Q490C,Q490E,Q490F,Q490G,Q490H,Q490K,Q490L,Q490P,Q490R,Q490S,Q490T,Q490V,Q490W,Q490Y,L492A,L492D,L492F,L492H,L492I,L492M,L492N,L492Q,L492R,L492T,L492W,L492Y,Q496G,Q496S,Q496W,V497A,V497C,V497I,V497M,V497T,K498A,K498C,K498E,K498F,K498G,K498H,K498I,K498L,K498M,K498N,K498Q,K498T,K498V,K498Y,D521A,D521C,D521E,D521F,D521G,D521H,D521I,D521K,D521L,D521M,D521P,D521R,D521S,D521T,D521V,D521W,D521Y,V522A,V522C,V522F,V522G,V522H,V522I,V522K,V522L,V522M,V522N,V522P,V522Q,V522R,V522S,V522T,V522W,V522Y,K534C,K534D,K534E,K534F,K534G,K534N,K534R,K534V,R542A,R542C,R542D,R542E,R542F,R542G,R542H,R542I,R542K,R542L,R542M,R542N,R542P,R542Q,R542S,R542T,R542V,R542W,R542Y,G547A,G547C,G547L,G547P,S548C,S548E,S548F,S548H,S548I,S548L,S548M,S548N,S548Q,S548R,S548T,S548V,S548W,S548Y,E553I,E553N,E553Y,G554A,G554C,G554D,G554F,G554H,G554L,G554M,G554Q,G554V,G554W,L555A,L555C,L555D,L555E,L555F,L555G,L555H,L555I,L555K,L555M,L555N,L555P,L555Q,L555T,L555V,L555W,L555Y,K560A,K560E,K560G,K560H,K560P,K560R,K560W,H561A,H561C,H561D,H561E,H561F,H561G,H561I,H561M,H561N,H561Q,H561S,H561T,H561V,H561W,D563A,D563C,D563E,D563F,D563I,D563L,D563M,D563Q,D563R,D563S,D563T,D563V,D563W,D563Y,D564A,D564C,D564F,D564G,D564K,D564L,D564M,D564N,D564Q,D564R,D564S,D564T,D564V,D564Y,R570A,R570C,R570D,R570E,R570G,R570H,R570I,R570M,R570Q,R570S,R570T,R570V,Y571H,Y571M,Y571N,Y571R,Y571W,K581A,K581C,K581D,K581E,K581F,K581G,K581H,K581I,K581L,K581M,K581N,K581P,K581R,K581S,K581T,K581V,K581W,K581Y,N583A,N583C,N583D,N583E,N583F,N583G,N583H,N583I,N583K,N583L,N583M,N583P,N583R,N583S,N583T,N583V,N583W,N583Y,R586D,R586E,R586F,R586G,R586H,R586L,R586N,R586P,R586V,R586W,R586Y,S591D,V603A,V603C,V603D,V603E,V603F,V603G,V603H,V603M,V603N,V603P,V603Q,V603R,V603S,V603T,V603W,V603Y,F611A,F611C,F611D,F611K,F611L,F611M,F611N,F611R,F611S,F611V,F611W,F611Y,Q612C,Q612D,Q612F,Q612G,Q612H,Q612I,Q612K,Q612L,Q612M,Q612R,Q612S,Q612W,A622D,A622E,A622F,A622G,A622H,A622I,A622K,A622L,A622M,A622N,A622P,A622R,A622S,A622T,A622V,A622Y,Q626E,Q626F,Q626G,Q626H,Q626L,Q626M,Q626T,Q626V,V627P,T638A,T638D,T638E,T638F,T638G,T638I,T638K,T638L,T638M,T638P,T638Q,T638R,T638S,T638V,T638W,T638Y,S642A,S642C,S642E,S642F,S642G,S642H,S642I,S642K,S642L,S642M,S642N,S642P,S642Q,S642R,S642T,S642V,S642W,S642Y,A643C,A643E,A643F,A643G,A643H,A643K,A643L,A643M,A643N,A643Q,A643R,A643S,A643T,A643V,A643W,A643Y,R645A,R645D,R645F,R645G,R645H,R645I,R645K,R645L,R645M,R645P,R645Q,R645T,R645V,R645W,R645Y,K649A,K649C,K649F,K649I,K649L,K649M,K649N,K649Q,K649S,K649T,K649W,K649Y,Q650A,Q650C,Q650D,Q650E,Q650F,Q650G,Q650H,Q650I,Q650K,Q650L,Q650M,Q650N,Q650R,Q650T,Q650V,Q650Y,T660C,T660D,T660F,T660I,T660N,T660S,T660W,T660Y,P661A,P661C,P661D,P661E,P661F,P661G,P661H,P661I,P661K,P661L,P661M,P661Q,P661R,P661S,P661T,P661V,P661W,G662A,G662C,G662D,G662F,G662H,G662I,G662K,G662N,G662R,G662T,G662W,G662Y,Q663A,Q663C,Q663D,Q663E,Q663F,Q663G,Q663H,Q663I,Q663K,Q663L,Q663M,Q663N,Q663R,Q663S,Q663V,Q663W,T666A,T666C,T666D,T666H,T666K,T666N,T666R,T666W,R672C,R672D,R672E,R672F,R672G,R672H,R672I,R672K,R672L,R672M,R672N,R672T,R672V,R672W,R672Y,R673A,R673C,R673E,R673G,R673H,R673I,R673K,R673L,R673M,R673N,R673Q,R673S,R673T,R673V,R673W,R674L,R674M,R674T,R674V,R674Y,D675C,D675E,D675H,D675L,D675S,D675Y,D680A,D680C,D680E,D680F,D680H,D680I,D680K,D680L,D680M,D680N,D680Q,D680R,D680S,D680V,D680W,D680Y,T681A,T681G,T681H,T681K,T681L,T681M,T681N,T681P,T681Q,T681R,T681S,T681V,T681W,T681Y,A682M,S683A,S683C,S683D,S683E,S683F,S683G,S683I,S683L,S683M,S683P,S683Q,S683R,S683V,S683W,Q684A,Q684C,Q684D,Q684G,Q684N,Q684P,K685A,K685E,K685F,K685G,K685I,K685L,K685M,K685N,K685Q,K685R,K685S,K685T,K685V,K685W,K685Y,S692C,S692E,S692H,S692I,S692K,S692L,S692M,S692N,S692P,S692Q,S692T,S692V,S692W,R702C,R702D,R702F,R702G,R702H,R702I,R702K,R702L,R702M,R702N,R702Q,R702S,R702T,R702V,R702W,R705C,R705F,R705H,R705I,R705L,R705M,R705P,R705S,R705T,R705V，和R705W，其中所述置换在每个所述位置仅仅由单个替换组成，并且其中所述位置是通过对应于SEQIDNO:3示出的参考β-葡萄糖苷酶1(BGL1)的氨基酸序列来编号。如在实验部分所描述，示例性的β-葡萄糖苷酶变体具有改善的β-葡萄糖苷酶活性(对于CNPG、PASC、PCS、pH5下G2，pH6下G2，或G2+CC，PI大于1)。在另外的实施方案中，本发明提供本“
发明内容”前述各段落中的任一段落的β-葡萄糖苷酶变体，其中所述变体是分离的。本发明提供包含所述β-葡萄糖苷酶变体的组合物。在一个优选的实施方案中，所述组合物富含所述β-葡萄糖苷酶变体。另外，本发明提供编码本“
发明内容”前述各段落中的任一段落的β-葡萄糖苷酶变体的分离核酸。在一个优选的实施方案中，本发明提供包含有效连接至调控序列的所述分离核酸的表达载体。在另一个实施方案中，本发明提供包含所述表达载体的宿主细胞。本发明还提供产生β-葡萄糖苷酶变体的方法，所述方法包括在合适的条件下在培养基中培养所述宿主细胞以产生所述β-葡萄糖苷酶变体。由此在另一个实施方案中，本发明提供包括所述宿主细胞和培养基的组合物。类似地，本发明还提供在所述培养基的上清液中包含所述β-葡萄糖苷酶变体的组合物。此外，本发明提供将生物质转化成糖的方法，所述方法包括使生物质与本“
发明内容”前述各段落中的任一段落的β-葡萄糖苷酶变体接触。本发明还提供产生燃料的方法，所述方法包括使生物质组合物与包含本“
发明内容”前述各段落中的任一段落的β-葡萄糖苷酶变体的组合物接触以产生糖溶液；和在足以产生燃料的条件下使糖溶液与发酵微生物一起培养。附图简述图1提供成熟形式的各种纤维素酶的氨基酸序列的比对：TrireBGL1，红褐肉座菌(也称为里氏木霉)Q12715β-D-葡萄糖苷葡萄糖水解酶1(SEQIDNO:3)；HananBglu，异常汉逊酵母P06835β-葡萄糖苷酶(SEQIDNO:4)；PirspBglu，厌气性瘤胃真菌E2Q875K3β-葡萄糖苷酶(SEQIDNO:5)；CocimBglu，厌酷球孢子菌O14424β-葡萄糖苷酶(SEQIDNO:6)；SacfiBglu2，扣囊复膜酵母β-葡萄糖苷酶2(SEQIDNO:7)；SacfiBglu1，扣囊复膜酵母P22506β-葡萄糖苷酶1(SEQIDNO:8)；SeplyBglu，番茄壳针孢菌Q99324β-1,2-D-葡萄糖苷酶(SEQIDNO:9)；KurcaBglu，髓囊毕赤酵母Q12653β-葡萄糖苷酶(SEQIDNO:10)；TrireBGL7，里氏木霉Q7Z9M0β-葡萄糖苷酶7(SEQIDNO:11)；UrofaBglu，蚕豆单胞锈菌Q70KQ7β葡萄糖苷酶(SEQIDNO:12)；AspteBglu，土曲霉(菌株NIH2624/FGSCA1156)Q0CEF3β-葡萄糖苷酶(SEQIDNO:13)；ChaglBglu，球毛壳菌Q2GZ54推定β-葡萄糖苷酶(SEQIDNO:14)；TrireBGL3，里氏木霉Q7Z9M5β-葡萄糖苷酶3(SEQIDNO:15)；PenbrBGL，巴西青霉GH3β-葡萄糖苷酶(SEQIDNO:16)；PerspBglu，黑团孢霉BCC2871A9UIG0β-葡萄糖苷酶(SEQIDNO:17)；PhaavBglu，PhaeosphaeriaavenariaQ9P879β-葡萄糖苷酶(SEQIDNO:18)；AspfuBGL，烟曲霉B0XPE1β-葡萄糖苷酶(SEQIDNO:19)；AsporBGL1，米曲霉Q2UUD6β-葡萄糖苷酶(SEQIDNO:20)；AspacBGL1，棘孢曲霉β-葡萄糖苷酶(SEQIDNO:21)；AspniBGL，黑曲霉Q9P8F4β-葡萄糖苷酶(SEQIDNO:22)；TalemBglu，埃默森篮状菌Q8TGI8β-葡萄糖苷酶(SEQIDNO:23)；和TheauBGL，嗜热子囊菌β-葡萄糖苷酶(SEQIDNO:24)。示出的各种纤维素酶的全长序列分别对应于SEQIDNOS:25-34,2,35-45图2描述本文所述的目的载体pTTT-pyrG13和表达载体pTTT-pyrG-bgl1。具体实施方式本发明总体涉及酶，具体地讲为β-葡萄糖苷酶变体。还描述了编码β-葡萄糖苷酶变体的核酸、包含β-葡萄糖苷酶变体的组合物、使用β-葡萄糖苷酶变体的方法和鉴定另外有用的β-葡萄糖苷酶变体的方法。应当了解，前述一般描述和以下详细描述都仅是示例性和解释性的，并不限制本文所述的组合物和方法。除非本文另外定义，否则本文使用的所有技术和科学术语具有本发明所属领域的普通技术人员通常理解的相同含义。在本专利申请中，除非另外特别说明，否则所用的单数包括复数。除非另外指出，否则所用的“或”是指“和/或”。同样，术语“包含”、“含有”、“具有”和“包括”并非意图进行限制。本文提及的所有专利和出版物，包括在这些专利和出版物中公开的所有氨基酸和核苷酸序列明确地以引用方式并入。本文提供的标题并不对本发明的各个方面或实施方案构成限制，可通过参考整篇说明书了解本发明的各个方面或实施方案。因此，通过参考整篇说明书，本文的术语得到更全面的定义。除非本文另外定义，否则本文使用的所有技术和科学术语具有本发明所属领域的普通技术人员通常理解的相同含义。Singleton等人，DICTIONARYOFMICROBIOLOGYANDMOLECULARBIOLOGY，第2版，JohnWileyandSons,NewYork(1994)和Hale&Marham,THEHARPERCOLLINSDICTIONARYOFBIOLOGY,HarperPerennial,NY(1991)向技术人员提供本发明所使用的许多术语的通用词典。虽然任何与本文所述的那些方法和材料相似或等同的方法和材料可用于实施或测试本发明，但描述的是优选的方法和材料。数值范围包括限定该范围的数字。除非另外指明，否则分别以5'至3'的方向从左至右书写核酸；以氨基至羧基的方向从左至右书写氨基酸序列。对于本领域的定义和术语，技术人员应特别依据如下文献：Sambrook等人，MOLECULARCLONING:ALABORATORYMANUAL(第二版)，ColdSpringHarborPress,Plainview,N.Y.,1989和AusubelFM等人，CurrentProtocolsinMolecularBiology,JohnWiley&Sons,NewYork,N.Y.,1993。应当了解，本发明不限于所描述的具体方法、方案和试剂，因为这些可变化。I.定义以下术语通过参考整篇说明书得到更全面的定义。如本文所用，术语“多肽”是指由通过肽键连接的氨基酸残基的单链组成的化合物。如本文所用，术语“蛋白质”可与术语“多肽”同义。“变体”是指通过以下一种或多种方式从前体蛋白质(例如，天然蛋白质)衍生而来的蛋白质：向C末端、N末端和氨基酸序列内的位点之一者或多者添加一个或多个氨基酸；置换氨基酸序列内的位点处的一个或多个氨基酸；以及去除C末端、N末端和氨基酸序列内的位点之一者或多者处的一个或多个氨基酸。β-葡萄糖苷酶变体的制备可通过本领域已知的任何方式进行。在优选的实施方案中，通过以下方式制备β-葡萄糖苷酶变体：修饰编码天然蛋白质的DNA序列，将经修饰的DNA序列转化到合适的宿主中，以及表达经修饰的DNA序列以形成变体酶。本发明的β-葡萄糖苷酶变体包括具有与前体酶氨基酸序列相比改变的氨基酸序列的肽，其中变体β-葡萄糖苷酶保留前体酶的特有β-葡萄糖苷酶活性，但其在某个特定方面可具有改变的特性。例如，相对于参考β-葡萄糖苷酶，变体β-葡萄糖苷酶可具有改变(增加或减少)的水平表达、活性和稳定性。预期，根据本发明的变体可衍自编码纤维素酶变体的DNA片段，其中所表达的纤维素酶变体的功能活性得到保留。例如，编码纤维素酶的DNA片段还可包括编码在5'端或3'端连接到纤维素酶DNA序列的铰链或接头的DNA序列或其部分，其中所编码的纤维素酶结构域的功能活性得到保持。术语变体和衍生物在本文可互换使用。此外，如本文所用，“变体”也可指任何具有与野生型多肽的序列不同的序列的多肽。例如，可根据本文所述的变体序列和一个或多个具体置换，从头合成BGL1变体多肽。由此，本发明的β-葡萄糖苷酶变体包括具有与野生型BGL1相比改变的氨基酸序列的多肽。出于本发明的目的，通常通过在特定氨基酸残基处的置换来提及变体。例如，可通过符号“X(#)Y”来提及变体，所述符号指在残基编号“#”处包含置换的变体，“#”在野生型多肽中是X残基但在变体的相同位置是Y残基。因此，BGL1变体X#Y指在位置或残基编号#处包含置换的BGL1变体，其中野生型BGL1参考酶的X残基被替换或置换成Y残基。包含多个置换的变体在变体的不同置换之间标示有“|”。将在功能上与红褐肉座菌BGL1的具体残基类似的等同残基定义为β-葡萄糖苷酶的那些可呈这样的构象的氨基酸，所述构象使得那些氨基酸以确定的且归因于红褐肉座菌BGL1的具体残基的方式来改变、修饰或促成蛋白质结构、底物结合或催化。在一些优选的实施方案中，“等同残基”是与红褐肉座菌BGL1的氨基酸序列对齐的残基。术语“核酸分子”包括RNA、DNA和cDNA分子。应当理解，由于遗传密码的简并性，可产生出多个编码给定蛋白质(如BGL1和/或其变体)的核苷酸序列。本发明设想了编码变体纤维素酶(如BGL1)的每种可能的变体核苷酸序列，考虑到遗传密码的简并性，所有这些变体核苷酸序列都是可能的。“异源”核酸构建体或序列，其序列的一部分不是表达其的细胞所天然具有。就控制序列而言的异源是指这样的控制序列(即启动子或增强子)，对于其目前调节表达的同一基因，在自然界中其是不起到这个调节作用的。一般来讲，异源核酸序列对其所在的细胞或基因组部分不是内源的，并且已通过感染、转染、转化、微注射、电穿孔等添加至细胞。“异源”核酸构建体可含有与天然细胞中存在的控制序列/DNA编码序列组合相同或不同的控制序列/DNA编码序列组合。如本文所用，术语“载体”是指设计用于在不同宿主细胞之间转移的核酸构建体。“表达载体”是指能够将异源DNA片段掺入外来细胞中并在该细胞中表达异源DNA片段的载体。许多原核和真核表达载体可商购获得。适当表达载体的选择在本领域技术人员的知识范围之内。因此，“表达盒”或“表达载体”是通过重组或合成方式产生的核酸构建体，其具有一系列能让特定核酸在靶细胞内转录的指定核酸元件。可将重组表达盒掺入质粒、染色体、线粒体DNA、质体DNA、病毒或核酸片段中。通常，表达载体的重组表达盒部分包括待转录的核酸序列和启动子等序列。如本文所用，术语“质粒”是指用作克隆载体的环状双链(ds)DNA构建体，并且其在许多细菌和一些真核生物中形成染色体外自我复制遗传元件。如本文所用，术语“编码选择性标记的核苷酸序列”是指能够在细胞中表达的核苷酸序列，并且在所述细胞中选择性标记的表达使得含有所表达基因的细胞能够在相应选择性试剂存在下或在相应选择性生长条件下生长。如本文所用，术语“启动子”是指起到指导下游基因转录的作用的核酸序列。启动子一般将适于靶基因在其中被表达的宿主细胞。启动子连同其他转录和翻译调控核酸序列(也称为“控制序列”)对于表达给定基因是必要的。一般来讲，转录和翻译调控核酸序列包括但不限于启动子序列、核糖体结合位点、转录起始和终止序列、翻译起始和终止序列以及增强子或激活序列。如本文所定义，“嵌合基因”或“异源核酸构建体”是指可由不同基因的部分(包括调控元件)构成的非天然基因(即已被引入到宿主中的基因)。用于转化宿主细胞的嵌合基因构建体通常由这样的转录调控区(启动子)组成，其有效连接至异源蛋白质编码序列，或者，在选择性标记嵌合基因中，其有效连接至编码能例如给被转化的细胞赋予抗生素抗性的蛋白质的选择性标记基因。用于转化宿主细胞的本发明的典型嵌合基因包含组成型或诱导型转录调控区、蛋白质编码序列和终止序列。如果期望分泌靶蛋白，嵌合基因构建体也可包含编码信号肽的第二DNA序列。某核酸当被设置成与另一核酸序列处于功能关系时，该核酸是“有效连接”的。例如，编码分泌前导序列的DNA有效连接到某多肽的DNA的条件是它被表达成参与该多肽的分泌的前体蛋白质；启动子或增强子有效连接至编码序列的条件是它影响该序列的转录；或者核糖体结合位点有效连接至编码序列的条件是它被定位成能促进翻译。一般来讲，“有效连接”是指被连接的DNA序列是邻接的，并且在分泌前导序列的情况下，被连接的DNA序列是邻接的并在阅读框中。然而，增强子不必是邻接的。通过在方便的限制性位点的连接反应来完成连接。如果不存在此类位点，那么根据常规做法使用合成的寡核苷酸衔接子、接头或PCR引物。如本文所用，术语“基因”是指DNA的参与产生多肽链的区段，其可包括或不包括在编码区之前和之后的区域，例如5'非翻译(5'UTR)或“前导”序列和3'UTR或“尾随”序列以及各个编码区段(外显子)之间的间插序列(内含子)。一般来讲，编码变体纤维素酶(如BGL1)的核酸分子将在中等至高度严格条件下与野生型序列(如本文提供的SEQIDNO:1)杂交。然而，在一些情况中，使用具有显著不同的密码子用法的BGL1编码核苷酸序列，而由BGL1编码核苷酸序列所编码的蛋白质具有与天然蛋白质相同或基本上相同的氨基酸序列。例如，根据宿主利用特定密码子的频率，可修改编码序列以促进BGL1在特定原核或真核表达系统中的更快表达(Te'o等人，FEMSMicrobiologyLetters,190:13-19,2000例如描述了用于在丝状真菌中表达的基因的优化)。如果某核酸序列与参考核酸序列在中等至高度严格杂交和洗涤条件下彼此特异性杂交，则认为这两个序列“选择性杂交”。杂交条件基于核酸结合复合物或探针的解链温度(Tm)。例如，“最严格”通常发生在约Tm-5℃(比探针Tm低5℃)；“高严格”发生在比Tm低约5-10℃；“中度”或“中等严格”发生在比探针Tm低约10-20℃；“低严格”发生在比探针Tm低约20-25℃。在功能上，最严格条件可用于鉴定与杂交探针具有严格同一性或近严格同一性的序列；而高严格条件用于鉴定与探针具有约80％或更高序列同一性的序列。中等和高严格杂交条件在本领域是熟知的(参见例如，Sambrook等人，1989，第9和11章，和Ausubel等人，1993，所述文献明确地以引用方式并入本文)。高严格条件的实例包括在约42℃下，在50％甲酰胺、5XSSC、5XDenhardt's溶液、0.5％SDS和100μg/ml变性载体DNA中杂交，然后在室温下在2XSSC和0.5％SDS中洗涤两次，并且在42℃下在0.1XSSC和0.5％SDS中再洗涤两次。当用来指涉例如细胞、或核酸、蛋白质或载体时，术语“重组”表示该细胞、核酸、蛋白质或载体已通过引入异源核酸或蛋白质或者改变天然核酸或蛋白质而被修饰，或者表示该细胞衍自经如此修饰的细胞。因此，例如，重组细胞可表达在该细胞的天然(非重组)形式中未发现的基因，或者表达本来会过表达、低表达或根本不表达的天然基因。如本文所用，涉及细胞的术语“转化”、“稳定转化”或“转基因”是指该细胞有非天然(异源)核酸序列整合进其基因组中或作为附加型质粒存在，该附加型质粒经过多个传代都得到保留。如本文所用，术语“表达”是指基于基因的核酸序列而产生多肽的过程。该过程包括转录和翻译二者。在将核酸序列插入细胞中的语境下的术语“引入”，是指“转染”或“转化”或“转导”，并且包括是指将核酸序列掺入真核或原核细胞，在其中核酸序列可被掺入细胞的基因组(例如，染色体、质粒、质体或线粒体DNA)、转变成自主复制子或瞬时表达(例如，转染的mRNA)。因此得出，术语“BGL1表达”是指bgl1基因或其变体的转录和翻译，其产物包括前体RNA、mRNA、多肽、翻译后加工的多肽和其衍生物，包括来自相关物种如康宁木霉(Trichodermakoningii)、红褐肉座菌(也称为长梗木霉(Trichodermalongibrachiatum)、里氏木霉或绿色木霉(Trichodermaviride))和施韦尼兹肉座菌(Hypocreaschweinitzii)的BGL1。以举例的方式，关于BGL1表达的检测分析包括对BGL1蛋白进行的蛋白质印迹和HPLC、对bgl1mRNA进行的RNA印迹分析和逆转录酶聚合酶链反应(RT-PCR)检测分析。术语“选择性剪接”是指从单个基因产生多个多肽同种型的过程，并且涉及在该基因的一些但不是全部转录物的加工过程中将不连续的外显子剪接在一起。因此，特定的外显子可被连接至若干选择性外显子(alternativeexon)的任何一个以形成信使RNA。选择性剪接得到的mRNA产生出其中一些部分相同而其他部分不同的多肽(“剪接变体”)。术语“信号序列”是指在蛋白质的N端部分的氨基酸序列，其促进该蛋白质的成熟形式分泌到细胞外部。成熟形式的该胞外蛋白质不存在信号序列，信号序列在分泌过程中即被切割掉。在本发明中使用的宿主细胞可以是原核细胞如大肠杆菌(E.coli)，或真核细胞如酵母、植物、昆虫、两栖动物或哺乳动物细胞。术语“丝状真菌”是指本领域技术人员所了解的任何和所有丝状真菌。优选的真菌选自曲霉属(Aspergillus)、木霉属(Trichoderma)、镰孢属(Fusarium)、金孢属(Chrysosporium)、青霉属(Penicillium)、腐质霉属(Humicola)、脉孢菌属(Neurospora)或其可选择的有性形式，诸如裸胞壳属(Emericella)、肉座菌属(Hypocrea)。现在已证明，无性工业真菌里氏木霉是子囊菌红褐肉座菌的克隆衍生物(参见Kuhls等人，PNAS,93:7755-7760,1996)。术语“纤维低聚糖”是指包含2至8个葡萄糖单位并且具有β-1,4键的低聚糖类，例如，纤维二糖。术语“纤维素酶”或“纤维素分解酶”是指一类能够将纤维素聚合物水解成较短的纤维低聚糖低聚物、纤维二糖和/或葡萄糖的酶。纤维素酶的许多实例，诸如外切葡聚糖酶、外切纤维二糖水解酶、内切葡聚糖酶和葡萄糖苷酶已经从纤维素分解生物中获得，尤其包括真菌、植物和细菌。由这些微生物产生的酶是蛋白质的混合物，其具有三类用于将纤维素转化成葡萄糖的作用：内切葡聚糖酶(EG)、纤维二糖水解酶(CBH)和β-葡萄糖苷酶。这三种不同类型的纤维素酶协同作用以将纤维素及其衍生物转化成葡萄糖。如本文所用，术语“β-葡萄糖苷酶活性”是指能够催化β-D-葡糖苷底物(如纤维二糖、昆布二糖或对硝基酚-β-D-葡萄糖)的水解从而释放β-D-葡萄糖的多肽。例如，β-葡萄糖苷酶和其活性变体能够从纤维低聚糖(例如，纤维二糖、纤维三糖和纤维四糖)中释放葡萄糖单体。可通过水解合成的葡萄糖苷底物(包括但不限于对硝基苯基-β-D-吡喃葡萄糖苷)以产生葡萄糖和对硝基酚，或通过水解纤维二糖以产生两个葡萄糖分子，来检测β-葡萄糖苷酶的活性。例如，可通过测量在氨预处理玉米芯(CC)存在下的纤维二糖酶活性或通过CC水解活性，来测定β-葡萄糖苷酶活性。许多微生物产生水解纤维素的酶，包括木腐真菌木霉属、堆肥细菌高温单孢菌属(Thermomonospora)、芽孢杆菌属(Bacillus)和纤维单胞菌属(Cellulomonas)；链霉菌属(Streptomyces)；以及真菌腐质霉属(Humicola)、曲霉属(Aspergillus)、金孢属(Chrysosporium)和镰孢属(Fusarium)。如本文所用，术语“纤维素结合域”是指纤维素酶的氨基酸序列的部分或该酶的某个区，其涉及纤维素酶或其衍生物的纤维素结合活性。纤维素结合域通常通过将纤维素酶与纤维素、纤维素衍生物或它们的其他多糖等同物非共价结合来发挥作用。纤维素结合域允许或促进纤维素纤维通过结构上不同的催化核心区水解，并且通常不依赖该催化核心而发挥作用。因此，纤维素结合域将不具有可归因于催化核心的显著的水解活性。换句话说，纤维素结合域是纤维素酶蛋白三级结构的结构元件，其不同于具有催化活性的结构元件。纤维素结合域和纤维素结合模块在本文可互换使用。如本文所用，术语“表面活性剂”是指通常在本领域被公认为具有表面活性特性的任何化合物。因此，例如，表面活性剂包括阴离子、阳离子和非离子表面活性剂，诸如一般在洗涤剂中存在的那些。阴离子表面活性剂包括直链或支链烷基苯磺酸盐；具有直链或支链烷基基团或烯基基团的烷基或烯基醚硫酸盐；烷基或烯基硫酸盐；烯烃磺酸盐；和链烷磺酸盐。两性表面活性剂包括季铵盐磺酸盐和甜菜碱型两性表面活性剂。此类两性表面活性剂在同一分子中具有带正电和负电的基团。非离子表面活性剂可包括聚氧化烯醚以及高级脂肪酸链烷醇酰胺或其亚烷基氧加合物、脂肪酸甘油单酯等。如本文所用，术语“含纤维素织物”是指任何由含棉或不含棉纤维素或含棉或不含棉纤维素共混物制成的缝制或非缝制织物、纱线或纤维，包括天然纤维素材料和人造纤维素材料(诸如黄麻、亚麻、苎麻、人造丝和莱赛尔纤维(lyocell))。如本文所用，术语“含棉织物”是指由纯棉或棉共混物制成的缝制或非缝制织物、纱线或纤维，包括棉机织物、棉针织品、粗斜纹棉布、棉纱线、原棉等。如本文所用，术语“石洗组合物”是指用于石洗含纤维素织物的制剂。石洗组合物用于在销售之前，即，在制造过程中修饰含纤维素织物。相反，洗涤剂组合物目的是用于清洗弄脏的衣服而不是在制造过程中使用。如本文所用，术语“洗涤剂组合物”是指目的是用在洗涤介质中用于洗涤弄脏的含纤维素织物的混合物。在本发明的上下文中，此类组合物除纤维素酶和表面活性剂外还可包含另外的水解酶、助剂、漂白剂、漂白活化剂、上蓝剂和荧光染料、抗结块剂、掩蔽剂、纤维素酶活化剂、抗氧化剂和增溶剂。如本文所用，术语“bgl1基因表达的降低或消除”是指bgl1基因已从基因组缺失，因而不能被重组宿主微生物表达；或bgl1基因或转录物已被修饰而使得功能性BGL1酶不能由宿主微生物产生或产生的水平显著小于未修饰的bgl1基因或转录物。术语“变体bgl1基因”是指已通过从编码序列去除、添加至编码序列和/或操纵编码序列，使来自红褐肉座菌的bgl1基因的核酸序列发生改变。如本文所用，术语“活性的”和“生物活性的”是指与特定蛋白质相关的生物活性并且在本文可互换使用。例如，与蛋白酶相关的酶活性是蛋白质水解，因此活性蛋白酶具有蛋白质水解活性。于是，给定的蛋白质的生物活性是指通常被本领域技术人员认为归因于该蛋白质的任何生物活性。如本文所用，术语“富含”是指β-葡萄糖苷酶(如BGL1)被发现其浓度相对于在野生型或天然存在的真菌纤维素酶组合物中发现的BGL1浓度而言更高。术语“富含”、“升高”和“提高”在本文可互换使用。野生型真菌纤维素酶组合物是由天然存在的真菌源产生的组合物，并且其包含一种或多种BGL、CBH和EG组分，其中每种这些组分以由该真菌源产生的比例存在。因此，富含BGL1组合物将具有改变比例的BGL1，其中BGL1与其他纤维素酶组分(即，EG、CBH和其他内切葡聚糖酶)的比例升高。可通过本领域已知的任何方式增加BGL1或减少(或消除)至少一种其他组分来增大该比例。如本文所用，术语“分离的”或“纯化的”是指从与其天然相关的至少一种组分中移除的核酸或氨基酸。出于本专利申请的目的，“分离的”是指不是文库(例如，筛选文库)的部分的核酸或氨基酸。因此，举例而言，通过文献中充分公开的公认的分离技术可将天然存在的纤维素酶体系纯化成基本上纯的组分，所述分离技术包括在合适pH下的离子交换色谱、亲和色谱、尺寸排阻等。例如，在离子交换色谱(通常是阴离子交换色谱)中，可以通过用pH梯度或盐梯度或pH梯度和盐梯度二者洗脱来分离纤维素酶组分。然后可将纯化的BGL1加入到酶溶液中，得到富含BGL1的溶液。也可以通过使用分子遗传学方法使编码BGL1的基因过表达，并且可能同时使一种或多种编码其他纤维素酶的基因缺失，来升高微生物产生的BGL1的量。真菌纤维素酶可包含多于一种的β-葡萄糖苷酶组分。不同的组分通常具有不同的等电点，这使得可通过离子交换色谱等来分离它们。在酶溶液中可使用单一BGL1组分或各BGL1组分的组合。当用在酶溶液中时，变体BGL1组分通常以足够使可溶性糖以最高速率从生物质释放的量加入。所加入的变体BGL1组分的量取决于待糖化的生物质的类型，技术人员可容易地对其进行确定。在组合物中存在的变体BGL1组分的总蛋白质的重量百分比优选地介于0.1和100之间，其中例证性的例子为约0.1、优选地约0.5、1、优选地约5、优选地约10、优选地约15、或优选地约20重量％至优选地约25、优选地约30、优选地约35、优选地约40、优选地约45或优选地约50重量％。此外，优选的范围可以是约0.5至约15重量％、约0.5至约20重量％、约1至约10重量％、约1至约15重量％、约1至约20重量％、约1至约25重量％、约5至约20重量％、约5至约25重量％、约5至约30重量％、约5至约35重量％、约5至约40重量％、约5至约45重量％、约5至约50重量％、约10至约20重量％、约10至约25重量％、约10至约30重量％、约10至约35重量％、约10至约40重量％、约10至约45重量％、约10至约50重量％、约15至约60重量％、约15至约65重量％、约15至约70重量％、约15至约75重量％、约15至约80重量％、约15至约85重量％、约15至约95重量％。然而，当使用时，相对于纤维素酶组合物中存在的任何(EG或CBH类型)酶组分，变体BGL1组分的重量百分比是优选地约1、优选地约5、优选地约10、优选地约15、或优选地约20重量％至优选地约25、优选地约30、优选地约35、优选地约40、优选地约45或优选地约50重量％。此外，优选的范围可以是约0.5至约15重量％、约0.5至约20重量％、约1至约10重量％、约1至约15重量％、约1至约20重量％、约1至约25重量％、约5至约20重量％、约5至约25重量％、约5至约30重量％、约5至约35重量％、约5至约40重量％、约5至约45重量％、约5至约50重量％、约10至约20重量％、约10至约25重量％、约10至约30重量％、约10至约35重量％、约10至约40重量％、约10至约45重量％、约10至约50重量％、约15至约20重量％、约15至约25重量％、约15至约30重量％、约15至约35重量％、约15至约40重量％、约15至约45重量％、约15至约50重量％。作为组合物的一部分，变体BGL1组分(占总蛋白质含量)的重量百分比优选地介于0.1和100之间，其中例证性的例子是约0.1、优选地约0.5、1、优选地约5、优选地约10、优选地约15、或优选地约20重量％至优选地约25、优选地约30、优选地约35、优选地约40、优选地约45或优选地约50重量％。此外，优选的范围可以是约0.5至约15重量％、约0.5至约20重量％、约1至约10重量％、约1至约15重量％、约1至约20重量％、约1至约25重量％、约5至约20重量％、约5至约25重量％、约5至约30重量％、约5至约35重量％、约5至约40重量％、约5至约45重量％、约5至约50重量％、约10至约20重量％、约10至约25重量％、约10至约30重量％、约10至约35重量％、约10至约40重量％、约10至约45重量％、约10至约50重量％、约15至约60重量％、约15至约65重量％、约15至约70重量％、约15至约75重量％、约15至约80重量％、约15至约85重量％、约15至约95重量％。然而，当使用时，相对于纤维素酶组合物中存在的任何(EG或CBH类型)酶组分，变体BGL1组分的重量百分比是优选地约1、优选地约5、优选地约10、优选地约15、或优选地约20重量％至优选地约25、优选地约30、优选地约35、优选地约40、优选地约45或优选地约50重量％。此外，优选的范围可以是约0.5至约15重量％、约0.5至约20重量％、约1至约10重量％、约1至约15重量％、约1至约20重量％、约1至约25重量％、约5至约20重量％、约5至约25重量％、约5至约30重量％、约5至约35重量％、约5至约40重量％、约5至约45重量％、约5至约50重量％、约10至约20重量％、约10至约25重量％、约10至约30重量％、约10至约35重量％、约10至约40重量％、约10至约45重量％、约10至约50重量％、约15至约20重量％、约15至约25重量％、约15至约30重量％、约15至约35重量％、约15至约40重量％、约15至约45重量％、约15至约50重量％。II.BGL1变体本发明特别提供具有多种相比野生型BGL1改善的活性的红褐肉座菌β-葡萄糖苷酶1(BGL1)变体。示例性的改善的活性包括但不限于(a)预处理玉米秸秆(PCS)水解活性、(b)纤维二糖酶活性、(c)蛋白质表达、(d)通过在氨预处理玉米芯(CC)存在下纤维二糖酶活性或通过在本文所述条件下的CC水解活性量度的β-葡萄糖苷酶活性、(e)在66摄氏度下的热稳定性、(f)磷酸溶胀纤维素(PASC)水解活性和(g)在葡萄糖存在下的水解活性。在一些方面，BGL1变体具有单个置换。在其他方面，BGL1变体具有两个或更多个置换。在其他方面，BGL1变体具有2、3、4、5、6、7、8、9或10或更多个置换。在这些方面的任何一个中，BGL1变体可具有不同的活性和活性组合。在一些方面，具有单个置换的BGL1变体具有至少两种相比野生型BGL1改善的活性(包括两种活性)，诸如(a)预处理玉米秸秆(PCS)水解活性、(b)纤维二糖酶活性、(c)蛋白质表达、(d)通过在氨预处理玉米芯(CC)存在下纤维二糖酶活性或通过如本文所述CC水解活性量度的β-葡萄糖苷酶活性、(e)热稳定性、(f)磷酸溶胀纤维素(PASC)水解活性和(g)在葡萄糖存在下的水解活性。在一些方面，具有单个置换的BGL1变体具有至少三种相比野生型BGL1改善的活性、至少四种相比野生型BGL1改善的活性、至少五种相比野生型BGL1改善的活性、至少六种相比野生型BGL1改善的活性、或至少七种相比野生型BGL1改善的活性。在其他方面，包含两个或更多个置换的BGL1变体具有至少两种相比野生型BGL1改善的活性(包括两种活性)，诸如(a)预处理玉米秸秆(PCS)水解活性、(b)纤维二糖酶活性、(c)蛋白质表达、(d)通过在氨预处理玉米芯(CC)存在下纤维二糖酶活性或通过根据本文所述方法的CC水解活性量度的β-葡萄糖苷酶活性、(e)在66摄氏度下的热稳定性、(f)磷酸溶胀纤维素(PASC)水解活性和(g)在葡萄糖存在下的水解活性。在其他方面，具有置换的组合的BGL1变体具有至少三种相比野生型BGL1改善的活性、至少四种相比野生型BGL1改善的活性、至少五种相比野生型BGL1改善的活性、至少六种相比野生型BGL1改善的活性、或至少七种相比野生型BGL1改善的活性。因此，在一个方面，本发明提供具有至少两种相比野生型BGL1改善的活性的β-葡萄糖苷酶1(BGL1)变体，所述至少两种改善的活性选自：(a)预处理玉米秸秆(PCS)水解活性、(b)纤维二糖酶活性、(c)蛋白质表达、(d)通过在氨预处理玉米芯(CC)存在下纤维二糖酶活性或通过CC水解活性量度的β-葡萄糖苷酶活性、(e)热稳定性、(f)磷酸溶胀纤维素(PASC)水解活性和(g)在葡萄糖存在下的水解活性，其中BGL1变体是如表4-8、3-2、4-2和4-3所示的任何变体。一些BGL1变体(例如，包括如下的变体：L266A,I567E,S283F,S283P,T258E,T258I,T258K,T258Q,P536T,P536W,I532Y,Y530T,P607D,Q406M,Q406S,V602T,G300M,A630S,A630T,T180H,T180M,A450M,I444E,I444F,I444N,I444W,I444Y,V500Q,A633I,S482P,A667V,A485L,A485W,Y678R,V603G,L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,Y530S,Q684N,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,F260W,Y530F,Q406D,G605C,N263T,P607I,A450P,T242H,A630Y,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,L293F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的(b)和(d)活性的组合。一些BGL1变体(例如，包括如下的变体：P607H,T011E,T011Y,N146E,I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,P536Q,N369E,N369W,N369Y,N146A,N146Q,P607K,N369T,A655N,I671K,F260T,P607S,F260D,F260G,F260Q,P607G,N400S,P607F,P607I,A450P,T242H,T568E,A630Y,A655D,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L，或L293F)可具有相比野生型BGL1改善的(b)和(e)活性。一些BGL1变体(例如，包括如下的变体：N261C,T258C,F392Q,S624E,P607C,P604M,A377Q,N461A,N461F,N461P,T436A,T436C,T436F,T436I,T436M,T436Q,T436Y,Q220C,A655L,T646H,Y678F,A468I,D177M,P661E,L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,P536Q,N369E,N369W,N369Y,T436E,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260W,Y530F,N461V,I671C,K206A,A450P,T242H,E170F,S507G,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,L293F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的(b)和(f)活性。一些BGL1变体(例如，包括如下的变体：I567Q,A565F,A565K,A565Q,A565V,F556E,F260I,P607E,G605R,G300C,A377C,A377D,S308C,N146H,N146S,A655C,A655G,P176L,T209I,L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,A565C,A655N,I671K,F260T,P607S,Y639V,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,P607F,Q406D,G605C,N263T,N461VI671C,K206A,T568E,E170F,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,A633C,S312C，或N455D)可具有相比野生型BGL1改善的(a)和(b)活性。一些BGL1变体(例如，包括如下的变体：I567K,I567R,A565E,A565S,A565Y,F392Y,Q406H,Q406T,P604C,N038F,T568A,N461G,Y639L,Y639M,T243A,T243C,Q245H,Q245M,Q245T,T646A,T646C,I671F,I671L,I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,A565C,Y639G,Y530F,N461V,I671C,K206A,T568E,A630Y,A655D,S507G,F260E,T568K，或F260L)可具有相比野生型BGL1改善的(b)和(c)活性。一些BGL1变体(例如，包括如下的变体：I567S,G606E,G606H,G606N,G606S,L293A,S308R,I444C,M201D,R542N,L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,N566F,L293M,Q220P,S692L,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,Q406D,G605C,N263T,S308E,A338D,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,A633C,S312C，或N455D)可具有相比野生型BGL1改善的(a)和(d)活性。一些BGL1变体(例如，包括如下的变体：L266F,I567Y,A270R,S384C,A630W,E128R,N146M,N146V,N146W,L181F,V043C,Y639P,S507F,Q245P,G662C,A630H,V466T,N146A,N146Q,P607K,N369T,S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D,P607F,A630Y,S308E,A655D，或L293F)可具有相比野生型BGL1改善的(e)和(g)活性。一些BGL1变体(例如，包括如下的变体：N261E,N261K,N400A,V602K,L293I,N461S,D457A,V043Q,Q303N,K320S,G662D,F260A,S474R,I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,A601D,S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D,D564T,T568E,A655D,A338D,F260E,T568K，或F260L)可具有相比野生型BGL1改善的(c)和(e)活性。BGL1变体(例如，包括如下的变体：N566L,N566P,N566W,A270K,A270N,F556H,F556K,P604N,N461D,N463E,K206G,A468Q,A468Y,N566F,N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,A468T,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,N461VI671C,K206A,E170F,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的(a)和(f)活性。一些BGL1变体(例如，包括如下的变体：S283D,A270D,N146Y,F260A,S474R,A565C,K206S,D564T,N461VI671C,K206A,T568E,A338D,F260E,T568K，或F260L)可具有相比野生型BGL1改善的(a)和(c)活性。其他BGL1变体(例如，包括如下的变体：F556G,F260S,P604E,P604V,N146D,Y639T,T221C,N473S,N583R,R645G,G662Y,F260A,S474R,A655N,I671K,F260T,P607S,S692L,D564T,F260D,F260G,F260Q,P607G,N400S,P607F,T568E,S308E,A338D,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或F260L)可具有相比野生型BGL1改善的(a)和(e)活性。一些BGL1变体(例如，包括如下的变体：D259S,T243V,Y530F,A338D，或F260L)可具有相比野生型BGL1改善的(c)和(d)活性。一些BGL1变体(例如，包括如下的变体：S550Q,P607R,N400Q,V602F,A601G,A601L,L293K,Y575C,Y575R,A450Q,I486C,I486Y,A655S,Q245F,D329A,P536G,P607Q,A655Q,Y575A,Y575K,A630H,V466T,S692L,F260D,F260G,F260Q,P607G,N400S,P607I,A450P,T242H,S308E,A630Y,A338D,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L，或L293F)可具有相比野生型BGL1改善的(d)和(e)活性。一些BGL1变体(例如，包括如下的变体：P536F,F392C,S624L,S624R,S624W,I486F,I486W,A667G,A667S,L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,N566F,Y575A,Y575K,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260W,P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,L293F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的(d)和(f)活性。一些BGL1变体(例如，包括如下的变体：S384G,S384W,N038E,N038M,N038P,V043H,V043W,Y068E,Y068G,Y068M,L110C,L110G,L110Q,L110W,A655H,N264L,S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D,Y639G,K206S,A655D，或S507G)可具有相比野生型BGL1改善的(c)和(g)活性。一些BGL1变体(例如，包括如下的变体：G606D,Y068V,L293M,Q220P,A630H,V466T,Y530S,Q684N,F260W,Q406D,G605C,N263T,S308E,A630Y,L293F,A633C,S312C，或N455D)可具有(d)和(g)活性。一些BGL1变体(例如，包括如下的变体：A377I,N461Y,N146A,N146Q,P607K,N369T,T436E,Y639G,Y530S,Q684N,Y639V,F260W,P607F,Q406D,G605C,N263T,A630Y,A655D,E170F,S507G,L293F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的(b)和(g)活性。一些BGL1变体(例如，包括如下的变体：K206D,A601D,Y530F,N461VI671C,K206A,S507G,F260E，或T568K)可具有相比野生型BGL1改善的(c)和(f)活性。其他BGL1(例如，包括如下的变体：A468G,P536Q,N369E,N369W,N369Y,A601D,Y575A,Y575K,P607I,A450P,T242H,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F)可具有相比野生型BGL1改善的(e)和(f)活性。此外，BGL1变体可具有至少三种相比野生型BGL1改善的活性。例如，一些BGL1变体(例如，包括如下的变体：L266C,I567F,S624P,P607L,G606I,G606K,G606L,G606M,G606Q,G606V,G605E,I444V,A633V,A655W,Y678H,V522Y,G554F,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260D,F260G,F260Q,P607G,N400S,Q406D,G605C,N263T,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(b)和(d)中的任何两种或所有三种的活性，而其他(例如，包括如下的变体：L266N,F556L,S550I,S550T,S550V,T258L,P536I,P536V,F392R,S624G,S624N,S624Q,S624T,A601M,A630V,N463S,A450F,A450T,A450V,A450W,I486V,S482I,Y678I,G427F,D564T,Q684C,Q684G,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,F260W,Y530F,P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C,N455D，或L293F)具有相比野生型BGL1改善的选自(b)、(d)和(f)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：F260A,S474R,D564T,T568E,A338D,F260E,T568K，或F260L)可具有相比野生型BGL1改善的选自(a)、(c)和(e)中的任何两种或所有三种的活性，而其他BGL1变体(例如，包括如下的变体：I567V,N566G,A630K,Y639K,Q245N,K320Y,A347Y,T568E,A655D,F260E,T568K，或F260L)可具有相比野生型BGL1改善的选自(b)、(c)和(e)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：N566F,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(d)和(f)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：N566H,F556V,P604Y,L293V,A630G,N461C,N463T,D457C,Q220M,T221A,T221G,T221I,A655R,A468F,A468S,Q216I,D564V,A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C,S683W,N461V,I671C,K206A,E170F,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(b)和(f)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：A565C,N461V,I671C,K206A,T568E,F260E,T568K，或F260L)可具有相比野生型BGL1改善的选自(a)、(b)和(c)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：P536G,P607Q,A655Q,F260D,F260G,F260Q,P607G,N400S,P607I,A450P,T242H,A630Y,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F,F260L，或L293F)可具有相比野生型BGL1改善的选自(b)、(d)和(e)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：P536Q,N369E,N369W,N369Y,P607I,A450P,T242H,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F)可具有相比野生型BGL1改善的选自(b)、(e)和(f)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：A601D,F260E或T568K)可具有相比野生型BGL1改善的选自(c)、(e)和(f)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：L293M,Q220P,Q406D,G605C,N263T,S308E,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(d)和(g)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：Y575A,Y575K,P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F)可具有相比野生型BGL1改善的选自(d)、(e)和(f)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：A630H,V466T,S308E,A630Y，或L293F)可具有相比野生型BGL1改善的选自(d)、(e)和(g)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：N146A,N146Q,P607K,N369T,P607F,A630Y,A655D，或L293F)可具有相比野生型BGL1改善的选自(b)、(e)和(g)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：S384E,L181M,V043A,V043G,V043N,Q060D,A655Y,T242S,S474D，或A655D)可具有相比野生型BGL1改善的选自(c)、(e)和(g)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：T436E,F260W,E170F,S507G,L293F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(b)、(f)和(g)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：Y639G,P607F,A655D，或S507G)可具有相比野生型BGL1改善的选自(b)、(c)和(g)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：A655N,I671K,F260T,P607S,F260D,F260G,F260Q,P607G,N400S,P607F,T568E,F260E,T568K,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或F260L)可具有相比野生型BGL1改善的选自(a)、(b)和(e)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：K206S或P607F)可具有相比野生型BGL1改善的选自(a)、(c)和(g)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：Y530S,Q684N,F260W,Q406D,G605C,N263T,A630Y,L293F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(b)、(d)和(g)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：A468T,E170F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(f)和(g)中的任何两种或所有三种的活性。一些BGL1变体(例如，包括如下的变体：S692L,F260D,F260G,F260Q,P607G,N400S,S308E,A338D,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或F260L)可具有相比野生型BGL1改善的选自(a)、(d)和(e)中的任何两种或所有三种的活性，而其他BGL1变体(例如，Y639V)可具有相比野生型BGL1改善的选自(a)、(b)和(g)中的任何两种或所有三种的活性。其他BGL1变体(例如，包括如下的变体：A565G,A270C,T258S,T258V,P536D,P536E,S624F,S624I,S624V,A601C,A601Y,S308H,A630C,A630D,N463K,N463R,A450E,S482A,A667F,A667L,A667R,A667Y,A485T,V466S,Y678A,Y678C,Y678Q,A468C,Q226W,Q226Y,N263C,N263S,N278F,S312Y,Q316T,K345E,G427C,P661F,P661L,P661Q,T666C，或S683W)可具有相比野生型BGL1改善的选自(a)、(b)、(d)和(f)中的任何两种、任何三种或所有四种的活性。一些BGL1变体(例如，包括如下的变体：F260D,F260G,F260Q,P607G，或N400S)可具有相比野生型BGL1改善的选自(a)、(b)、(d)和(e)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：F260W,L293F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(b)、(d)、(f)和(g)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：Y530F)可具有相比野生型BGL1改善的选自(b)、(c)、(d)和(f)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：P607F)可具有相比野生型BGL1改善的选自(a)、(b)、(e)和(g)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：Q406D,G605C,N263T,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(b)、(d)和(g)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：N461V,I671C,K206A,F260E，或T568K)可具有相比野生型BGL1改善的选自(a)、(b)、(c)和(f)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：P607I,A450P,T242H,P536C,A630Q,D215S,G372A,G547A,F611A,G662C,G662F，或L293F)可具有相比野生型BGL1改善的选自(b)、(d)、(e)和(f)中的任何两种、任何三种或所有四种的活性。一些BGL1变体(例如，包括如下的变体：T568E,F260E,T568K，或F260L)可具有相比野生型BGL1改善的选自(a)、(b)、(c)和(e)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：S308E)可具有相比野生型BGL1改善的选自(a)、(d)、(e)和(g)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：A630Y或L293F)可具有相比野生型BGL1改善的(b)、(d)、(e)和(g)。其他BGL1变体(例如，包括如下的变体：A655D)可具有相比野生型BGL1改善的选自(b)、(c)、(e)和(g)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：E170F,A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(b)、(f)和(g)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：A338D，或F260L)可具有相比野生型BGL1改善的选自(a)、(c)、(d)和(e)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：S507G)可具有相比野生型BGL1改善的选自(b)、(c)、(f)和(g)中的任何两种、任何三种或所有四种的活性。其他BGL1变体(例如，包括如下的变体：F260E或T568K)可具有相比野生型BGL1改善的选自(a)、(b)、(c)、(e)和(f)中的任何两种、任何三种、任何四种或所有五种的活性。其他BGL1变体(例如，包括如下的变体：P536C,A630Q,D215S,G372A,G547A,F611A,G662C，或G662F)可具有相比野生型BGL1改善的选自(a)、(b)、(d)、(e)和(f)中的任何两种、任何三种、任何四种或所有五种的活性。其他BGL1变体(例如，包括如下的变体：F260L)可具有相比野生型BGL1改善的(a)、(b)、(c)、(d)和(e)活性。其他BGL1变体(例如，包括如下的变体：L293F)可具有相比野生型BGL1改善的选自(b)、(d)、(e)、(f)和(g)中的任何两种、任何三种、任何四种或所有五种的活性。其他BGL1变体(例如，包括如下的变体：A633C,S312C，或N455D)可具有相比野生型BGL1改善的选自(a)、(b)、(d)、(f)和(g)中的任何两种、任何三种、任何四种或所有五种的活性。在一个方面，合适的BGL1变体可以是以下的任何一种：L266Y,I567S,A270D,S550D,T258S,P536D,P536V,F260D,F260G,Y530F,S624N,P607Q,G606M,Q406H,N400Q,G300M,N038L,N038M,A601Y,L293V,T568K,S308E,A630Y,N461D,N146D,A450E,V043L,Q220A,A655Q,S482A,A667L,A485T,K206A，或Y678Q。本发明还提供具有至少两种相比野生型BGL1改善的活性的BGL1变体，所述至少两种改善的活性选自：(a)预处理玉米秸秆(PCS)水解活性、(b)纤维二糖酶活性、(c)蛋白质表达、(d)通过氨预处理玉米芯(CC)水解活性量度的β-葡萄糖苷酶活性、(e)热稳定性、(f)磷酸溶胀纤维素(PASC)水解活性和(g)在葡萄糖存在下的水解活性，其中BGL1变体包含两个或更多个表5-1列出的置换。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(a)和(c)并且置换是：L167W|D225Q,T242S|S312Y,D178K|A338K|S474D|G662L,K345E|N369T|G372A|K428N|P661L|S683W,D177M|D225Q|D564V|Q684G，和D178N|N264K|A338D|S474R|G662K,D177M|D564T|Q626F|Q684A,K428N|S683W,K345E|K428N|S683W,Q226Y|G372A|V603G|T666C,L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662D,L167W|D225Q|D564V|Q626F|Q684N,D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,K345E|N369E|G372A|P661E,N369T|P661L|S683W,R265M|K560S,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(b)和(f)并且置换是：L167W|D177M|D225Q|Q626F|Q684G,L167W|D177M|D564V|Q684G,D215S|S312Y,E170F|S312Y|N369Y,L167W|D225Q|Q626F|Q684R,L167W|D564T|Q626F,P176L|Q226W|Q316T|K320S|V522Y|G662C,R363E|V522Y|G662F,Q316T|K320S|V522Y|G662F,Q226W|K320Y|V522Y,Q316T|K320S|V522Y，和Q226W|K320S|R363E|V522Y|G662F,L167W|D177M|D225Q|D564V,D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,P176L|K320S|V522Y|G662C,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,E170F|Q226Y|N369Y|G372A|P661F，和L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|G372A|S683W,N369E|S683W,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(e)和(g)并且置换是：L167W|D225Q|Q626F|Q684D,L167W|D225Q|Q684N,L167W|D225Q|D564T|,Q626F|Q684C,Q626F|Q684D,N264M|R265P|N369I|D370W,R179V|N238F|D370W,R179V|N238F|K656R,R179V|N264M|D370W,R179V|N238F|R265M,R179V|R265P|D370W|K656R,R179V|N238W|N264M|R265M|N369I,R179V|N369I|D370W|K656R,R179V|N264M|R265P|K656R,R179V|R265M|N369I,R179V|N264M|R265M|D370W|K656R,R179V|N264M|R265M|N369I,R179V|N238W|N264M,N238W|N264M|R265M|D370W,R179V|N238W|R265P|D370W,R179V|N238W|N264M|D370W|K656R,N264M|R265P,R265P|D370W(任选地也包含G662F),R179V|N264M|R265P|N369I|D370W,R265M|N369I,R179V|R265M|D370W,N238W|N264M|R265P,R179V|N238W|N264M|R265P,N264M|N369I,N238F|R265M|N369I,N263C|K345E|N369E|G372A|K428N|P661E|S683W,N263C|K345E|N369T|G372A|K428N|P661E|S683W,N263C|K345E|N369E|G372A,N263C|P661L|S683W,N263C|K345E|N369T|G372A|K428N,K345E|G372A|K428N|P661E,E170F|Q226Y|N369Y|G372A,Q226Y|T242S|G372A|P661F,Q216E|T282I|S312D|S692K,Q216I|T282K|S312K|A622K,P176L|Q316T|G662C,Q226W|Q316T|V522Y|G662F,P176L|Q316T,A347Y|R542N,N238F|N264M|R265M|N369I,L167W|D225Q|D564V|Q626F|Q684N,E170F|V603G,L167W|D177M|D564T|Q684N,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(d和(f)并且置换是：L167W|D177M|D564V|Q684R,L167W|D225Q|D564V,D177M|D225Q|D564T|Q626F|Q684N,L167W|Q626F,D225Q|D564V|Q626F|Q684R,D177M|D225Q|D564V|Q684R,Q226W|K320Y,P176L|V522Y,R363E|G662C,L167W|D225Q|Q626F|Q684R,L167W|D564T|Q626F,P176L|Q226W|Q316T|K320S|V522Y|G662C,R363E|V522Y|G662F,Q316T|K320S|V522Y|G662F,Q226W|K320Y|V522Y,Q316T|K320S|V522Y,Q226W|K320S|R363E|V522Y|G662F,L167W|D177M|D564T|Q626F|Q684N,L167W|Q626F|Q684D,L167W|D177M|D564T|Q684R,L167W|D177M|D225Q|Q684D,R179V|R265P|N369I,Q316T|K320Y|R363E|V522Y|G662F,L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,P176L|K320S|V522Y|G662C,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(b)和(e)并且置换是：K345E|N369E|K428N|P661L,Q316T|K320Y|V522Y,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,P176L|K320S|V522Y|G662C,K345E|N369E|P661L,L167W|D177M|D564T|Q684N,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(d)和(e)并且置换是：N263C|K345E|N369E|P661L,N238F|N264M|R265M|N369I,P176L|K320S|V522Y|G662C,K345E|N369E|P661L,E170F|V603G,L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(b)和(g)并且置换是：E170F|T242S|N369Y|G372A|V603G|T666C,E170F|Q226Y|N369Y|V603G|T666C,E170F|Q226Y|S312Y,L167W|D177M|D225Q|D564V,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,L167W|D177M|D564T|Q684N,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性是(d)和(g)并且置换是：D178I|Q303E|A338I,Q316T|K320Y|G662F,L167W|D177M|D564T|Q626F|Q684N,L167W|Q626F|Q684D,L167W|D177M|D564T|Q684R,L167W|D177M|D225Q|Q684D,R179V|R265P|N369I,Q316T|K320Y|R363E|V522Y|G662F,N238F|N264M|R265M|N369I,N238W|R265P|K656R,N264M|R265P,(任选地也包含G662F),N264L|A338I|S474R|G662D,L167W|D177M|Q626F|Q684G，和L167W|D177M|D564V|Q626F|Q684A,E170F|V603G,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(d)和(f)中的任何两种或所有三种并且置换是：L167W|D225Q|Q626F|Q684R,L167W|D564T|Q626F,P176L|Q226W|Q316T|K320S|V522Y|G662C,R363E|V522Y|G662F,Q316T|K320S|V522Y|G662F,Q226W|K320Y|V522Y,Q316T|K320S|V522Y,Q226W|K320S|R363E|V522Y|G662F,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,P176L|K320S|V522Y|G662C,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(d)、(f)和(g)中的任何两种或所有三种并且置换是：L167W|D177M|D564T|Q626F|Q684N,L167W|Q626F|Q684D,L167W|D177M|D564T|Q684R,L167W|D177M|D225Q|Q684D,R179V|R265P|N369I,Q316T|K320Y|R363E|V522Y|G662F,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)和(e)中的任何两种或所有三种并且置换是：K345E|N369T|G372A|K428N|P661L|S683W,L167W|D225Q|D564V|Q626F|Q684N,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369E|P661L,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(f)和(g)中的任何两种或所有三种并且置换是：L167W|D177M|D225Q|D564V,L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)和(d)中的任何两种或所有三种并且置换是：D177M|D225Q|D564V|Q684G,D178N|N264K|A338D|S474R|G662K,L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662D,K345E|N369E|G372A|P661E,N369T|P661L|S683W,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,E170F|V603G,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)和(g)中的任何两种或所有三种并且置换是：D177M|D564T|Q626F|Q684A,N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662D,|D225Q|D564V|Q626F|Q684N,R265M|K560S,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(d)、(e)和(g)中的任何两种或所有三种并且置换是：N238F|N264M|R265M|N369I,E170F|V603G,L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)和(c)中的任何两种或所有三种并且置换是：K428N|S683W,K345E|K428N|S683W,Q226Y|G372A|V603G|T666C,D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,K345E|N369E|G372A|P661E,N369T|P661L|S683W,N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(d)和(f)中的任何两种、任何三种或所有四种并且置换是：L167W|D177M|Q626F,L167W|D177M|D225Q|D564V|Q684G,D177M|D225Q|D564T|Q684N,D177M|Q626F|Q684R,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(d)和(g)中的任何两种、任何三种或所有四种并且置换是：N238W|R265P|K656R,N264M|R265P(任选地也包含G662F),N264L|A338I|S474R|G662DE170F|V603G,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(e)和(g)中的任何两种、任何三种或所有四种并且置换是：L167W|D225Q|D564V|Q626F|Q684N,E170F|V603G,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)和(f)中的任何两种、任何三种或所有四种并且置换是：D177M|D225Q|D564T|Q684A,D177M|D225Q|D564V|Q626F|Q684N,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|G372A|S683W,N369E|S683W,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)和(d)中的任何两种、任何三种或所有四种并且置换是：K345E|N369E|G372A|P661E,N369T|P661L|S683W,K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661L,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(d)、(f)和(g)中的任何两种、任何三种或所有四种并且置换是：L167W|D177M|Q626F|Q684G,L167W|D177M|D564V|Q626F|Q684A,E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(f)和(g)中的任何两种、任何三种或所有四种并且置换是：R265M|K560S,K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)和(e)中的任何两种、任何三种或所有四种并且置换是：N369T|G372A|P661L|S683W,P176L|Q226W|K320Y|R363E,K345E|N369E|P661L,K345E|N369E|G372A|S683W,N369E|S683W,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(b)、(d)、(e)和(f)中的任何两种、任何三种或所有四种并且置换是：P176L|K320S|V522Y|G662C,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)和(f)中的任何两种、任何三种、任何四种或所有五种并且置换是：K345E|N369T|G372A|P661E|S683W,K320S|R363E,E170F|Q226Y|T242S|S312Y|G372A|V603G|P661F|T666C,T242S|T666C,Q226Y|T666C,Q216E|S312K|S692K,P176L|G662F,P176L|Q226W|Q316T|K320Y|R363E,P176L|Q226W|K320S|R363E|G662F,P176L|Q226W|Q316T|K320Y|V522Y,P176L|Q226W|K320Y|R363E|V522Y,Q226W|K320Y|R363E,Q316T|K320Y|R363E|G662F,Q226W|Q316T|R363E|V522Y|G662F,P176L|K320S|R363E|G662C,R363E|G547A|G662C,Q226W|K320S|G662C,P176L|Q226W|Q316T|K320Y|R363E|G662F,P176L|Q226W|Q316T|K320S|G662F,P176L|Q316T|K320S|R363E|V522Y|G662C,K320Y|R363E|G662C,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)和(e)中的任何两种、任何三种、任何四种或所有五种并且置换是：K345E|N369E|P661L,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(c)、(d)、(e)和(g)中的任何两种、任何三种、任何四种或所有五种并且置换是：E170F|V603G,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，和P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(d)、(f)和(g)中的任何两种、任何三种、任何四种或所有五种并且置换是：E170F|Q226Y|N369Y|G372A|P661F,L167W|D177M|D564T|Q626F|Q684G,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(d)、(e)和(g)中的任何两种、任何三种、任何四种或所有五种并且置换是：L167W|D177M|D564T|Q684N,G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(e)、(f)和(g)中的任何两种、任何三种、任何四种、任何五种或所有六种并且置换是：K345E|N369E|G372A|S683W,N369E|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)、(e)和(g)中的任何两种、任何三种、任何四种、任何五种或所有六种并且置换是：G372A|P661E|S683W,P176L|Q316T|K320S|R363E|G662F,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)、(e)和(f)中的任何两种、任何三种、任何四种、任何五种或所有六种并且置换是：K345E|N369E|P661E|S683W,K345E|P661E|S683W,N263C|K345E|N369E,N263C|N369T|P661E,K345E|N369E|S683W,N263C|K345E|N369T|K428N,N263C|N369E|K428N|P661E,N263C|N369T|S683W,N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。在其他方面，本发明提供BGL1变体，如以上和本说明书通篇所述，其中相比野生型BGL1改善的活性选自(a)、(b)、(c)、(d)、(e)、(f)和(g)中的任何两种、任何三种、任何四种、任何五种、任何六种或所有七种并且置换是：N263C|N369T,N369T|G372A|K428N|S683W,N263C|G372A,N263C|K345E|N369E|G372A|P661E，或P176L|Q226W|G547A|G662C。III.纤维素酶纤维素酶在本领域已知为水解纤维素(β-1,4-葡聚糖或βD-糖苷键)导致形成葡萄糖、纤维二糖、纤维低聚糖等的酶。如以上所示，传统上将纤维素酶分成三个主要类型：内切葡聚糖酶(EC3.2.1.4)(“EG”)、外切葡聚糖酶或纤维二糖水解酶(EC3.2.1.91)(“CBH”)和β-葡萄糖苷酶(EC3.2.1.21)(“BG”)。某些真菌产生完整的纤维素酶体系，其包括外切纤维二糖水解酶或CBH型纤维素酶、内切葡聚糖酶或EG型纤维素酶和β-葡萄糖苷酶或BG型纤维素酶。然而，有时这些体系缺乏CBH型纤维素酶，并且细菌纤维素酶也通常很少包括或不包括CBH型纤维素酶。此外，已证实EG组分和CBH组分协同地相互作用来更有效地降解纤维素。多组分的或完全的纤维素酶体系中的不同组分，即，各种内切葡聚糖酶和外切纤维二糖水解酶，通常具有不同的特性，诸如等电点、分子量、糖基化程度、底物特异性和酶作用模式。据信内切葡聚糖酶型纤维素酶水解纤维素的低结晶度区域中的内部β-1,4-糖苷键，而外切纤维二糖水解酶型纤维素酶从纤维素的还原端或非还原端水解纤维二糖。因此，内切葡聚糖酶组分的作用可通过产生被外切纤维二糖水解酶组分识别的新链端来极大地促进外切纤维二糖水解酶的作用。另外，已证实β-葡萄糖苷酶型纤维素酶催化烷基和/或芳基β-D-葡萄糖苷(如甲基β-D-葡萄糖苷和对硝基苯基葡萄糖苷)以及仅含有碳水化合物残基的葡萄糖苷(如纤维二糖)的水解。这产生出葡萄糖作为唯一产物以供微生物，并减少或消除抑制纤维二糖水解酶和内切葡聚糖酶的纤维二糖。纤维素酶也在洗涤剂组合物中有许多用途，包括增强清洁能力、作为软化剂和改善棉织品的触感(Hemmpel,ITBDyeing/Printing/Finishing3:5-14,1991；Tyndall,TextileChemistandColorist24:23-26,1992；和Kumar等人，TextileChemistandColorist,29:37-42,1997)。虽然机制不属本发明的一部分，但如美国专利5,648,263、5,691,178和5,776,757所例证，纤维素酶的软化和颜色恢复特性已被归因于纤维素酶组合物中的碱性内切葡聚糖酶组分，所述专利公开说明，包含富含了特定碱性内切葡聚糖酶组分的纤维素酶组合物的洗涤剂组合物与未富含此种组分的纤维素酶组合物相比，为经处理的衣服赋予了颜色恢复和改善的软化。此外，已证实此类碱性内切葡聚糖酶组分在洗涤剂组合物中的使用与洗涤剂组合物的pH要求互补(例如，通过在碱性pH7.5至10下表现最大活性而互补，如在美国专利5,648,263、5,691,178和5,776,757中所述)。还已证实纤维素酶组合物会降解含棉织物，导致织物的强度损失降低(美国专利4,822,516)，这使人们不愿将纤维素酶组合物用在商业洗涤剂应用中。已表明，与含有完全的纤维素酶体系的组合物相比，包含内切葡聚糖酶组分的纤维素酶组合物对含棉织物表现出强度损失降低。还已证实纤维素酶可用于将纤维素生物质降解成乙醇(其中纤维素酶将纤维素降解成葡萄糖，并且酵母或其他微生物进一步将葡萄糖发酵成乙醇)、用于处理机械纸浆(Pere等人，Proc.TappiPulpingConf.,Nashville,Tenn.,27-31，第693-696页，1996)、用作饲料添加剂(WO91/04673)和用于谷物湿磨。大多数CBH和EG具有多域结构，该多域结构由核心域和通过连接肽与之间隔开的纤维素结合域(CBD)组成(Suurnakki等人，2000)。核心域含有活性位点，而CBD通过将酶与纤维素结合来与纤维素相互作用(vanTilbeurgh等人，FEBSLett.204:223-227,1986；Tomme等人，Eur.J.Biochem.170:575-581,1988)。CBD在结晶纤维素的水解中尤其重要。已证实当CBD不存在时，纤维二糖水解酶降解结晶纤维素的能力明显降低(Linder和Teeri,J.Biotechnol.57:15-28,1997)。然而，CBD的准确功能和作用机制仍然只是推测。有人提出，CBD仅仅通过增加纤维素表面的有效酶浓度(Stahlberg等人，Bio/Technol.9:286-290,1991)，和/或通过从纤维素表面松开单条纤维素链(Tormo等人，EMBOJ.，第15卷，第21期，第5739-5751页，1996)来增强酶活性。大多数关于纤维素酶域对不同底物的作用的研究都是用纤维二糖水解酶的核心蛋白进行的，因为它们的核心蛋白可通过木瓜蛋白酶的有限蛋白水解而容易地产生(Tomme等人，1988)。许多纤维素酶在科学文献中已有描述，其实例包括：来自里氏木霉的纤维素酶：Shoemaker等人，Bio/Technology,1:691-696,1983，该文献公开了CBH1；Teeri等人，Gene,51:43-52,1987，该文献公开了BGL1。来自除木霉属之外的菌种的纤维素酶也已有描述，例如，Ooi等人，NucleicAcidsResearch,18(19)1990，该文献公开了编码由棘孢曲霉(Aspergillusaculeatus)产生的内切葡聚糖酶F1-CMC的cDNA序列；Kawaguchi等人，Gene,173:287-8,1996，该文献公开了编码来自棘孢曲霉的β-葡萄糖苷酶1的cDNA的克隆和测序；Sakamoto等人，Curr.Genet.27:435-439,1995，该文献公开了编码来自白曲霉(Aspergilluskawachii)IFO4308的内切葡聚糖酶CMCase-1的cDNA序列；Saarilahti等人，Gene,90:9-14,1990，该文献公开了来自胡萝卜软腐欧文氏菌(Erwiniacarotovara)的内切葡聚糖酶；Spilliaert等人，EurJBiochem.224:923-30,1994，该文献公开了编码来自海洋红嗜热盐菌(Rhodothermusmarinus)的热稳定β-葡聚糖酶的bglA的克隆和测序；和Halldorsdottir等人，ApplMicrobiolBiotechnol.,49:277-84,1998，该文献公开了编码糖基水解酶家族12的热稳定纤维素酶的海洋红嗜热盐菌基因的克隆、测序和过表达。然而，仍需要鉴定和表征具有改善的特性的新型纤维素酶，所述改善的特性诸如在热应激条件下或在表面活性剂存在下改善的性能、增强的特异性活性、改变的底物切割模式和/或体外高水平表达。开发包含不同数量的CBH型、EG型和BG型纤维素酶的新型改善的纤维素酶组合物，对于以下用途是受关注的：(1)用在洗涤剂组合物中，使之表现增强的清洁能力、起到软化剂的作用和/或改善棉织物的触感(例如，“石洗”或“生物抛光”)；(2)用在组合物中将木浆或其他生物质降解成糖(例如，用于生物燃料生产)；和/或(3)用在饲料组合物中。还提供包含一种或多种β-葡萄糖苷酶变体的酶共混物。在某些方面，所述酶共混物包含一种或多种β-葡萄糖苷酶变体和完全纤维素酶。如本文所用，“完全纤维素酶”是指含有天然存在的和非天然存在的纤维素酶的组合物，其包含至少两种不同的酶类型：(1)内切葡聚糖酶，其切割内部β-1,4键，产生较短的葡萄糖低聚糖，(2)纤维二糖水解酶，其以“外切”的方式作用，释放纤维二糖单位(β-1,4葡萄糖-葡萄糖二糖)，和任选地(3)β-葡萄糖苷酶，从短的纤维低聚糖(例如，纤维二糖)释放葡萄糖单体。“天然存在的”组合物是通过天然存在的来源产生的组合物，其包含一种或多种纤维二糖水解酶型、一种或多种内切葡聚糖酶型和一种或多种β-葡萄糖苷酶组分，其中这些组分中的每种都以由所述来源产生的比例存在。天然存在的组合物是由未针对纤维素分解酶进行修饰的生物体所产生的组合物，使得所述组分酶的比例与由天然生物体所产生的比例相比不变。“非天然存在的”组合物涵盖通过以下方式产生的那些组合物：(1)以天然存在的比例或非天然存在的(即，改变的)比例来将各组分纤维素分解酶进行组合；或(2)修饰生物体以过表达或低表达一种或多种纤维素分解酶；或(3)修饰生物体使得至少一种纤维素分解酶缺失。因此，在一些实施方案中，完全纤维素酶制剂可使各种EG和/或CBH和/或β-葡萄糖苷酶中的一种或多种被缺失或过表达。在本发明中，完全纤维素酶制剂可来自任何可用于水解纤维素材料的微生物。在一些实施方案中，完全纤维素酶制剂是丝状真菌完全纤维素酶。在一些实施方案中，完全纤维素酶制剂来自枝顶孢霉属(Acremonium)、曲霉属(Aspergillus)、金孢霉属(Chrysosporium)、裸胞壳属(Emericella)、镰孢属(Fusarium)、腐质霉属(Humicola)、毛霉属(Mucor)、毁丝霉属(Myceliophthora)、脉孢菌属(Neurospora)、青霉属(Penicillium)、节格孢属(Scytalidium)、梭孢壳霉属(Thielavia)、弯颈霉属(Tolypocladium)或木霉属(Trichoderma)菌种。在一些实施方案中，完全纤维素酶制剂是棘孢曲霉(Aspergillusaculeatus)、泡盛曲霉(Aspergillusawamori)、臭曲霉(Aspergillusfoetidus)、日本曲霉(Aspergillusjaponicus)、构巢曲霉(Aspergillusnidulans)、黑曲霉(Aspergillusniger)或米曲霉(Aspergillusoryzae)完全纤维素酶。在另一方面，完全纤维素酶制剂是拟杆镰孢菌(Fusariumbactridioides)、纹枯镰孢菌(Fusariumcerealis)、弯钩镰孢菌(Fusariumcrookwellense)、黄色镰孢菌(Fusariumculmorum)、禾谷镰孢菌(Fusariumgraminearum)、禾赤镰孢菌(Fusariumgraminum)、异孢镰孢菌(Fusariumheterosporum)、荆条镰孢菌(Fusariumnegundi)、尖孢镰孢菌(Fusariumoxysporum)、多枝镰孢菌(Fusariumreticulatum)、粉红镰孢菌(Fusariumroseum)、接骨木镰孢菌(Fusariumsambucinum)、肤色镰孢菌(Fusariumsarcochroum)、拟枝孢镰孢菌(Fusariumsporotrichioides)、硫色镰孢菌(Fusariumsulphureum)、簇囊镰孢菌(Fusariumtorulosum)、拟丝孢镰孢菌(Fusariumtrichothecioides)或镶片镰孢(Fusariumvenenatum)完全纤维素酶。在另一方面，完全纤维素酶制剂是特异腐质霉(Humicolainsolens)、柔毛腐质霉(Humicolalanuginosa)、米赫毛霉(Mucormiehei)、嗜热毁丝霉(Myceliophthorathermophila)、粗糙脉孢菌(Neurosporacrassa)、产紫青霉(Penicilliumpurpurogenum)、绳状青霉(Penicilliumfuniculosum)、嗜热革节孢(Scytalidiumthermophilum)或太瑞斯梭孢壳霉(Thielaviaterrestris)完全纤维素酶。在又一方面，完全纤维素酶制剂是哈茨木霉(Trichodermaharzianum)、康宁木霉、长梗木霉、里氏木霉(例如，RL-P37(Sheir-Neiss等人，Appl.Microbiol.Biotechnology,20:46-53,1984；和Montenecourt,Can.,1-20,1987)、QM9414(ATCCNo.26921)、NRRL15709、ATCC13631、56764、56466、56767)或绿色木霉(Trichodermaviride)，例如，ATCC32098和32086完全纤维素酶。在一些实施方案中，完全纤维素酶制剂是里氏木霉RutC30完全纤维素酶，其以里氏木霉ATCC56765得自美国典型培养物保藏中心。在一些实施方案中，完全纤维素酶是绳状青霉，其以绳状青霉ATCC号：10446得自美国典型培养物保藏中心。完全纤维素酶制剂也可获自商业来源。适用于本发明的商业纤维素酶制剂的实例包括，例如，CELLUCLASTTM和CELLICTM(得自NovozymesA/S)以及LAMINEXTMBG、INDIAGETM44L、PRIMAFASTTM100、PRIMAFASTTM200、SPEZYMETMCP、1000和1500(DaniscoUS.Inc.,Genencor)。在本发明中，完全纤维素酶制剂可来自本领域已知的任何导致能够水解纤维素材料的酶得以表达的微生物培养方法。发酵可包括摇瓶培养、小或大规模发酵，诸如在允许纤维素酶表达或分离的条件下，在实验室或工业发酵罐中在适当的培养基中进行的连续、分批、分批补料或固态发酵。一般来讲，在适于产生能够水解纤维素材料的酶的细胞培养基中培养微生物。使用本领域已知的程序，在包含碳源和氮源以及无机盐的合适的营养培养基中进行培养。适于生长和纤维素酶产生的合适培养基、温度范围和其他条件在本领域是已知的。作为非限制性例子，里氏木霉产生纤维素酶的正常温度范围是24℃至28℃。一般来讲，通过发酵制备完全纤维素酶制剂后，即按原样使用，不需要回收和/或纯化或只需要最低程度地回收和/或纯化。例如，一旦细胞将纤维素酶分泌到细胞培养基中，就可使用含有纤维素酶的细胞培养基。在一些实施方案中，完全纤维素酶制剂包括未分开的发酵材料内容物，其包括细胞培养基、胞外酶和细胞。作为另外一种选择，完全纤维素酶制剂可通过任何便利的方法来加工，例如，通过沉淀、离心、亲和性、过滤或本领域已知的任何其他方法。在一些实施方案中，例如，可将完全纤维素酶制剂浓缩后使用而不进行进一步纯化。在一些实施方案中，完全纤维素酶制剂包含能降低细胞活力或杀死细胞的化学试剂。在一些实施方案中，使用本领域已知的方法对细胞进行裂解或透化。可使用羧甲基纤维素(CMC)作为底物来测定完全纤维素酶制剂的内切葡聚糖酶活性。根据CMC活性来量度的完全纤维素酶活性的测定。该方法测定酶混合物对CMC作用所产生的还原末端的产量，其中1个单位是释放lμmol产物/分钟的酶量(Ghose,MeasurementofCellulseActivities,PureAppl.Chem.,59:257-268,1987)。在某些方面，纤维素酶是富含β-葡萄糖苷酶的纤维素酶。β-葡萄糖苷酶增强型完全纤维素酶一般包含β-葡萄糖苷酶和完全纤维素酶制剂。然而，应当了解，β-葡萄糖苷酶增强型完全纤维素酶组合物可通过重组方式产生。例如，在能够产生完全纤维素酶的微生物中表达β-葡萄糖苷酶。在一些实施方案中，β-葡萄糖苷酶增强型完全纤维素酶组合物包含完全纤维素酶制剂和β-葡萄糖苷酶。在具体的实施方案中，按蛋白质重量计，β-葡萄糖苷酶增强型完全纤维素酶组合物包含至少5％、至少7％、至少10％、至少15％、或至少20％，并且至多25％、30％、35％、至多40％、或至多50％的β-葡萄糖苷酶。IV.产生变体bgl1核酸序列的方法在一个实施方案中，本发明提供变体bgl1基因在丝状真菌功能性启动子控制下的表达。因此，本发明依赖重组遗传学领域的常规技术(参见例如，Sambrook等人，MolecularCloning,ALaboratoryManual，第2版，1989；Kriegler,GeneTransferandExpression:ALaboratoryManual,1990；以及Ausubel等人编辑，CURRENTPROTOCOLSINMOLECULARBIOLOGY,GreenePublishingandWiley-Interscience,NewYork,1994)。本发明设想了本领域已知的任何可引入突变的方法。本发明涉及变体BGL1的表达、纯化和/或分离和使用。这些酶优选地利用来自红褐肉座菌的bgl1基因通过重组方法来制备。可在纯化或不纯化的情况下使用发酵液。从红褐肉座菌分离和克隆bgl1基因后，使用本领域已知的其他方法如定点诱变来作出与所表达bgl1变体中的被置换氨基酸对应的置换、添加或缺失。此外，定点诱变和在DNA水平上使表达的蛋白质中掺入氨基酸变化的其他方法是本领域已知的(Sambrook等人，上文；和Ausubel等人，上文)。通过本领域已知的多种方法制备编码红褐肉座菌BGL1的氨基酸序列变体的DNA。这些方法包括但不限于通过对早期制备的编码红褐肉座菌BGL1的DNA进行定点(或寡核苷酸介导的)诱变、PCR诱变和盒诱变来制备。定点诱变是制备置换变体的优选方法。这种技术在本领域是熟知的(参见，例如，Carter等人，NucleicAcidsRes.13:4431-4443,1985；和Kunkel等人，Proc.Natl.Acad.Sci.USA82:488；1987)。简而言之，在进行DNA的定点诱变时，通过如下方式改变起始DNA：首先将编码所需的突变的寡核苷酸与该起始DNA的单链杂交。杂交之后，使用杂交的寡核苷酸作为引物，并且使用起始DNA的该条单链作为模板，用DNA聚合酶合成完整的第二链。从而将编码所需的突变的寡核苷酸掺入所得的双链DNA。PCR诱变也适于制备起始多肽(即，红褐肉座菌BGL1)的氨基酸序列变体(参见，例如，Higuchi，PCRProtocols，第177-183页，AcademicPress,1990；Vallette等人，Nuc.AcidsRes.17:723-733,1989；和Cadwell等人，PCRMethodsandApplications,2:28-33,1992)。简而言之，当少量的模板DNA用作PCR中的起始材料时，可使用在序列上与模板DNA中的对应区域稍微不同的引物来产生相对大量的特定DNA片段，所述DNA片段仅在引物不同于模板的位置处与模板序列不同。用于制备变体的另一种方法，即盒诱变，基于由如下文献描述的技术：Wells等人，Gene34:315-323,1985。起始材料是包含待突变的起始多肽DNA的质粒(或其他载体)。鉴定待突变的起始DNA中的密码子。所鉴定的突变位点的每侧必须存在唯一的限制性内切核酸酶位点。如果不存在此类限制性位点，那么它们可这样生成：使用上述寡核苷酸介导的诱变方法将它们引入在起始多肽DNA中的适当位置处。在这些位点对质粒DNA进行切割以使其线性化。使用标准程序合成编码限制性位点之间的DNA序列但包含所需的突变的双链寡核苷酸，其中该寡核苷酸的两条链先单独合成，然后再使用标准技术杂交在一起。该双链寡核苷酸被称为盒。该盒被设计为具有与线性化质粒的末端相容的5'和3'端，使得其可被直接连接至质粒。该质粒现在便含有突变的DNA序列。作为另外一种选择或除此以外，可确定编码变体BGL1的所需的氨基酸序列，并且可通过合成法产生编码此氨基酸序列变体的核酸序列。通常可根据纤维素酶的预期用途，对如此制备的变体BGL1进行进一步修饰。此类修饰可涉及氨基酸序列的进一步改变、与异源多肽的融合和/或共价修饰。V.bgl1核酸和BGL1多肽A.变体bgl1核酸野生型bgl1的核酸序列以SEQIDNO:1示出。本发明涵盖编码本文所述变体β-葡萄糖苷酶的核酸分子。该核酸可以是DNA分子。本发明还提供分离的、合成的或重组的核酸，其包含的核酸序列与本文所述的编码变体β-葡萄糖苷酶的核酸序列在至少约10、15、20、25、30、35、40、45、50、75、100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100、1150、1200、1250、1300、1350、1400、1450、1500、1550、1600、1650、1700、1750、1800、1850、1900、1950、2000个或更多个残基上具有70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高或完全(100％)序列同一性。本发明提供包含本发明的核酸或其亚序列的表达盒。在一个方面，该表达盒可包含有效连接至启动子的核酸。启动子可以是真菌、病毒、细菌、哺乳动物或植物启动子。启动子可以是组成型启动子或诱导型启动子。在一个方面，启动子在丝状真菌(例如，里氏木霉)中可表达。在具体的实施方案中，启动子来自丝状真菌(例如，里氏木霉)纤维二糖水解酶I(“CBHI”)基因启动子。本发明提供重组细胞(例如，宿主细胞)，该细胞经工程改造而表达本发明的核酸或本发明的表达盒。在某些方面，重组细胞是细菌细胞、哺乳动物细胞、真菌细胞、酵母细胞、昆虫细胞或植物细胞。在一个具体的方面，重组细胞是丝状真菌细胞。本发明提供包含本发明的核酸或本发明的表达盒的转基因植物。在编码BGL1变体的DNA序列已被克隆进DNA构建体后，该DNA用于转化微生物。出于表达根据本发明的变体bgl1的目的要进行转化微生物可有利地包括衍自木霉菌的菌株。因此，根据本发明的变体BGL1纤维素酶的优选制备方式包括用DNA构建体转化木霉菌宿主细胞，所述DNA构建体包含编码该变体BGL1一部分或整体的DNA的至少一个片段。DNA构建体将一般被功能连接到启动子。然后使经转化的宿主细胞在表达所需的蛋白质的条件下生长。随后，可将所需的蛋白质产物纯化到基本上同质。然而，事实上对于编码变体BGL1的给定DNA而言最佳的表达载体可能与红褐肉座菌不同。因此，可能将最有利的是在与变体BGL1的来源生物体具有系统发育学相似性的转化宿主中表达蛋白质。在一个另选的实施方案中，黑曲霉可用作表达媒介。对于用黑曲霉进行的转化技术的描述，参见WO98/31821，其公开内容全部以引用的方式并入。因此，本发明对曲霉属表达系统的描述仅为说明性目的而提供，并且作为表达本发明的变体BGL1的一种选择。然而，本领域的技术人员可能倾向于在另一不同的宿主细胞(如果适用的话)中表达编码变体BGL1的DNA，并且应当理解在确定最佳表达宿主时应考虑变体BGL1的来源。此外，本领域的技术人员将能够利用本领域可获得的工具通过常规技术来选择适合特定基因的最佳表达系统。B.变体BGL1多肽本发明提供分离的、合成的或重组的多肽，其包含的氨基酸序列与变体β-葡萄糖苷酶的多肽序列在至少约10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、125、150、175、200、225、250、275、300、325、350个或更多个残基上或者在全长不成熟多肽或全长成熟多肽上具有至少约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更高或完全(100％)序列同一性。本发明的变体β-葡萄糖苷酶具有衍自前体BGL1的氨基酸序列的氨基酸序列。通过前体氨基酸序列的一个或多个氨基酸的置换、缺失或插入，而使BGL1变体的氨基酸序列不同于前体BGL1氨基酸序列。在一个优选的实施方案中，前体BGL1是红褐肉座菌BGL1。红褐肉座菌BGL1的成熟氨基酸序列在实施例2(SEQIDNO:3)中示出。因此，本发明涉及这样的BGL1变体，其在与红褐肉座菌BGL1中具体的经鉴定的残基对等的位置处含有氨基酸残基。BGL1同源物的残基(氨基酸)与红褐肉座菌BGL1的残基等同的条件是，它与红褐肉座菌BGL1中的特定残基或该残基的部分是同源的(即，一级结构或三级结构中的位置相对应)或者在功能上是类似的(即，具有相同或相似的功能能力以在化学上或结构上结合、反应或相互作用)。如本文所用，编号旨在对应于成熟BGL1氨基酸序列(SEQIDNO:3)的编号。用以确定同源性的氨基酸序列的比对优选地通过使用“序列比较算法”来确定。用于比较的序列的最佳比对可例如通过Smith&Waterman,Adv.Appl.Math.2:482(1981)的局部同源算法、通过Needleman&Wunsch,J.Mol.Biol.48:443(1970)的同源比对算法、通过Pearson&Lipman,Proc.Nat'lAcad.Sci.USA85:2444(1988)的寻找相似性方法、通过这些算法(WisconsinGeneticsSoftwarePackage(GeneticsComputerGroup,575ScienceDr.,Madison,Wis.)中的GAP、BESTFIT、FASTA和TFASTA)的计算机化执行或通过目测来进行，目测可利用绘图软件包，例如由ChemicalComputingGroup(MontrealCanada)开发的MOE。适于确定序列相似性的算法的例子是BLAST算法，其描述于Altschul等人，J.Mol.Biol.215:403-410(1990)。通过国家生物技术信息中心(NationalCenterforBiotechnologyInformation)(www.ncbi.nlm.nih.gov)可公开获得用于进行BLAST分析的软件。该算法涉及首先通过如下方式来鉴定高分值序列对(HSP)：鉴定查询序列中字长W的短字，所述短字在与数据库序列中相同长度的字比对时，匹配或满足某一正值阈值分数T。以这些初始邻近字匹配(wordhit)作为起始点寻找包含它们的更长HSP。只要累积比对分数可以增加，将这些字匹配沿两个所比较的序列各自的两个方向扩展。当发生以下情况时停止字匹配的延伸：累积比对分数比最大达到的值低数量X；累积分数趋于零或更低；或到达任一序列的末端。BLAST算法参数W、T和X决定比对的灵敏度和速度。BLAST程序使用的默认值是字长(W)为11，BLOSUM62计分矩阵(参见Henikoff&Henikoff,Proc.Natl.Acad.Sci.USA89:10915,1989)比对(B)为50，期望值(E)为10，M'5，N'-4，以及对两条链的比较。BLAST算法然后进行两条序列之间相似性的统计分析(参见，例如，Karlin和Altschul,Proc.Nat'l.Acad.Sci.USA90:5873-5787,1993)。BLAST算法所提供的一种相似性量度是最小和概率(P(N))，其提供两条核苷酸或氨基酸序列之间可能偶然发生匹配的概率的指标。例如，如果在试验氨基酸序列与蛋白酶氨基酸序列的比较中最小和概率小于约0.1、更优选小于约0.01、最优选小于约0.001，则认为氨基酸序列与蛋白酶相似。出于本发明的目的，可借助于本领域已知的计算机程序适当地确定同一性的程度，所述计算机程序诸如GCG程序包中提供的GAP(ProgramManualfortheWisconsinPackage，第8版，1994年8月，GeneticsComputerGroup,575ScienceDrive,Madison,Wis.,USA53711)(Needleman和Wunsch，JournalofMolecularBiology,48,443-45,1970)，用以下用于多核苷酸序列比较的设置来应用GAP：空位形成罚分为5.0和空位延伸罚分为0.3。可使用里氏木霉BGL1与其他纤维素酶之间的结构比对来鉴定其他纤维素酶中与里氏木霉BGL1(SEQIDNO:3)具有中等至高度同源性的等同/对应位置，所述同源性例如约50％、55％、60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％,、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或甚至99％。一种获得结构比对的方法是使用来自GCG软件包的PileUp程序，其使用空位罚分默认值，即，空位形成罚分为3.0和空位延伸罚分为0.1。其他结构比对方法包括疏水簇分析(Gaboriaud等人，FEBSLetters,224:149-155,1987)和反向穿带(reversethreading)(Huber和Torda，ProteinScience,7:142-149,1998)。图1提供各种参考β-葡萄糖苷酶的成熟形式的示例性比对。参考纤维素酶包括：TrireBGL1，红褐肉座菌(也称为里氏木霉)Q12715β-D-葡萄糖苷葡萄糖水解酶1(SEQIDNO:3)；HananBglu，异常汉逊酵母P06835β-葡萄糖苷酶(SEQIDNO:4)；PirspBglu，厌气性瘤胃真菌E2Q875K3β-葡萄糖苷酶(SEQIDNO:5)；CocimBglu，厌酷球孢子菌O14424β-葡萄糖苷酶(SEQIDNO:6)；SacfiBglu2，扣囊复膜酵母β-葡萄糖苷酶2(SEQIDNO:7)；SacfiBglu1，扣囊复膜酵母P22506β-葡萄糖苷酶1(SEQIDNO:8)；SeplyBglu，番茄壳针孢菌Q99324β-1,2-D-葡萄糖苷酶(SEQIDNO:9)；KurcaBglu，髓囊毕赤酵母Q12653β-葡萄糖苷酶(SEQIDNO:10)；TrireBGL7，里氏木霉Q7Z9M0β-葡萄糖苷酶7(SEQIDNO:11)；UrofaBglu，蚕豆单胞锈菌Q70KQ7β葡萄糖苷酶(SEQIDNO:12)；AspteBglu，土曲霉(菌株NIH2624/FGSCA1156)Q0CEF3β-葡萄糖苷酶(SEQIDNO:13)；ChaglBglu，球毛壳菌Q2GZ54推定β-葡萄糖苷酶(SEQIDNO:14)；TrireBGL3，里氏木霉Q7Z9M5β-葡萄糖苷酶3(SEQIDNO:15)；PenbrBGL，巴西青霉GH3β-葡萄糖苷酶(SEQIDNO:16)；PerspBglu，黑团孢霉BCC2871A9UIG0β-葡萄糖苷酶(SEQIDNO:17)；PhaavBglu，PhaeosphaeriaavenariaQ9P879β-葡萄糖苷酶(SEQIDNO:18)；AspfuBGL，烟曲霉B0XPE1β-葡萄糖苷酶(SEQIDNO:19)；AsporBGL1，米曲霉Q2UUD6β-葡萄糖苷酶(SEQIDNO:20)；AspacBGL1，棘孢曲霉β-葡萄糖苷酶(SEQIDNO:21)；AspniBGL，黑曲霉Q9P8F4β-葡萄糖苷酶(SEQIDNO:22)；TalemBglu，埃默森篮状菌Q8TGI8β-葡萄糖苷酶(SEQIDNO:23)；和TheauBGL，嗜热子囊菌β-葡萄糖苷酶(SEQIDNO:24)。使用ClustalW和MUSCLE多序列比对算法来比对序列。表1提供了显示图1的序列比对的β-葡萄糖苷酶同一性百分比的矩阵。以粗体示出的数字表示与里氏木霉BGL1的同一性百分比。表1.β-葡萄糖苷酶同一性百分比矩阵**顶行和左列的数字对应图1的比对序列的各SEQIDNO。(+)表示100％氨基酸序列同一性。当将给定的核酸序列相对于GenBankDNASequences和其他公用数据库中的核酸序列进行评估时，通常使用BLASTN程序进行序列搜索。BLASTX程序优选用于针对GenBankProteinSequences和其他公用数据库中的氨基酸序列搜索在所有阅读框中翻译的核酸序列。使用默认参数开放空位罚分11.0和延伸空位罚分1.0来运行BLASTN和BLASTX，并利用BLOSUM-62矩阵。(参见，例如，Altschul等人，1997。)VI.重组bgl1变体的表达本发明还提供产生重组β-葡萄糖苷酶变体的方法，所述方法包括以下步骤：(a)培养宿主细胞，所述细胞经工程改造而表达本发明的β-葡萄糖苷酶变体；和(b)回收β-葡萄糖苷酶变体。步骤(b)可能需要回收包含β-葡萄糖苷酶变体的发酵液，并且任选地可包括进一步纯化步骤。本发明的方法依赖使用细胞来表达变体bgl1，但对具体的bgl1表达方法不作要求。变体BGL1优选地从细胞分泌出来。本发明提供已用包含变体BGL1编码核酸序列的表达载体转导、转化或转染的宿主细胞。培养条件(例如温度、pH等)是以前在转导、转化或转染之前用于亲本宿主细胞的那些条件，并且对于本领域技术人员将显而易见。在一种方法中，用表达盒转染丝状真菌细胞或酵母细胞，所述表达盒具有启动子或生物活性启动子片段或一个或多个(例如，一系列)增强子，其在宿主细胞系中起作用，有效连接至编码变体BGL1的DNA片段，使得变体bgl1在所述细胞系中表达。A.核酸构建体/表达载体可将编码变体BGL1的天然或合成多核苷酸片段(“BGL1编码核酸序列”)掺入异源核酸构建体或载体中，这些异源核酸构建体或载体能够引入丝状真菌或酵母细胞中或在其中复制。本文所公开的载体和方法适用于在宿主细胞中表达变体BGL1。任何载体都可以使用，只要它能够在其被引入的细胞中复制并且存活。大量合适的载体和启动子是本领域技术人员已知的，并且可商购获得。克隆和表达载体在以下文献中也有所描述：Sambrook等人，1989；AusubelFM等人，1989和Strathern等人，TheMolecularBiologyoftheYeastSaccharomyces，1981，所述每篇文献明确地以引用方式并入本文。适用于真菌的表达载体描述于vandenHondel等人(1991)，载于：Bennett和Lasure(编著)，MoreGeneManipulationsinFungi，AcademicPress，第396-428页。可通过多种程序将适当的DNA序列插入到质粒或载体(本文统称为“载体”)中。一般来讲，通过标准程序将DNA序列插入到适当的限制性内切核酸酶位点中。此类程序和相关的亚克隆程序被认为是在本领域的技术人员的知识范围内。可通过将包含变体bgl1编码序列的异源核酸构建体引入所选的丝状真菌菌株的细胞中，来产生包含变体bgl1的编码序列的重组丝状真菌。一旦获得所需形式的变体bgl1核酸序列，其可以多种方式进行修饰。当该序列包含非编码侧翼区时，可对侧翼区进行切除、诱变等。因此，可对天然存在的序列进行转换、颠换、缺失和插入。可根据熟知的重组技术将所选择的变体bgl1编码序列插入合适的载体中，并用于转化能够表达bgl1的丝状真菌。由于遗传密码固有的简并性，编码基本上相同或功能上等同的氨基酸序列的其他核酸序列也可用于克隆和表达变体bgl1。因此，应理解，编码区中的此类置换属于本发明所涵盖的序列变体。任何和所有这些序列变体可按与本文所述相同的方式用于亲本BGL1编码核酸序列。本发明还包括重组核酸构建体，该构建体包含如上所述的变体BGL1编码核酸序列中的一种或多种。该构建体包含其中已正向或反向插入了本发明序列的载体，例如质粒或病毒载体。异源核酸构建体可包含如下形式的变体bgl1编码序列。(i)处于分离状态；(ii)与另外的编码序列相组合；例如融合蛋白或信号肽编码序列，其中bgl1编码序列是主要的编码序列；(iii)与能有效地使编码序列在合适的宿主中得到表达的非编码序列相组合，所述非编码序列例如内含子和控制元件(例如，启动子和终止子元件或5'和/或3'非翻译区)；和/或(iv)在载体或宿主环境中，其中bgl1编码序列是异源基因。在本发明的一个方面，采用异源核酸构建体将变体BGL1编码核酸序列在体外转移到细胞中，其中优选已构建的丝状真菌和酵母细胞系。对于变体BGL1的长期制备，稳定表达是优选的。因此，任何能有效产生稳定的转化株的方法都可用于实施本发明。合适的载体通常配有编码选择性标记的核酸序列、插入位点和合适的控制元件，例如启动子和终止序列。载体可包含调控序列，包括例如非编码序列，如内含子和控制元件，即，启动子和终止子元件或5'和/或3'非翻译区，其有效连接至编码序列，并且能有效地使编码序列在宿主细胞中得到表达(和/或在其中通常不表达经修饰的可溶性蛋白质抗原编码序列的载体或宿主细胞环境中)。大量合适的载体和启动子是本领域技术人员已知的，其中许多可商购获得并且/或描述于Sambrook等人(上文)。示例性的启动子包括组成型启动子和诱导型启动子，其例子包括CMV启动子、SV40早期启动子、RSV启动子、EF-1.α.启动子、如所述含有tet-on或tet-off系统中的四环素应答元件(TRE)的启动子(ClonTech和BASF)、β肌动蛋白启动子以及可通过加入某些金属盐进行上调的金属硫蛋白启动子。启动子序列是被特定丝状真菌识别以实现表达目的的DNA序列。它有效连接至编码变体BGL1多肽的DNA序列。这种连接包括将启动子针对编码变体BGL1多肽的DNA序列的起始密码子在所公开的表达载体中进行定位。启动子序列含有介导变体BGL1多肽的表达的转录和翻译控制序列。例子包括来自如下基因的启动子：黑曲霉、泡盛曲霉或米曲霉葡糖淀粉酶、α-淀粉酶或α-葡萄糖苷酶编码基因；构巢曲霉gpdA或trpC基因；粗糙脉孢菌cbh1或trp1基因；黑曲霉或米黑根毛霉Rhizomucormiehei)天冬氨酸蛋白酶编码基因；红褐肉座菌(里氏木霉)bgl1、cbh1、cbh2、egl1、egl2或其他纤维素酶编码基因。适当的选择性标记的选择将取决于宿主细胞，并且适合不同宿主的标记是本领域熟知的。典型的选择性标记基因包括来自构巢曲霉或里氏木霉的argB、来自构巢曲霉的amdS、来自粗糙脉孢菌或里氏木霉的pyr4、来自黑曲霉或构巢曲霉的pyrG。另外的示例性的选择性标记包括但不限于trpc、trp1、oliC31、niaD或leu2，其包含在用于转化突变株如trp-、pyr-、leu-等的异源核酸构建体中。此类选择性标记赋予转化株利用通常不被丝状真菌代谢的代谢物的能力。例如，来自红褐肉座菌的amdS基因编码乙酰胺酶这种酶，从而允许转化株细胞以乙酰胺作为氮源生长。选择性标记(例如pyrG)可恢复营养缺陷型突变株在选择性基本培养基上生长的能力，或者选择性标记(例如olic31)可赋予转化株在抑制性药物或抗生素存在下生长的能力。使用本领域一般采用的方法将选择性标记编码序列克隆到任何合适的质粒中。示例性质粒包括pUC18、pBR322、pRAX和pUC100。pRAX质粒含有来自构巢曲霉的AMAL序列，这使其可以在黑曲霉中复制。除非另外指明，否则本发明的实施将采用分子生物学、微生物学、重组DNA和免疫学的常规技术，这些技术在本领域技术范围内。此类技术在文献中充分阐明。参见，例如，Sambrook等人，1989；Freshney,AnimalCellCulture,1987；Ausubel等人，1993；和Coligan等人，CurrentProtocolsinImmunology,1991。B.用于产生BGL1的宿主细胞和培养条件(i)丝状真菌因此，本发明提供丝状真菌，其包含已被相对于对应的非转化亲本真菌以能有效引起变体BGL1产生或表达的方式进行了修饰、选择和培养的细胞。可为了变体bgl1表达而进行处理和/或修饰的亲本丝状真菌菌种的例子包括但不限于木霉属，例如，里氏木霉、长梗木霉、绿色木霉、康宁木霉；青霉、腐质霉，包括特异腐质霉；曲霉、金孢霉、镰孢、肉座菌和裸胞壳。在通常用于培养亲本真菌细胞系的条件下对表达bgl1的细胞进行培养。一般来讲，在含有生理盐和营养素的标准培养基中培养细胞，例如Pourquie,J.等人，BiochemistryandGeneticsofCelluloseDegradation，Aubert等人编辑，AcademicPress，第71-86页，1988和Ilmen等人，Appl.Environ.Microbiol.63:1298-1306,1997中所描述。培养条件也是标准的，例如，将培养物在振荡培养器或发酵罐中在28℃下温育，直到达到所需的bgl1表达水平。对于给定的丝状真菌而言优选的培养条件可在科学文献中找到和/或得自真菌的来源，例如美国典型培养物保藏中心(ATCC；www.atcc.org/)。真菌生长确立后，使细胞暴露于能有效引起或允许变体bgl1表达的条件。在BGL1编码序列处于诱导型启动子的控制下的情况中，向培养基中以能有效诱导bgl1表达的浓度加入诱导剂，例如糖、金属盐或抗生素。在一个实施方案中，菌株包括黑曲霉，它是可用于获得过表达的蛋白质的有用菌株。例如已知黑曲霉变种泡盛曲霉(A.nigervarawamori)dgr246所分泌的分泌型纤维素酶的量提高(Goedegebuur等人，Curr.Genet(2002)41:89-98)。黑曲霉变种泡盛曲霉的其他菌株如GCDAP3、GCDAP4和GAP3-4(Ward等人，1993,Appl.Microbiol.Biotechnol.39:738-743)也是已知的。在另一个实施方案中，菌株包括里氏木霉，它是可用于获得过表达的蛋白质的有用菌株。例如，已知由Sheir-Neiss等人，Appl.Microbiol.Biotechnol.20:46-53(1984)描述的RL-P37所分泌纤维素酶的量提高。RL-P37的功能等同物包括里氏木霉菌株RUT-C30(ATCCNo.56765)和菌株QM9414(ATCCNo.26921)。预期这些菌株也可用于过表达变体bgl1。若期望在不存在潜在的有害的天然纤维素分解活性的情况下获得变体BGL1，有用的是获得这样的木霉属宿主细胞株，其在引入含有编码变体BGL1的DNA片段的DNA构建体或质粒之前，已使一个或多个纤维素酶基因缺失。可通过美国专利5,246,853和WO92/06209中所公开的方法制备此类菌株，所述专利的公开内容据此以引用方式并入本文。通过在缺失一个或多个纤维素酶基因的宿主微生物中产生变体BGL1纤维素酶，简化了鉴定和随后的纯化程序。任何来自木霉菌的已被克隆的基因均可被缺失，例如，bgl1、cbh1、cbh2、egl1和egl2基因以及编码EGIII和/或EGV蛋白质的那些基因(分别参见例如，美国专利5,475,101和WO94/28117)。可通过本领域已知的方法将某种形式的待缺失或破坏的所需基因插入质粒，来完成基因缺失。然后在适当的限制性酶位点(在所需的基因编码区的内部)切割该缺失质粒，并且用选择性标记替换基因编码序列或其部分。来自待缺失或破坏的基因的基因座的侧翼DNA序列(优选地约0.5至2.0kb)保留在选择性标记基因的任一侧。适当的缺失质粒将通常具有存在于其中的唯一限制性酶位点，以使得含有该缺失基因的片段(包括侧翼DNA序列)和选择性标记基因能作为单一线性块被去除。必须对选择性标记加以挑选，以使得能够检测经转化的微生物。任何在所选择的微生物中被表达的选择性标记基因将是合适的。例如，对于曲霉菌，挑选选择性标记，使得该选择性标记在转化株中的存在不会显著影响转化株的特性。这样的选择性标记可以是编码可检测分析的产物的基因。例如，可使用曲霉菌基因的功能拷贝，如果其在宿主菌株中缺乏，则会导致产生显示营养缺陷型表型的宿主菌株。类似地，对于木霉菌而言也存在选择性标记。在一个实施方案中，用功能性pyrG基因转化曲霉菌的pyrG-衍生菌株，从而提供用于转化的选择性标记。通过选择对氟乳清酸(FOA)有抗性的曲霉菌菌株可获得pyrG-衍生菌株。pyrG基因编码乳清酸核苷-5'-单磷酸脱羧酶，该酶是尿苷生物合成所需要的酶。具有完整pyrG基因的菌株能在缺乏尿苷的培养基中生长但对氟乳清酸敏感。通过选择FOA抗性，可以选出缺乏功能性乳清酸核苷单磷酸脱羧酶并且需要尿苷进行生长的pyrG-衍生菌株。使用FOA选择技术，还可以获得缺乏功能性乳清酸焦磷酸核糖基转移酶的需要尿苷的菌株。可以用编码该酶的基因的功能性拷贝来转化这些细胞(Berges和Barreau，Curr.Genet.19:359-365,1991；和vanHartingsveldt等人，Mol.Gen.Genet.206:71-75,1986)。使用以上提及的FOA抗性技术容易进行衍生菌株的选择，因此pyrG基因优选被用作选择性标记。在第二实施方案中，用功能性pyr4基因转化肉座菌(木霉菌)的pyr4-衍生菌株，从而提供用于转化的选择性标记。通过选择对氟乳清酸(FOA)有抗性的肉座菌(木霉菌)菌株，可获得pyr4-衍生菌株。pyr4基因编码乳清酸核苷-5'-单磷酸脱羧酶，该酶是尿苷生物合成所需要的酶。具有完整pyr4基因的菌株能在缺乏尿苷的培养基中生长但对氟乳清酸敏感。通过选择FOA抗性，可以选出缺乏功能性乳清酸核苷单磷酸脱羧酶并且需要尿苷进行生长的pyr4-衍生菌株。使用FOA选择技术，还可以获得缺乏功能性乳清酸焦磷酸核糖基转移酶的需要尿苷的菌株。可以用编码该酶的基因的功能性拷贝来转化这些细胞(Berges和Barreau，1991)。使用以上提及的FOA抗性技术容易进行衍生菌株的选择，因此pyr4基因优选被用作选择性标记。为转化pyrG-曲霉菌或pyr4-肉座菌(木霉菌)使之缺乏表达一种或多种纤维素酶基因的能力，于是从该缺失质粒中分离包含被破坏或缺失的纤维素酶基因的单个DNA片段并且用于转化适当的pyr-曲霉菌或pyr-木霉菌宿主。然后转化株基于它们分别表达pyrG或pyr4基因产物的能力进行鉴定和选择，从而补足宿主菌株的尿苷营养缺陷型。然后对所得的转化株进行Southern印迹分析以鉴定和确认双交换整合事件，该事件用适当的pyr选择性标记替换待缺失的基因的基因组拷贝的部分或全部编码区。虽然以上描述的特定质粒载体涉及pyr-转化株的制备，但本发明不限于这些载体。使用以上技术可在曲霉菌和肉座菌(木霉菌)菌株中缺失和替换多个基因。此外，如上所讨论，可使用任何可用的选择性标记。事实上，任何已被克隆并且因此被鉴定的宿主(例如，曲霉菌或肉座菌)基因可使用上述策略从基因组中缺失。如上所述，所使用的宿主菌株可以是这样的肉座菌(木霉菌)的衍生物，该菌缺乏或具有与所选的选择性标记对应的一个或多个非功能性基因。例如，如果为曲霉菌选择的选择性标记为pyrG，那么特定的pyrG-衍生菌株被用作转化程序中的受体。同样，例如，如果为肉座菌选择的选择性标记为pyr4，那么特定的pyr4-衍生菌株被用作转化程序中的受体。类似地，可使用包括与构巢曲霉基因amdS、argB、trpC、niaD等同的肉座菌(木霉菌)基因的选择性标记。因此对应的受体菌株必须是衍生菌株，例如分别是argB-、trpC-、niaD-。然后制备编码BGL1变体的DNA以用于插入到适当的微生物中。根据本发明，编码BGL1变体的DNA包括编码具有功能性纤维素分解活性的蛋白质所必需的DNA。可将编码BGL1变体的DNA片段在功能上连接到真菌启动子序列，例如，曲霉属中glaA基因的启动子或木霉属中cbh1或egl1基因的启动子。还预期可将编码BGL1变体的DNA的一个以上的拷贝重组到菌株中以有利于过表达。通过构建携带编码该变体的DNA的表达载体来制备编码BGL1变体的DNA。携带所插入的编码BGL1变体的DNA片段的表达载体，可以是任何能够在给定宿主生物中自主复制或能够整合进宿主的DNA中的载体，通常是质粒。在优选的实施方案中，设想了两种类型的表达载体用于获得基因表达。第一种含有这样的DNA序列，其中启动子、基因编码区和终止子序列都源于待表达的基因。可根据需要缺失非所需的DNA序列(例如，编码不需要的结构域的DNA序列)来获得基因截短，以使待表达的结构域处于其自身转录和翻译调控序列的控制下。选择性标记也可包含在载体上，以便对多个拷贝的新型基因序列在宿主中的整合情况进行选择。第二种类型的表达载体是预先装配的，并且含有高水平转录所需的序列及选择性标记。预期，基因或其部分的编码区可插入到该通用的表达载体中，使得其处于表达盒启动子和终止子序列的转录控制下。例如，在曲霉属中，pRAX是这种通用表达载体。可将基因或其部分插入到glaA强启动子的下游。例如，在肉座菌属中，pTREX是这种通用表达载体。可将基因或其部分插入到cbh1强启动子的下游。在该载体中，应该使编码本发明的BGL1变体的DNA序列有效连接至转录和翻译序列，即，阅读框中与结构基因相连的合适启动子序列和信号序列。启动子可以是任何在宿主细胞中显示转录活性的DNA序列，并且可衍自编码对宿主细胞而言同源或异源的蛋白质的基因。任选的信号肽使得BGL1变体可以产生到细胞外。编码信号序列的DNA优选地是与待表达的基因天然缔合的DNA，然而本发明中设想了来自任何合适来源的信号序列，例如来自木霉属的外切纤维二糖水解酶或内切葡聚糖酶。用于将编码本发明的变体BGL1的DNA序列与启动子连接在一起并插入合适的载体中的程序是本领域熟知的。可根据已知的技术如转化、转染、微注射、微穿孔、生物射弹轰击等，将上述DNA载体或构建体引入宿主细胞。在优选的转化技术中，必须考虑到肉座菌(木霉菌)中细胞壁对DNA的渗透性非常低。因此，所需的DNA序列、基因或基因片段的摄取充其量是微乎其微的。有许多方法可在转化过程之前增加衍生菌株(即，缺乏对应于所使用的选择性标记的功能基因)中肉座菌(木霉菌)细胞壁的渗透性。应当理解，在某些情况中，与使用质粒进行表达相比，可通过染色体整合达到更高或更有效的表达。本文也设想了通过染色体整合进行的表达。本发明中制备用于转化的曲霉菌或肉座菌(木霉菌)的优选方法涉及从真菌菌丝体制备原生质体(参见Campbell等人，Curr.Genet.16:53-56；1989)。菌丝体可从萌发的营养孢子获得。用能消化细胞壁的酶处理菌丝体，得到原生质体。然后通过在悬浮介质中存在渗透稳定剂来保护原生质体。这些稳定剂包括山梨醇、甘露糖醇、氯化钾、硫酸镁等。通常这些稳定剂的浓度在0.8M和1.2M之间变化。优选在悬浮介质中使用约1.2M山梨醇溶液。DNA向宿主菌株(曲霉菌或肉座菌(木霉菌))中的摄取取决于钙离子浓度。一般在摄取溶液中使用约10mMCaCl2和50mMCaCl2之间的浓度。除了在摄取溶液中需要钙离子外，通常包括的其他项为缓冲体系，例如TE缓冲液(10MmTris,pH7.4；1mMEDTA)或10mMMOPSpH6.0缓冲液(吗啉丙磺酸)和聚乙二醇(PEG)。据信，聚乙二醇的作用是融合细胞膜，从而允许该介质的内容物递送至宿主细胞(例如曲霉菌或肉座菌菌株)的细胞质中，并且质粒DNA得以转移到细胞核。这种融合在很多情况下使质粒DNA的多个拷贝整合到宿主染色体中。通常，将含有已进行过渗透性处理的密度为105至106/mL、优选2×105/mL的曲霉菌原生质体或细胞的悬浮液用于转化。类似地，将含有已进行过渗透性处理的密度为108至109/mL、优选2×108/mL的肉座菌(木霉菌)原生质体或细胞的悬浮液用于转化。将体积为100μL的这些原生质体或细胞在适当的溶液中(例如，1.2M山梨醇；50mMCaCl2)与所需的DNA混合。一般向摄取溶液加入高浓度的PEG。可向原生质体悬浮液加入0.1至1体积的25％PEG4000。然而，优选向原生质体悬浮液加入约0.25体积。也可向摄取溶液加入添加剂例如二甲基亚砜、肝素、亚精胺、氯化钾等，促进转化。通常，然后在大约0℃下温育混合物10至30分钟之间的时间。然后向混合物加入另外的PEG以进一步增强所需的基因或DNA序列的摄取。一般以转化混合物体积的5至15倍的体积加入25％PEG4000；然而，更多或更少的体积可能是合适的。25％PEG4000优选地是转化混合物体积的约10倍。加入PEG后，接着在加入山梨醇和CaCl2溶液之前，使转化混合物在室温下或冰上温育。然后将原生质体悬浮液进一步加入到生长培养基的熔化等分试样中。该生长培养基仅允许转化株的生长。在本发明中可使用任何适于所需的转化株生长的生长培养基。然而，如果正在选择pyr+转化株，优选使用不含有尿苷的生长培养基。随后的菌落在去除尿苷的生长培养基上进行转移并纯化。在这个阶段，稳定的转化株可以按以下方面与不稳定的转化株区分开来：其生长速率较快，并且例如在木霉属中，在缺乏尿苷的固体培养基上形成具有光滑而不是粗糙轮廓的圆形菌落。此外，在一些情况下，通过以下方式进行进一步稳定性测试：使转化株在固体非选择性培养基(即，含有尿苷)上生长，从此培养基收获孢子，并测定随后将在缺乏尿苷的选择性培养基上萌发和生长的这些孢子的百分比。在上述方法的一个具体实施方案中，由于BGL1变体的适当翻译后加工，在液体培养基中生长后以活性形式从宿主细胞回收到BGL1变体。(ii)酵母本发明还设想到使用酵母作为产生BGL1的宿主细胞。已在酿酒酵母的多个菌株中表达了编码水解酶的若干个其他基因。这些基因包括编码以下酶的序列：来自里氏木霉的两种内切葡聚糖酶(Penttila等人，Yeast,3:175-185,1987)、两种纤维二糖水解酶(Penttila等人，Gene,63:103-112,1988)和一种β-葡萄糖苷酶(Cummings和Fowler，Curr.Genet.29:227-233,1996)、来自出芽短梗霉菌(Aureobasidliumpullulans)的木聚糖酶(Li和Ljungdahl，Appl.Environ.Microbiol.62:209-213,1996)、来自小麦的α-淀粉酶(Rothstein等人，Gene55:353-356,1987)等。此外，在酿酒酵母的实验室菌株中成功表达了编码溶纤维丁酸弧菌(Butyrivibriofibrisolvens)内切-[β]-1,4-葡聚糖酶(END1)、黄孢原毛平革菌(Phanerochaetechrysosporium)纤维二糖水解酶(CBH1)、黄色瘤胃球菌(Ruminococcusflavefaciens)纤维糊精酶(CEL1)和扣囊内孢霉(Endomycesfibrilizer)纤维二糖酶(BGL1)的纤维素酶基因盒(VanRensburg等人，Yeast,14:67-76,1998)。C.BGL1编码核酸序列向宿主细胞中的引入。本发明还提供经基因修饰以包含外源提供的变体BGL1编码核酸序列的细胞和细胞组合物。可用克隆载体或表达载体对亲本细胞或细胞系进行基因修饰(即，转导、转化或转染)。如上进一步描述，载体可以是例如质粒、病毒粒子、噬菌体等的形式。本发明的转化方法可导致转化载体的全部或部分稳定整合到丝状真菌的基因组中。然而，还设想到使自主复制的染色体外转化载体得以维持的转化。可使用许多标准转染方法来产生表达大量异源蛋白质的里氏木霉细胞系。一些公布的用于将DNA构建体引入产纤维素酶的木霉属菌株中的方法包括Lorito、Hayes、DiPietro和Harman，1993,Curr.Genet.24:349-356；Goldman、VanMontagu和Herrera-Estrella，1990,Curr.Genet.17:169-174；Penttila、Nevalainen、Ratto、Salminen和Knowles，1987,Gene6:155-164；对于曲霉属，Yelton、Hamer和Timberlake，1984,Proc.Natl.Acad.Sci.USA81:1470-1474；对于镰孢属，Bajar、Podila和Kolattukudy，1991,Proc.Natl.Acad.Sci.USA88:8202-8212；对于链霉菌属，Hopwood等人，1985,TheJohnInnesFoundation,Norwich,UK；以及对于芽孢杆菌属，Brigidi、DeRossi、Bertarini、Riccardi和Matteuzzi，1990,FEMSMicrobiol.Lett.55:135-138)。其他用于将异源核酸构建体(表达载体)引入丝状真菌(例如，红褐肉座菌)中的方法包括但不限于使用粒子枪或基因枪、在转化过程之前使丝状真菌细胞壁透化(例如，通过使用高浓度碱，例如，0.05M至0.4MCaCl2或乙酸锂)、原生质体融合或农杆菌介导转化。有文献描述了通过用聚乙二醇和CaCl2处理原生质体或原生质球而转化丝状真菌的示例性方法(Campbell等人，Curr.Genet.16:53-56,1989；和Penttila等人，Gene,63:11-22,1988)。可使用任何公知的用于将外来核苷酸序列引入宿主细胞中的程序。这些程序包括使用磷酸钙转染、聚凝胺(polybrene)、原生质体融合、电穿孔、生物导弹术(biolistics)、脂质体、微注射、质粒载体(plasmavector)、病毒载体和任何其他熟知的用于将克隆的基因组DNA、cDNA、合成DNA或其他外来遗传材料引入宿主细胞中的方法(参见，例如，Sambrook等人，上文)。同样有用的是在美国专利6,255,115中描述的农杆菌介导转染方法。只需要所使用的特定基因工程程序能够将至少一种基因成功地引入到能够表达异源基因的宿主细胞中。此外，可使包含变体BGL1编码核酸序列的异源核酸构建体在体外转录，并且将所得的RNA通过熟知的方法(例如通过注射)引入宿主细胞中。本发明还包括新型和有用的丝状真菌(诸如红褐肉座菌和黑曲霉)转化株，用于产生真菌纤维素酶组合物。本发明包括包含变体bgl1编码序列或内源bgl1编码序列已缺失的丝状真菌(尤其是真菌)转化株。在引入包含变体bgl1的编码序列的异源核酸构建体后，可将经基因修饰的细胞在常规营养培养基中培养，所述常规营养培养基经改进为适于激活启动子、选择转化株或扩增变体BGL1编码核酸序列的表达。培养条件(例如温度、pH等)是之前用于使所选宿主细胞表达的那些条件，并且对于本领域的技术人员将显而易见。已向其中引入此类异源核酸构建体的细胞的子代，一般被认为包含存在于异源核酸构建体中的变体BGL1编码核酸序列。本发明还包括新型和有用的丝状真菌(诸如红褐肉座菌)转化株，用于产生真菌纤维素酶组合物。黑曲霉也可用于产生变体BGL1。本发明包括包含变体bgl1编码序列或内源bgl1编码序列已缺失的丝状真菌(尤其是真菌)转化株。丝状真菌的稳定转化株一般可以按以下方面与不稳定的转化株区分开来：其生长速率较快，并且例如在木霉属中，在固体培养基上形成具有光滑而不是粗糙的轮廓的圆形菌落。此外，在一些情况下，通过以下方式进行进一步稳定性测试：使转化株在固体非选择性培养基上生长，从此培养基收获孢子，并测定随后将在选择性培养基上萌发和生长的这些孢子的百分比。VII.重组BGL1蛋白的分离和纯化一般来讲，在细胞培养中产生的变体BGL1蛋白被分泌到培养基中，并且可例如通过从细胞培养基中除去不需要的组分将其纯化或分离。然而，在一些情况下，变体BGL1蛋白可以细胞形式产生，这就需要从细胞裂解物中回收。在此类情况下，使用本领域的技术人员通常采用的技术从产生变体BGL1蛋白的细胞纯化变体BGL1蛋白。例子包括但不限于亲和色谱法(Tilbeurgh等人，FEBSLett.16:215,1984)、离子交换色谱法(Goyal等人，BioresourceTechnol.36:37-50,1991；Fliess等人，Eur.J.Appl.Microbiol.Biotechnol.17:314-318,1983；Bhikhabhai等人，J.Appl.Biochem.6:336-345,1984；Ellouz等人，J.Chromatography396:307-317,1987)，包括使用具有高拆分力的材料的离子交换(Medve等人，J.ChromatographyA808:153-165,1998)、疏水相互作用色谱法(Tomaz和Queiroz，J.ChromatographyA865:123-128,1999)和两相分配(Brumbauer等人，Bioseparation7:287-295,1999)。通常，使变体BGL1蛋白进行分级分离以分开具有选定的特性(如与特定结合剂(例如，抗体或受体)的结合亲和力)的蛋白质；或具有选定的分子量范围或等电点范围的蛋白质。给定的变体BGL1蛋白的表达一旦实现，将由此产生的BGL1蛋白从细胞或细胞培养物中纯化。适于这种纯化的示例性程序包括如下：抗体-亲和柱色谱法、离子交换色谱法；乙醇沉淀；反相HPLC；在二氧化硅或阳离子交换树脂诸如DEAE上的色谱法；色谱聚焦；SDS-PAGE；硫酸铵沉淀；和使用例如SephadexG-75进行的凝胶过滤。有多种蛋白质纯化方法可采用，此类方法在本领域是已知的并描述于例如Deutscher,MethodsinEnzymology,182:779,1990；Scopes,MethodsEnzymol.90:479-91,1982。所选择的纯化步骤将例如取决于所使用的生产过程和所产生的特定蛋白质的性质。VIII.bgl1和BGL1的效用可以理解，变体bgl1核酸、变体BGL1蛋白和具有变体BGL1蛋白活性的组合物具有广泛的应用，其中一些应用在以下描述。本发明还提供变体β-葡萄糖苷酶和能降解木质纤维素材料的酶共混物。此类酶组合或混合物包括多酶组合物，其含有至少一种本发明的变体β-葡萄糖苷酶。设想了协同的酶组合和相关方法。由于大部分生物质来源的复杂性(生物质来源可包含纤维素、半纤维素、果胶、木质素、蛋白质和灰分以及其他组分)，在某些方面，本发明的酶共混物可包含具有一系列底物特异性的酶，所述酶协同工作以最有效的方式将生物质降解为可发酵糖。用于木质纤维素糖化的多酶复合物的一个例子是纤维二糖水解酶、木聚糖酶、内切葡聚糖酶、β-葡萄糖苷酶、β-木聚糖苷酶和任选的辅助蛋白质的混合物。因此，本发明提供包含木聚糖水解酶、半纤维素和/或纤维素水解酶的混合物(或“共混物”)的组合物(包括制品、全套酶或“共混物”)，所述木聚糖水解酶、半纤维素和/或纤维素水解酶的混合物包括以下的至少一种、几种或全部：纤维素酶、葡聚糖酶；纤维二糖水解酶；L-α-阿拉伯呋喃糖苷酶；木聚糖酶；任选的β-葡萄糖苷酶；β-木聚糖苷酶，优选包括本发明的至少一种β-葡萄糖苷酶变体。本发明提供非天然存在的酶共混物。如本文所用，术语“共混物”是指：(1)将各个组分酶进行组合而制备的组合物，所述各个组分酶或者为发酵液的形式，或者是部分地或完全地分离或纯化的；(2)由经修饰以表达一种或多种组分酶的生物体产生的组合物；任选地，该生物体也可被修饰成缺失一个或多个基因，任选地为编码影响木聚糖水解、半纤维素水解和/或纤维素水解的蛋白质的基因；(3)通过在糖化或发酵反应过程中同时、分别或顺序地组合各个组分酶而制备的组合物；(4)例如在糖化或发酵反应过程中原位产生的酶混合物；和(5)上述(1)-(4)中任何一种或全部的组合。如本文所用，术语“发酵液”是指通过发酵产生并且不经过回收和/或纯化或者经过最低限度的回收和/或纯化的酶制剂。例如，使微生物培养物生长至饱和，在碳源限制条件下温育，从而允许蛋白质合成(例如酶的表达)，并且一旦酶分泌到细胞培养基中，就可使用发酵液。发酵液可含有在发酵结束时衍生的未经分级分离的或经分级分离的发酵材料内容物。通常，发酵液未经分级分离并且包含消耗的培养基以及微生物细胞(例如，丝状真菌细胞)后存在的细胞碎片。在一些实施方案中，发酵液含有消耗的细胞培养基、胞外酶以及存活的或死亡的微生物细胞。在一些实施方案中，发酵液被分级分离以除去微生物细胞，并且包含消耗的细胞培养基和胞外酶。还应当理解，可将本文具体描述的任何酶与本文所述的任何一种或多种酶，或与任何其他可用和合适的酶组合，来制备多酶组合物。本发明不受限于或局限于下列的具体示例性组合。本发明提供使用本发明的酶(包括本发明的酶混合物或“共混物”)进行生物质糖化的方法和工艺。生物质可包括任何包含纤维素和/或半纤维素(木质纤维素生物质还包含木质素)的组合物，例如，种子、谷物、块茎、食品加工或工业加工的植物废料或副产物(例如，茎秆)、玉米(包括芯、秸秆等)、草(例如，印度草，如黄假高梁(Sorghastrumnutans)；或软枝草，例如，黍属(Panicum)物种，如柳枝稷(Panicumvirgatum))、木材(包括木屑、加工废料)、纸、纸浆、回收纸(例如，报纸)。其他生物质材料包括但不限于马铃薯、大豆(油菜籽)、大麦、黑麦、燕麦、小麦、甜菜或蔗渣。本发明提供糖化方法，所述方法包括使包含木聚糖、半纤维素、纤维素或可发酵糖的组合物与本发明的β-葡萄糖苷酶、或与本发明的核酸编码的多肽、或与本发明的混合物或“共混物”或制品中的任何一种进行接触。可将经糖化的生物质(例如，通过本发明的酶加工的木质纤维素材料)通过微生物发酵和/或通过化学合成制成生物基产品。如本文所用，发酵微生物可以是任何适用于所需的生产生物基产品的发酵工艺的微生物。发酵微生物的合适的非限制性例子包括丝状真菌、酵母和细菌。在一些实施方案中，可通过发酵和/或通过化学合成将经糖化的生物质制成燃料(例如，生物燃料，如生物乙醇、生物丁醇、生物甲醇、生物丙醇、生物柴油、喷气燃料等)。在一些实施方案中，可通过发酵和/或通过化学合成将经糖化的生物质制成日用化学品(例如，抗坏血酸、异戊二烯、1,3-丙二醇、脂质、氨基酸、蛋白质和酶)。除了生物质的糖化外，本发明的酶和酶共混物还可用于工业、农业、食品与饲料以及食品与饲料补充剂加工工艺。以下描述酶的示例性应用。本发明的酶可用于木材、木制品、木材废料或副产物、纸材、纸制品、纸浆或木浆、牛皮纸浆、或木材或纸回收处理或工业工艺，例如，任何木材、木浆、纸废料、纸或纸浆处理或木材或纸脱墨工艺。在一个方面，本发明的酶可用于处理/预处理纸浆或回收的纸或纸浆等。在一个方面，本发明的酶通过其用于处理/预处理纸浆或回收的纸或纸浆等来提高纸的“亮度”。纸的级别越高，亮度越好；纸亮度可影响光学扫描设备的扫描性能；因此，本发明的酶和工艺可用于制造用于例如光学扫描设备的高级别“亮度”纸，包括喷墨、激光和照片打印质量的纸。本发明的酶可用于加工或处理任何纤维素材料，例如，来自木材、棉花、大麻、亚麻或亚麻布的纤维。在一个方面，本发明提供使用本发明的酶的木材、木浆、纸、纸浆、纸废料或者木材或纸回收处理工艺。本发明的酶可用于对印刷废纸(如报纸)进行脱墨，或用于对非接触印刷废纸(例如，静电印刷和激光印刷纸)以及接触和非接触打印废纸的混合物进行脱墨(如美国专利6,767,728和6,426,200；和Neo,J.WoodChem.Tech.6:147,1986所述)。本发明的酶可用于从纸张级阔叶木浆产生木糖的工艺中，方式是将含在木浆中的木聚糖萃取到液相中，将含在所获得的液相中的木聚糖置于足以使木聚糖水解成木糖的条件下，以及回收木糖，其中所述萃取步骤包括用酶对木浆或碱溶性材料的水性悬浮液进行至少一次处理，如在例如美国专利6,512,110中所述。本发明的酶可用于从纤维素纤维溶出纸浆的工艺中，所述纤维素纤维诸如由阔叶木纤维、阔叶木纤维和针叶木纤维的混合物、废纸制成的回收纸产品，所述废纸例如来自未印刷信封、脱墨信封、未印刷帐薄纸、脱墨帐薄纸等，如在例如美国专利6,254,722中所述。本发明提供使用本发明的一种或多种酶处理纤维和织物的方法。酶可用于任何纤维或织物处理方法中，其在本领域是熟知的，参见，例如，美国专利6,261,828；6,077,316；6,024,766；6,021,536；6,017,751；5,980,581；美国专利公开20020142438A1。例如，本发明的酶可用于纤维和/或织物脱浆。在一个方面，通过包括使织物与本发明的酶在溶液中接触的方法来改善织物的触感和外观。在一个方面，在压力下用该溶液处理织物。例如，本发明的酶可用于去除污渍。本发明的酶可用于处理任何纤维素材料，包括纤维(例如，来自棉花、大麻、亚麻或亚麻布的纤维)、缝制或非缝制织物，例如，由棉花、棉共混物或天然或人造纤维素材料或其共混物制成的针织品、机织物、斜纹粗棉布、纱线和毛巾料。本发明的纺织物处理工艺(使用本发明的酶)可与其他纺织物处理(例如，擦洗和漂白)联合使用。擦洗是从棉纤维中除去非纤维素材料，例如，表皮(主要由蜡构成)和初生细胞壁(主要由果胶、蛋白质和木葡聚糖构成)。本发明的酶在食品加工工业具有许多应用。例如，在一个方面，本发明的酶用于改进油从富含油的植物材料(例如，富含油的种子)的萃取，例如，从大豆萃取大豆油、从橄榄萃取橄榄油、从油菜籽萃取油菜籽油和/或从向日葵籽萃取向日葵油。本发明的酶可用于分离植物细胞材料的组分。例如，本发明的酶可用于将植物细胞分离成组分。在一个方面，本发明的酶可用于将农作物分离成有价值的蛋白质和油以及外壳部分。可通过使用本领域已知的方法进行所述分离过程。本发明的酶可用于在水果汁或蔬菜汁、糖浆、萃取物等的制备中增加产量。本发明的酶可用于多种植物细胞壁来源的材料或废料(例如来自谷物、粮食、酒或果汁生产)或农业废弃物(诸如蔬菜皮、菜豆外壳、糖用甜菜浆、橄榄浆、马铃薯浆等)的酶处理。本发明的酶可用于改变所加工水果或蔬菜的稠度和外观。本发明的酶可用于处理植物材料以有利于植物材料(包括食物)的加工、有利于植物组分的纯化或萃取。本发明的酶可用于提高饲料价值、降低水结合能力、改进在废水处理厂中的降解能力和/或改进植物材料向青贮饲料的转化等。在一个方面，本发明的酶用于烘焙应用，例如，曲奇饼干和薄脆饼干。在一个方面，本发明的酶用于制作非粘性面团，从而可以容易地切削并减小饼干大小。本发明的酶可用于水解阿糖基木聚糖以防止烘焙产品迅速再水化，导致丧失松脆性并缩短货架期。在一个方面，本发明的酶用作面团加工中的添加剂。本发明提供使用本发明的酶处理动物饲料和食品以及食品或饲料添加剂(补充剂)的方法，动物包括哺乳类(例如，人)、鸟类、鱼类等。本发明提供包含本发明的酶的动物饲料、食品和添加剂(补充剂)。在一个方面，用本发明的酶处理动物饲料、食品和添加剂可有助于动物饲料或添加剂(补充剂)中的营养素如淀粉、蛋白质等的利用度(availability)。通过分解难以消化的蛋白质或间接或直接暴露淀粉(或其他营养素)，所述酶使得营养素更易被其他内源或外源酶接触到。所述酶也可仅仅引起易消化和易吸收的营养素和糖的释放。当加入到动物饲料时，本发明的酶改进植物细胞壁材料的体内分解，这部分是由于肠道粘度的降低(参见，例如，Bedford等人，Proceedingsofthe1stSymposiumonEnzymesinAnimalNutrition,1993，第73-77页)，从而实现动物更好地利用植物营养素。因此，通过在饲料中使用本发明的酶，动物的生长速率和/或饲料转化率(即所摄取饲料的重量相对于体重增加的比例)得到改进。本发明的动物饲料添加剂可以是可与饲料组分容易混合的粒状酶制品。作为另外一种选择，本发明的饲料添加剂可形成预混合物的组分。本发明的粒状酶制品可以是包衣的或不包衣的。酶颗粒的粒度可与饲料和预混合物组分的粒度相容。这提供了将酶掺入饲料中的安全且方便的方法。作为另外一种选择，本发明的动物饲料添加剂可以是稳定化的液体组合物。这可以是水基或油基浆液。参见，例如，美国专利6,245,546。在另一方面，本发明的酶可通过在转基因饲料作物(例如，转基因植物、种子等)中直接表达所述酶来提供，所述转基因饲料作物诸如粮食、谷物、玉米、大豆、油菜籽、羽扇豆等。如上所讨论，本发明提供包含编码本发明的多肽的核酸序列的转基因植物、植物部分或植物细胞。在一个方面，表达所述核酸，使得以可回收的量产生本发明的酶。可从任何植物或植物部分回收木聚糖酶。作为另外一种选择，含有重组多肽的植物或植物部分可原样使用，以改进食品或饲料的质量，例如，改进营养价值、口味和流变特性，或破坏抗营养因子。在一个方面，本发明提供用于在动物受试者消耗之前使用本发明的酶从饲料中去除低聚糖的方法。在这个过程中，形成具有提高的代谢能值的饲料。除了本发明的酶外，还可使用半乳糖苷酶、纤维素酶、木聚糖酶以及它们的组合。在另一方面，本发明提供利用本发明的酶作为动物饮食中的营养补充剂的方法，利用方式是制备含有本发明的重组酶的营养补充剂，并对动物施用所述营养补充剂以提高由动物摄取的食物中含有的半纤维素酶的利用。本发明的酶可用于多种其他工业应用，例如，废物处理。例如，在一个方面，本发明提供使用本发明的酶进行的固体废物消化工艺。该方法可包括减少基本上未处理固体废物的质量和体积。可在控制的温度下，在酶溶液(包括本发明的酶)存在下用酶消化工艺处理固体废物。这就使得反应时不会发生所加入的微生物的明显细菌发酵。固体废物被转化为液化废物和任何残余固体废物。可将所得的液化废物从所述任何残余固化废物中分离出来。参见例如，美国专利5,709,796。本发明提供包含本发明的一种或多种酶的洗涤剂、消毒剂或清洁剂(清洁或清洗)组合物，以及制备和使用这些组合物的方法。本发明结合了所有制备和使用洗涤剂、消毒剂或清洁剂组合物的方法，参见，例如，美国专利6,413,928；6,399,561；6,365,561；6,380,147。在具体的实施方案中，洗涤剂、消毒剂或清洁剂组合物可以是一组分和二组分水性组合物、非水性液体组合物、浇注固体、颗粒状形式、微粒形式、压片、凝胶和/或糊料和浆料形式。本发明的酶还可以固体或液体形式用作洗涤剂、消毒剂或清洁剂添加剂产品。此类添加剂产品旨在补充或增强常规洗涤剂组合物的性能并且可以在清洁过程的任何阶段加入。本发明提供清洁组合物，包括用于清洁硬质表面的洗涤剂组合物、用于清洁织物的洗涤剂组合物、盘碟洗涤组合物、口腔清洁组合物、假牙清洁组合物和隐形眼镜清洁溶液。当本发明的酶是适用于洗衣机洗涤方法的组合物的组分时，除本发明的酶之外，所述组合物还可包含表面活性剂和助洗化合物。它们可另外包含一种或多种洗涤剂组分，例如，有机聚合化合物、漂白剂、另外的酶、抑泡剂、分散剂、钙皂分散剂、土壤悬浮液以及抗再沉积剂和腐蚀抑制剂。本发明的衣物洗涤组合物还可含有软化剂作为另外的洗涤剂组分。此类含碳水化合物分解酶的组合物当作为衣物洗涤剂组合物配制时，可提供织物清洁、污渍去除、白度保持、软化、颜色外观、染料转移抑制和消毒。包含不同量的BG型、EG型和变体CBH型纤维素酶的新型改进的纤维素酶组合物在洗涤剂组合物、在用于将木浆降解为糖的组合物(例如，用于生物乙醇生产)和/或在饲料组合物中具有效用，所述洗涤剂组合物表现增强的清洁能力，用作软化剂和/或改进棉织物的触感(例如，“石洗”或“生物抛光”)。对每种类型的纤维素酶的分离和表征使得能够控制此类组合物的各个方面。由于可通过使用具有插入到基因组的bgl1基因的至少一个另外拷贝的转化株来提高纤维素产物的水解速率，所以含有纤维素或杂聚糖的产物可以更快的速率并且以更大的程度降解。由纤维素制成的产品如纸、棉、纤维素尿片等可以在垃圾填埋场更有效地降解。因此，可在组合物中使用可从转化株获得的发酵产物或单独使用转化株，以有助于通过液化而降解多种丢弃在过分堆积的垃圾填埋场的纤维素产品。纤维素基原料包括农业废弃物、草和木材以及其他低价值生物质，如城市垃圾(例如，回收纸、庭院修剪废料等)。可由这些纤维素原料中的任何一种的发酵产生乙醇。然而，纤维素在可转化为乙醇之前，必须首先转化为糖。很多种原料可与本发明变体BGL1一起使用，并且选择使用的那种原料可取决于进行该转化的区域。例如，在美国中西部，可能以农业废弃物如小麦秸秆、玉米秸秆和蔗渣为主，而在加利福尼亚，可能以稻秆为主。然而，应当理解，任何可利用的纤维素生物质都可用在任何区域。本发明的方法可用于产生单糖、二糖和多糖，作为微生物生产有机产品、化学品和燃料、塑料和其他产品或中间体的化学或发酵原料。具体地讲，可通过纤维素或半纤维素的部分或完全溶解来提高加工残余物(干酒糟、来自酿造的废糟、甘蔗渣等)的价值。除了乙醇之外，一些可由纤维素生产的化学品还包括丙酮、乙酸酯、甘氨酸、赖氨酸、有机酸(例如，乳酸)、1,3-丙二醇、丁二醇、甘油、乙二醇、糠醛、聚羟基烷酸酯、顺，顺-已二烯二酸、动物饲料和木糖。此外，可从纤维素产生蛋白质和细胞。另外，变体bgl1核酸序列在相关核酸序列的鉴定和表征中具有效用。许多用于测定(预测或确认)相关基因或基因产物的功能的技术包括但不限于(A)DNA/RNA分析，如(1)过表达、异位表达和在其他物种中表达；(2)基因敲除(反向遗传学、靶向敲除、病毒诱导的基因沉默(VIGS，参见Baulcombe,100YearsofVirology,Calisher和Horzinek编辑，Springer-Verlag,NewYork,N.Y.15:189-201,1999)；(3)基因的甲基化状态的分析，尤其是侧翼调控区；和(4)原位杂交；(B)基因产物分析，如(1)重组蛋白质表达；(2)抗血清产生；(3)免疫定位；(4)催化活性或其他活性的生物化学检测分析；(5)磷酸化状态；和(6)通过酵母双杂交分析确定与其他蛋白质的相互作用；(C)途径分析，如基于其过表达表型或通过与相关基因的序列同源性，将基因或基因产物置于特定生化途径或信号传导途径中；和(D)其他分析，也可进行这些其他分析以测定或确认所分离基因和其产物在特定代谢或信号传导途径中的参与状况并且帮助确定基因功能。实施例在以下实施例中更详细地描述本发明，所述实施例不是旨在以任何方式限制受权利要求书保护的本发明的范围。附图意在被认为是说明书的组成部分并且是对本发明的描述。提供以下实施例以说明但不限制受权利要求书保护的本发明。在随后的实验公开中，应用以下缩写：M(摩尔每升)；mM(毫摩尔每升)；μM(微摩尔每升)；nM(纳摩尔每升)；mol(摩尔)；mmol(毫摩尔)；μmol(微摩尔)；nmol(纳摩尔)；g和gm(克)；mg(毫克)；μg(微克)；pg(皮克)；L(升)；ml和mL(毫升)；μl和μL(微升)；cm(厘米)；mm(毫米)；μm(微米)；nm(纳米)；U(单位)；V(伏特)；MW(分子量)；sec(秒)；min(s)(分钟)；h(s)和hr(s)(小时)；℃(摄氏度)；QS(足量)；ND(未进行)；NA(不适用)；rpm(每分钟转数)；H2O(水)；dH2O(去离子水)；HCl(盐酸)；aa(氨基酸)；bp(碱基对)；kb(千碱基对)；kD(千道尔顿)；cDNA(拷贝或互补DNA)；DNA(脱氧核糖核酸)；ssDNA(单链DNA)；dsDNA(双链DNA)；dNTP(脱氧核糖核苷三磷酸)；RNA(核糖核酸)；MgCl2(氯化镁)；NaCl(氯化钠)；w/v(重量体积比)；v/v(体积体积比)；g(重力)；OD(光密度)；ABTS(2,2’-联氮-双(3-乙基苯并-噻唑啉-6-磺酸)二铵盐；APB(酸预处理蔗渣)；BGL(β-葡萄糖苷酶)；CNP(2-氯-4-硝基酚)；CNPG(氯-硝基-苯基-β-D-葡萄糖苷)；HPLC(高压液相色谱)；PAGE(聚丙烯酰胺凝胶电泳)；PASC(磷酸溶胀纤维素)PCR(聚合酶链式反应)；PCS(酸预处理玉米秸秆)；Pi或PI(性能指数)；RT-PCR(逆转录PCR)；和SEL(位点评估库)。实施例1检测分析以下检测分析是用在下文所描述的实施例的标准检测分析。有时具体方案要求与这些标准检测分析有一定差异。在那些情况下，实施例中标识了与下面这些标准检测分析方案的差异。在这些实验中，使用分光光度计测量反应完成后形成的产物的吸光度。葡萄糖的测量A.用于测量残余葡萄糖的己糖激酶检测分析使用己糖激酶检测分析来测量来自表达BGL变体的红褐肉座菌培养物上清液的残余葡萄糖。将五(5)μL的上清液加入到195μL在96孔微量滴定板(CostarFlatBottomPS)中的葡萄糖己糖激酶检测分析混合物(InstrumentationLaboratory,Breda，荷兰)中。使板在室温下温育15min。温育之后，在340nm测量上清液的吸光度。含有残余葡萄糖的培养物上清液不进行合并以用于进一步研究。B.用于测量葡萄糖的ABTS检测分析用ABTS检测分析来检测在β-葡萄糖苷酶活性检测分析中产生的单体葡萄糖。检测分析缓冲液含有在50mM醋酸钠缓冲液(pH5.0)中的2.74g/L2,2’-联氮-双(3-乙基苯并-噻唑啉-6-磺酸)二铵盐(ABTS，Sigma，目录号A1888)、0.1U/mL辣根过氧化物酶VI-A型(Sigma，目录号P8375)和1单位/毫升食品级葡萄糖氧化酶(GENENCOR)。将十(10)μL(稀释的)样品加入到100μLABTS检测分析溶液中。在环境温度22℃下，于OD420下对反应进行动力学跟踪5min。通常包括适合每种检测分析条件的葡萄糖校准曲线。用于测定蛋白质含量的HPLC检测分析通过配备有ShodexHICPH-814PHM75×8mm凝胶柱(Phenomenex)的Agilent1200(AgilentTechnologies)HPLC，来测定来自合并的培养物上清液的BGL变体蛋白质的浓度。将五十(50)μL样品与50μL1.6M(NH4)2SO4混合并在5分钟后在0.22μmMilliporeMultiscreenHTS96孔过滤系统上真空过滤。在柱上注入四十(40)μL经过滤的样品。采用两种洗脱缓冲液来建立洗脱梯度：(1)缓冲液A：16mMNaH2PO4(pH6.75)、800mM(NH4)2SO4；和(2)缓冲液B：16mMNaH2PO4(pH6.75)。使用以下程序以1.8mL/min的流速进行洗脱：0％至50％的缓冲液B持续0.25min至1.5min，然后是50％至100％梯度的缓冲液B持续1.5min至4min。在柱上泵送100％缓冲液B4至4.5min。由使用纯化的野生型BGL1(15.625、31.25、62.5、125、250、500μg/mL)得到的校准曲线计算BGL变体的蛋白质浓度。为计算性能指数(PI)，用BGL变体的浓度除以相同板中野生型BGL1(例如，参考酶)的平均浓度。CNPG酶活性检测分析测定了BGL变体对氯-硝基酚-β-D-葡糖苷(CNPG)的活性。在50mM醋酸钠缓冲液(pH5.0)中将表达BGL变体的培养物上清液稀释10倍。将二十五(25)μL等份的稀释上清液加入到一式四份的75μL的在50mM醋酸钠缓冲液(pH5.0)中的1.33mMCNPG(最终浓度为1mMCNPG)。在微量滴定板读板机(Spectramax,MolecularDevices)中记录OD405下的CNP释放动力学3min。通过将平均CNPG水解活性除以BGL浓度来计算野生型BGL1和BGL变体的平均比活性。通过将BGL变体的比活性除以相同板上野生型BGL1(例如，参考酶)的平均比活性来计算性能指数(PI)。热稳定性检测分析使用CNPG检测分析来测定热温育后BGL1多肽(包括野生型和变体)的残余活性。在50mM醋酸钠缓冲液(pH5.0)中将表达BGL1多肽(包括野生型和变体)的培养物上清液稀释10倍。在66℃下，使一式四份的五十(50)μL等分试样在带裙边96孔PCR板中在热循环仪中温育1h。温育后，如上所述测定BGL1多肽的残余比活性。通过温育后的平均比活性与温育前的平均比活性的比率来确定变体和野生型酶的残余活性。通过将BGL变体的残余活性除以野生型BGL1(例如，参考酶)的残余活性来确定BGL变体的性能指数(PI)。葡萄糖抑制检测分析通过在3.75mM葡萄糖存在下，按如上所述方法重复CNPG酶活性检测分析来确定葡萄糖对β-葡萄糖苷酶的水解活性的影响。通过在葡萄糖存在下的平均比活性与在葡萄糖不存在时的平均比活性的比率来确定变体和野生型蛋白质的残余活性。通过将BGL变体的残余活性除以野生型BGL1(例如，参考酶)的残余活性来确定BGL变体的性能指数(PI)。磷酸溶胀纤维素(PASC)水解检测分析中的比活性根据公布的方法(参见，例如，Walseth,Tappi,35:228,1971；和Wood,BiochemJ.,121:353-362,1971)从Avicel制备磷酸溶胀纤维素(PASC)。用缓冲液和水稀释该材料以获得1％w/v混合物，其中醋酸钠的最终浓度为50mM(pH5.0)。将一百五十(150)μL的在50mM醋酸钠缓冲液(pH5.0)中的1％PASC悬浮液分配到96孔微量滴定板(CostarFlatBottomPS)中。将十(10)μL来自bgl1缺失菌株的含有0.75mg/mL蛋白质的培养物上清液加入到PASC悬浮液。然后将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经40X稀释(在50mM醋酸钠缓冲液(pH5.0)中)后取5、10、20或40μL加入到PASC/缺失突变体上清液混合物。加入补偿体积的醋酸盐缓冲液以补足在总体积方面的差异。将微量滴定板密封并且在50℃恒温培养箱中温育，并以900rpm连续振动。2h后，通过向每孔加入100μL100mM甘氨酸缓冲液(pH10)来终止水解反应。将板密封并且在室温下以3,500rpm离心5min。通过ABTS检测分析来分析上清液中的水解反应产物。建立野生型BGL1的剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。稀酸预处理玉米秸秆(PCS)水解检测分析中的比活性用2％w/wH2SO4预处理玉米秸秆(参见，Schell等人，J.Appl.Biochem.Biotechnol.,105:69-86,2003)，然后用去离子水多次洗涤以获得pH为4.5的浆料。然后加入醋酸钠缓冲液(pH5.0)(至最终浓度为50mM醋酸钠)，并且如果必要的话，使用1NNaOH将此混合物滴定至pH5.0。反应混合物中的纤维素浓度为约7％。在96孔微量滴定板(NuncFlatBottomPS)中每孔加入六十五(65)μL的此纤维素悬浮液。将十(10)μL来自bgl1缺失菌株的含有10mg/mL蛋白质的培养物上清液加入到PCS。然后将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经5X稀释(在50mM醋酸钠缓冲液(pH5.0)中)后取5、10、15或20μL的加入到PCS/缺失突变体上清液混合物。加入补偿体积的醋酸钠缓冲液以补足在总体积方面的差异。密封后，将板置于50℃恒温培养箱中，并以900rpm连续振动。16h后，将板放在冰上5min，并且通过向每孔加入100μL的100mM甘氨酸缓冲液(pH10)来终止水解反应。将板密封并且在室温下以3,000rpm离心5min。通过ABTS检测分析来分析上清液中的水解反应产物。建立野生型BGL1蛋白质的剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。纤维二糖酶活性检测分析(pH5)测试了野生型BGL1和BGL变体在pH5.0下的纤维二糖水解能力。将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经20X稀释(在50mM醋酸钠缓冲液(pH5.0)中)后取不同的数量(例如，5、10、15或20μL)加入到80μL的在50mM醋酸钠缓冲液(pH5.0)中的16.4mM(5.63mg/mL)纤维二糖溶液。加入补偿体积的醋酸纳缓冲液以补足在总体积方面的差异。将微量滴定板密封并且在50℃恒温培养箱中温育，并以900rpm连续振动。30min后，通过向每孔加入100μL100mM甘氨酸缓冲液(pH10)来终止水解反应。通过ABTS检测分析来分析水解反应产物。建立野生型BGL1的剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。纤维二糖酶活性检测分析(pH6)测试了野生型BGL1和BGL1变体在pH6.0下的纤维二糖水解能力。将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经20X稀释(在50mM柠檬酸钠缓冲液(pH6.0)中)后取不同的数量(5、10、15或20μL)加入到80μL的在50mM柠檬酸钠缓冲液(pH6.0)中的16.4mM(5.63mg/mL)纤维二糖溶液。加入补偿体积的柠檬酸盐缓冲液以补足在总体积方面的差异。将微量滴定板密封并且在50℃恒温培养箱中温育，并以900rpm连续振动。30min后，通过向每孔加入100μL100mM甘氨酸缓冲液(pH10)来终止水解反应。通过ABTS检测分析来分析水解反应产物。建立野生型BGL1蛋白质的剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。通过在氨预处理玉米芯(CC)存在下进行纤维二糖酶活性检测分析来测定β-葡萄糖苷酶活性研磨玉米芯以通过0.9mm筛，并如PCT专利申请公布WO2006110901中所描述进行预处理。将经预处理CC作为在50mM醋酸钠缓冲液(pH5.0)中的7％纤维素悬浮液来使用。向96孔微量滴定板(NuncFlatBottomPS)每孔加入六十五(65)μL的悬浮液。将四十五(45)μL的35.1mM(12.0mg/mL)纤维二糖溶液加入到预处理玉米芯，并将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经20X稀释(在50mM醋酸钠缓冲液(pH5.0)中)后取不同的数量(5、10、15或20μL)加入。加入补偿体积的醋酸盐缓冲液以补足在总体积方面的差异。将微量滴定板密封并且在50℃恒温培养箱中温育，并以900rpm连续振动。30min后，通过向每孔加入100μL100mM甘氨酸缓冲液(pH10)来终止水解反应。混合后，使板在3,500rpm下离心5min。通过ABTS检测分析来分析水解反应产物。建立野生型BGL1蛋白质的剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。实施例2构建红褐肉座菌BGL1位点评估库(“SEL”)将含有红褐肉座菌BGL1蛋白编码序列(SEQIDNO:1)的pTTTpyrG-bgl1质粒送至多个供应商，例如BASEClear(荷兰莱顿)、GeneArtAG(德国雷根斯堡)和SloningBioTechnologyGmbH(德国Puchheim)，以用于构建位点评估库(SEL)。SEQIDNO:2示出了全长BGL1蛋白质的氨基酸序列。供应商构建了表2-1中示出的BGL1成熟蛋白(SEQIDNO:3)中每个位点的位置库。SEQIDNO:1示出参考红褐肉座菌bgl1编码DNA序列：atgcgctaccgcaccgctgccgctttagccttagccaccggccccttcgccagagccgatagccacagcacctccggcgctagtgctgaagctgttgtccctcctgctggcaccccttggggcaccgcctacgacaaggccaaggccgccctcgccaagctcaacctccaggacaaggtcggcatcgtcagcggcgtcggctggaacggcggtccctgcgtcggcaacaccagccccgccagcaagatcagctaccccagcctctgcctccaggacggccccctcggcgtccgctacagcaccggcagcaccgccttcacccctggcgtccaggccgccagcacctgggacgtcaacctcatccgcgagcgcggccagttcatcggcgaagaggtcaaggccagcggcatccacgtcatcctcggtcccgttgctggtcccttaggcaagaccccccagggcggtcgcaactgggagggcttcggcgtcgacccctacctcaccggcattgccatgggccagaccatcaacggcatccagagcgtcggcgtccaggccaccgccaagcactacatcctcaacgagcaagagttaaaccgcgagactatcagcagcaaccccgacgaccgcaccctccacgagttatacacctggcccttcgccgacgccgtccaggccaacgtcgccagcgtcatgtgcagctacaacaaggtcaacaccacctgggcctgcgaggaccagtacaccctccagaccgtcctcaaggaccagctcggcttccccggctacgtcatgaccgactggaacgcccagcacaccaccgtccagagcgccaacagcggcctcgacatgagcatgcccggcaccgacttcaacggcaacaaccgcctctggggccctgccctcaccaacgccgtcaacagcaaccaggtccccacctcccgcgtcgacgacatggtcacccgcatcctcgccgcctggtacttaaccggccaagaccaggctggctatcccagcttcaacatcagccgcaacgtccagggcaaccacaagaccaacgtccgcgccattgcccgcgacggcatcgtcctcctcaagaacgacgccaacatcctccccctcaagaagcccgcctctatcgccgtcgtcggcagcgccgccatcatcggcaaccacgcccgcaacagccccagctgcaacgacaagggctgcgatgacggtgccctcggcatgggctggggctctggcgccgtcaactacccctacttcgtcgccccctacgacgccatcaacacccgcgccagcagccagggcacccaggtcaccctcagcaacaccgacaatacttcttctggcgcttctgctgctagaggcaaggacgtcgccatcgtttttatcactgccgattctggcgaaggctacatcaccgtcgagggcaacgccggcgaccgcaacaacctcgacccctggcacaacggcaatgccctcgtccaggccgttgctggtgctaacagcaacgtcatcgtcgtcgtccacagcgtcggcgccatcatcctcgagcagatcctcgccctcccccaggtcaaggccgtcgtctgggccggcttacccagccaggaaagcggcaacgccttagtcgacgtcctctggggtgacgtttccccctctggcaagctcgtctacaccattgccaagagccccaacgactacaacacccgcattgtcagcggcggcagcgacagcttcagcgagggcctcttcatcgactacaagcacttcgacgacgccaacattaccccccgctacgagttcggctacggcctcagctacaccaagttcaactacagccgcctcagcgtcctcagcaccgccaagagcggccctgccactggtgctgtcgtccctggtggcccttctgacctcttccagaacgtcgccacggtcaccgtcgacattgccaactccggccaggtcactggcgccgaggtcgcccagctctacatcacctaccccagcagcgcccctcgcactcctcccaagcagctcagaggcttcgctaagttaaacttaacccctggccagagcggcaccgccacctttaacatccgcagacgcgacctcagctactgggacaccgccagccagaagtgggtcgtccccagcggcagcttcggcatctccgtcggcgccagctcccgcgacatccgcctcaccagcaccctcagcgtcgcctgatga*SEQIDNO:2示出红褐肉座菌BGL1全长蛋白的序列：MRYRTAAALALATGPFARADSHSTSGASAEAVVPPAGTPWGTAYDKAKAALAKLNLQDKVGIVSGVGWNGGPCVGNTSPASKISYPSLCLQDGPLGVRYSTGSTAFTPGVQAASTWDVNLIRERGQFIGEEVKASGIHVILGPVAGPLGKTPQGGRNWEGFGVDPYLTGIAMGQTINGIQSVGVQATAKHYILNEQELNRETISSNPDDRTLHELYTWPFADAVQANVASVMCSYNKVNTTWACEDQYTLQTVLKDQLGFPGYVMTDWNAQHTTVQSANSGLDMSMPGTDFNGNNRLWGPALTNAVNSNQVPTSRVDDMVTRILAAWYLTGQDQAGYPSFNISRNVQGNHKTNVRAIARDGIVLLKNDANILPLKKPASIAVVGSAAIIGNHARNSPSCNDKGCDDGALGMGWGSGAVNYPYFVAPYDAINTRASSQGTQVTLSNTDNTSSGASAARGKDVAIVFITADSGEGYITVEGNAGDRNNLDPWHNGNALVQAVAGANSNVIVVVHSVGAIILEQILALPQVKAVVWAGLPSQESGNALVDVLWGDVSPSGKLVYTIAKSPNDYNTRIVSGGSDSFSEGLFIDYKHFDDANITPRYEFGYGLSYTKFNYSRLSVLSTAKSGPATGAVVPGGPSDLFQNVATVTVDIANSGQVTGAEVAQLYITYPSSAPRTPPKQLRGFAKLNLTPGQSGTATFNIRRRDLSYWDTASQKWVVPSGSFGISVGASSRDIRLTSTLSVA*SEQIDNO:3示出红褐肉座菌BGL1成熟蛋白的序列：VVPPAGTPWGTAYDKAKAALAKLNLQDKVGIVSGVGWNGGPCVGNTSPASKISYPSLCLQDGPLGVRYSTGSTAFTPGVQAASTWDVNLIRERGQFIGEEVKASGIHVILGPVAGPLGKTPQGGRNWEGFGVDPYLTGIAMGQTINGIQSVGVQATAKHYILNEQELNRETISSNPDDRTLHELYTWPFADAVQANVASVMCSYNKVNTTWACEDQYTLQTVLKDQLGFPGYVMTDWNAQHTTVQSANSGLDMSMPGTDFNGNNRLWGPALTNAVNSNQVPTSRVDDMVTRILAAWYLTGQDQAGYPSFNISRNVQGNHKTNVRAIARDGIVLLKNDANILPLKKPASIAVVGSAAIIGNHARNSPSCNDKGCDDGALGMGWGSGAVNYPYFVAPYDAINTRASSQGTQVTLSNTDNTSSGASAARGKDVAIVFITADSGEGYITVEGNAGDRNNLDPWHNGNALVQAVAGANSNVIVVVHSVGAIILEQILALPQVKAVVWAGLPSQESGNALVDVLWGDVSPSGKLVYTIAKSPNDYNTRIVSGGSDSFSEGLFIDYKHFDDANITPRYEFGYGLSYTKFNYSRLSVLSTAKSGPATGAVVPGGPSDLFQNVATVTVDIANSGQVTGAEVAQLYITYPSSAPRTPPKQLRGFAKLNLTPGQSGTATFNIRRRDLSYWDTASQKWVVPSGSFGISVGASSRDIRLTSTLSVA*表2-1.选择用于构建SEL的成熟BGL1蛋白中的位置对于表2-1中列出的178个位点中的每个，通常获得14-16个置换变体。各SEL变体以分别纯化的质粒接收，每个纯化的质粒编码在指定位置发生置换的BGL1变体序列。BGL1变体的产生为了使BGL1和变体BGL蛋白能在里氏木霉中表达，通过LR重组反应将bgl1编码序列克隆到Gateway相容性目的载体pTTT-pyrG13中(图2)。该载体含有源于里氏木霉cbhI的启动子和终止子区域，允许所关注的基因的强诱导型表达，构巢曲霉amdS和pyrG选择性标记使转化株在作为唯一氮源的乙酰胺上生长，并且里氏木霉端粒区域允许非染色体质粒在真菌细胞中维持。此外，该载体允许用尿苷营养缺陷型选择里氏木霉菌株的转化株。cbhI启动子和终止子区域被氯霉素抗性基因CmR和致死大肠杆菌基因ccdB分开，其两侧为基于λ噬菌体的特异性重组位点attR1、attR2。此类构型允许通过重组反应来直接选择含有在正确取向的cbhI调控元件控制下的bgl1基因的重组子。最终的表达载体pTTT-pyrG-bgl1示于图2中。从以上列出的供应商获得表达编码BGL1变体序列的基因的纯化pTTTpyrG-bgl1质粒(pcbh1，AmpR，乙酰胺酶)。如之前在例如PCT专利申请公布WO2009/048488中所述，用各个pTTTpyrG构建体转化红褐肉座菌菌株(Δeg1、Δeg2、Δcbh1、Δcbh2、Δbgl1)的原生质体(每次转化用单个BGL1变体)，并使其在28℃下在含有乙酰胺的选择性琼脂上生长7天。产生出红褐肉座菌的原生质体，将其收获、接种在乙酰胺琼脂上并在28℃下温育7天。孢子收获在15％甘油中并在-20℃下保存。为了产生BGL1变体，将体积为10μL的孢子悬浮液加入到在PVDF过滤板中的200μL补充有2％葡萄糖/槐糖混合物的甘氨酸基本培养基。使每种BGL1变体一式四份生长。在用透氧膜密封板之后，使板在28℃下温育6天，并伴以220rpm的振动。通过在真空下将培养基转移到微量滴定板来收获滤液。使用如实施例1A中所述的己糖激酶检测分析来测量残余葡萄糖。实施例3BGL1变体的表达、活性和性能测试了红褐肉座菌BGL1SEL变体蛋白的多种所关注的特性。具体地讲，测试了β-葡萄糖苷酶变体的如下特性：使用HPLC检测分析来测定的蛋白质表达(HPLC)、CNPG水解活性(CNPG)、葡萄糖对活性的影响(Gluc)、热稳定性(Heat)、PASC的水解(PASC)、PCS的水解(PCS)、pH5.0下的纤维二糖酶活性(G2pH5)、pH6.0下的纤维二糖酶活性(G2pH6)、以及如实施例1所述通过在氨预处理玉米芯存在下的纤维二糖酶活性(G2CC)测量的β-葡萄糖苷酶活性。表3-1中示出的BGL1变体的性能指数四舍五入到百分位。性能指数(PI)是变体性能与野生型BGL1性能的比率。小于或等于0.05的性能指数一般固定为0.05。然而，对于HPLC蛋白值0.0，所有值都固定为0.04。将具有野生型残基的SEL酶的PI值设为1.00。四舍五入前大于1的PI值以粗斜体示于表3-1中。表3-1.BGL1SEL变体(3,153)的性能指数数据变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK022A1.171.071.101.391.060.951.070.991.05K022C0.040.040.040.040.040.040.040.040.04K022E1.221.081.151.771.041.001.141.021.06K022F0.661.190.931.161.141.191.201.231.10K022G0.871.060.931.010.961.020.990.940.90K022H0.470.840.790.570.970.970.851.020.71K022I0.780.990.931.030.960.970.950.930.86K022K1.001.001.001.001.001.001.001.001.00K022L0.040.040.040.040.040.040.040.040.04K022M0.040.040.040.040.040.040.040.040.04K022N0.580.910.830.790.910.850.780.980.66K022P0.891.171.001.241.040.901.091.071.05变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK022Q0.821.120.961.271.000.841.061.040.98K022R0.610.980.840.900.970.890.931.030.74K022S1.321.031.111.510.950.870.990.900.89K022T0.050.050.050.050.050.050.050.050.05K022V0.751.020.921.020.931.000.950.980.66K022W1.111.050.901.200.891.010.880.830.78K022Y0.981.120.991.300.930.820.991.000.87N024A1.541.391.001.570.991.020.990.951.00N024C0.741.760.941.831.211.351.361.431.20N024D1.080.701.060.960.911.080.890.910.76N024E1.180.961.191.480.891.090.890.940.77N024F0.480.381.130.611.061.220.940.880.75N024G0.810.301.240.570.931.090.930.920.97N024K0.590.311.020.490.990.970.960.970.51N024L1.100.781.020.960.881.110.880.910.87N024M0.650.851.161.160.790.890.760.800.96N024N1.001.001.001.001.001.001.001.001.00N024P1.000.301.280.580.981.031.000.960.96N024Q0.340.280.050.681.681.601.811.791.30N024R0.670.050.050.221.020.920.900.730.77N024S0.960.300.050.790.881.090.850.880.88N024T0.960.351.240.670.920.820.840.860.82N024V0.620.300.980.540.931.060.860.780.60N024Y0.570.320.050.051.021.060.910.920.72L025A0.701.200.870.790.990.911.031.031.06L025D0.681.130.970.721.020.891.031.091.02L025F0.711.080.930.780.960.860.910.920.83L025G0.511.000.910.570.930.940.890.830.85L025H0.040.040.040.040.040.040.040.040.04L025I1.001.100.941.170.960.901.000.990.91L025K0.531.040.860.530.941.080.890.990.74L025L1.001.001.001.001.001.001.001.001.00L025N0.270.800.820.370.901.250.901.000.58L025P0.050.050.050.050.050.050.050.050.05L025Q0.641.100.930.920.920.940.980.920.84L025R0.601.040.890.630.930.990.910.850.84L025S0.851.010.960.890.910.820.890.881.00L025T0.961.041.011.110.921.080.950.910.82L025V0.681.090.950.830.921.180.920.890.92L025W1.071.060.990.980.980.770.950.920.97L025Y0.491.030.880.410.921.010.960.960.75Q026A0.940.900.800.740.890.890.850.890.88Q026C0.401.430.990.611.331.301.321.401.41Q026D0.820.991.000.751.001.070.990.981.07Q026E0.710.970.920.661.071.140.940.921.05Q026F0.040.040.040.040.040.040.040.040.04变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ026G0.480.990.930.390.931.050.920.930.96Q026H0.551.130.971.321.081.000.991.020.85Q026I0.471.090.980.721.221.021.041.070.91Q026K0.431.480.961.301.251.301.341.341.37Q026L0.671.150.860.771.131.261.061.040.81Q026M0.040.040.040.040.040.040.040.040.04Q026N0.040.040.040.040.040.040.040.040.04Q026P0.421.121.110.511.081.191.041.070.89Q026Q1.001.001.001.001.001.001.001.001.00Q026R0.471.181.021.141.131.121.101.100.88Q026S0.461.260.891.261.141.101.141.151.14Q026T0.171.600.890.671.651.881.631.581.32Q026V0.531.050.970.611.021.160.980.970.88Q026W0.391.381.100.561.381.201.321.371.19Q026Y0.700.861.150.600.841.020.800.820.73D027A0.811.091.000.741.090.700.931.001.03D027C0.281.070.990.3433.470.220.921.030.92D027E0.711.150.990.881.080.860.920.990.82D027F0.050.050.050.050.050.050.050.050.55D027G0.200.900.930.210.220.080.760.791.00D027I0.170.870.920.170.470.060.710.730.83D027K0.050.050.050.050.050.050.050.050.05D027L0.341.170.930.440.600.240.840.921.28D027M0.291.110.890.390.220.210.830.881.14D027P0.060.050.050.050.050.050.050.050.48D027Q0.331.070.960.300.050.260.880.901.02D027R0.120.670.890.120.050.050.520.570.79D027S0.581.160.920.600.370.530.850.911.07D027T0.421.090.890.440.480.350.810.810.97D027V0.241.110.750.190.520.160.610.680.77D027W0.140.650.850.070.390.050.560.560.86D027Y0.230.780.940.170.370.120.580.710.91K028C0.440.600.900.570.910.760.730.930.73K028E0.440.560.950.500.840.790.700.900.64K028F0.040.040.040.040.040.040.040.040.04K028G0.040.040.040.040.040.040.040.040.04K028I0.200.290.890.200.750.750.540.950.46K028K1.001.001.001.001.001.001.001.001.00K028L0.190.291.090.200.790.740.590.980.58K028M0.700.821.020.950.880.920.800.920.75K028N0.090.131.000.080.460.500.430.880.39K028P0.040.040.040.040.040.040.040.040.04K028Q0.420.620.960.490.900.940.800.980.67K028R0.530.760.900.690.910.970.840.960.73K028S0.190.280.980.180.710.600.561.000.48K028T0.040.040.040.040.040.040.040.040.04变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK028V0.150.260.910.180.720.630.551.030.41K028W0.270.050.050.050.180.050.050.140.18K028Y0.040.040.040.040.040.040.040.040.04S033A0.110.350.820.050.620.890.640.640.36S033C0.421.290.880.491.161.341.361.421.18S033D0.210.580.750.050.750.940.730.810.61S033E0.110.050.050.050.050.330.050.050.37S033F0.080.210.710.050.490.460.400.540.34S033G0.461.080.890.391.000.971.071.151.00S033H0.200.600.820.090.840.700.800.820.64S033I0.090.190.750.050.380.430.260.300.29S033K0.120.430.760.050.760.240.710.800.40S033L0.090.210.670.050.360.380.220.280.31S033M0.210.700.750.050.850.720.710.880.64S033N0.180.620.810.050.890.900.780.910.60S033P0.090.050.050.050.050.110.050.050.11S033Q0.090.300.910.050.720.600.550.660.30S033R0.150.520.770.050.800.540.740.960.50S033S1.001.001.001.001.001.001.001.001.00S033T0.710.940.970.840.921.050.890.910.84S033V0.330.990.770.300.930.940.880.970.70S033W0.100.150.830.050.270.310.190.240.17S033Y0.100.150.050.050.240.210.170.260.26V035C0.771.420.900.851.021.071.161.221.05V035D0.040.040.040.040.040.040.040.040.04V035E0.771.310.920.770.951.021.021.111.09V035F0.260.050.050.070.200.090.120.100.10V035G0.991.170.900.360.860.660.981.031.09V035H0.811.190.860.310.960.720.981.070.83V035K0.591.400.870.740.990.890.971.080.91V035L0.781.220.951.030.980.870.981.030.79V035N0.691.210.880.640.901.050.971.070.85V035P0.441.340.880.051.080.330.920.920.79V035Q0.751.340.920.960.980.821.091.150.92V035R0.761.400.870.790.990.911.051.090.94V035S1.101.200.881.140.910.841.031.020.93V035T0.971.150.861.081.050.961.051.130.83V035V1.001.001.001.001.001.001.001.001.00V035W1.170.970.980.250.890.800.880.930.96V035Y0.831.200.990.090.960.711.031.000.90G036A0.330.530.740.050.740.700.730.650.70G036C0.180.600.770.051.000.701.121.070.83G036D0.521.020.860.051.210.671.281.151.12G036E0.461.080.880.051.140.631.231.151.01G036F0.090.410.710.050.700.171.161.130.65G036H0.040.040.040.040.040.040.040.040.04变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCG036I0.100.430.750.050.700.281.141.010.68G036K0.270.890.730.051.070.851.161.140.82G036N0.100.430.700.050.860.550.781.040.64G036P0.040.040.040.040.040.040.040.040.04G036Q0.330.730.760.050.830.630.880.860.72G036R0.421.080.740.111.090.801.041.120.99G036S0.671.340.900.141.481.161.471.451.53G036V0.270.710.770.050.720.180.950.910.71G036W0.090.400.650.050.830.091.331.210.82G036Y0.070.830.710.052.080.153.082.991.25W037A1.500.850.881.400.660.790.730.630.78W037C0.460.700.640.350.620.850.840.750.82W037E1.330.910.981.110.600.820.670.660.68W037F1.211.001.091.390.590.680.680.630.65W037G0.930.720.700.450.530.670.660.550.62W037H1.400.850.831.410.610.750.690.710.71W037I0.070.311.050.050.050.270.140.050.31W037K0.800.680.650.630.450.680.560.420.49W037L0.110.320.840.080.440.900.560.390.57W037M0.990.910.971.140.560.690.720.610.69W037P0.880.900.720.840.590.710.730.620.71W037R0.690.870.880.730.530.780.630.530.59W037S1.080.930.871.050.630.870.750.670.82W037T0.640.910.800.630.540.690.660.550.75W037V0.651.070.980.900.560.760.650.640.70W037W1.001.001.001.001.001.001.001.001.00W037Y1.111.020.861.450.620.850.700.850.66S050A0.991.020.991.100.990.961.000.850.63S050C0.610.921.100.641.051.261.140.961.02S050F0.780.851.030.730.861.240.850.750.62S050G0.670.801.050.600.871.250.870.760.86S050I0.510.830.940.600.861.120.920.790.77S050K0.640.771.020.670.910.990.910.820.79S050L0.320.730.990.401.011.300.860.741.05S050M0.650.970.910.810.951.230.790.660.98S050N0.630.830.950.570.871.270.860.680.78S050P0.600.901.020.740.971.221.000.710.90S050Q0.650.860.980.730.890.980.930.710.64S050R0.770.911.000.810.921.070.950.800.82S050S1.001.001.001.001.001.001.001.001.00S050T0.550.751.090.630.911.140.890.800.83S050V0.730.990.960.790.881.080.950.860.86S050W0.740.910.980.750.930.970.910.900.92S050Y0.720.961.040.810.900.930.700.990.93K051A1.070.991.061.030.971.111.100.940.93K051C0.461.210.960.571.301.161.181.451.27变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK051D0.981.051.050.800.991.101.121.111.09K051E0.641.030.960.751.140.910.960.920.88K051F0.040.040.040.040.040.040.040.040.04K051G0.661.071.020.711.050.951.081.080.97K051H0.781.171.011.021.021.031.121.071.02K051I0.450.940.950.641.021.101.001.050.91K051K1.001.001.001.001.001.001.001.001.00K051L0.510.970.930.650.941.050.821.330.78K051M0.801.161.081.041.010.921.071.010.97K051N0.550.990.970.490.980.980.941.020.80K051P0.270.710.870.290.980.910.940.920.83K051Q0.551.140.940.771.071.091.150.990.81K051R0.641.070.960.780.991.060.981.090.81K051S0.340.840.910.490.941.060.910.900.74K051T0.751.201.030.931.031.021.081.171.21K051V0.501.071.030.701.061.101.061.040.90K051W0.440.770.950.230.890.850.860.950.82I052A0.270.211.190.050.620.440.440.831.19I052C0.320.120.050.110.240.070.090.520.33I052D0.100.190.840.101.010.690.621.861.53I052F0.260.171.010.050.450.250.280.820.54I052I1.001.001.001.001.001.001.001.001.00I052K0.100.220.570.060.740.370.311.690.64I052L0.270.280.870.120.610.430.500.920.80I052M0.170.180.870.050.610.340.341.170.86I052N0.090.171.030.050.770.290.401.921.17I052P0.100.130.050.050.880.260.411.741.00I052Q0.100.200.790.050.980.510.521.850.53I052R0.310.190.050.160.270.150.120.570.40I052S0.300.131.150.080.330.130.160.640.41I052T0.100.210.820.051.010.880.702.040.95I052V0.230.260.880.110.670.450.421.041.17I052W0.320.120.050.080.240.120.100.580.38I052Y0.320.180.650.050.320.160.150.610.56D061A0.920.060.120.050.050.050.050.050.07D061C0.950.070.050.050.050.050.050.050.07D061D1.001.001.001.001.001.001.001.001.00D061E1.310.100.600.050.190.540.110.070.12D061F0.810.050.050.050.050.050.050.050.10D061G1.170.050.050.050.050.070.050.050.05D061H0.780.220.490.120.230.300.180.080.15D061I0.330.100.050.070.050.050.070.050.19D061K0.380.110.050.130.050.050.060.050.11D061L0.970.070.050.080.050.070.050.050.07D061M0.960.050.050.050.050.060.050.050.05D061N1.730.050.050.050.050.050.050.050.05变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCD061P1.060.060.050.360.050.050.050.050.05D061R0.490.090.050.110.050.050.050.050.06D061T0.520.070.050.160.050.050.050.050.09D061V0.190.100.050.310.050.190.160.050.18D061W0.240.220.490.190.280.240.220.100.29D061Y0.690.070.050.050.050.050.050.050.12R067A0.441.481.250.050.050.050.060.150.49R067C0.281.451.150.050.050.250.080.180.43R067D0.163.111.310.050.060.220.110.260.45R067E0.450.751.460.050.050.070.080.120.35R067F0.431.151.280.050.050.060.110.190.42R067G1.201.041.220.050.050.050.050.090.26R067I0.940.661.080.050.050.050.070.080.21R067K0.710.050.050.060.050.050.050.060.12R067L1.500.750.990.050.050.050.050.050.18R067M1.110.631.210.050.050.050.050.070.23R067N0.295.241.030.050.050.050.070.130.43R067P1.071.031.110.050.050.050.050.070.34R067Q0.191.430.050.100.050.050.080.230.98R067R1.001.001.001.001.001.001.001.001.00R067S0.851.051.410.050.050.140.070.120.24R067T2.110.771.400.110.050.050.050.080.22R067V1.730.781.390.050.050.050.050.100.25R067W0.311.521.190.070.050.050.140.210.25R067Y1.340.791.140.080.050.090.050.120.23R091A0.202.011.220.230.820.960.930.940.77R091C0.080.050.050.170.590.530.801.100.73R091D0.401.221.090.361.091.371.130.961.01R091E0.880.961.040.810.981.010.980.861.13R091F0.571.081.040.510.931.160.960.901.12R091G0.960.861.170.831.101.251.010.870.90R091H0.610.931.110.520.881.120.910.850.83R091I1.020.911.010.850.961.170.940.860.90R091K0.930.971.040.871.041.291.060.960.97R091L0.291.391.070.340.841.030.970.970.90R091M0.040.040.040.040.040.040.040.040.04R091N0.980.941.080.940.991.211.000.921.05R091P0.070.050.050.160.070.960.260.710.44R091Q0.631.051.210.611.011.241.061.020.80R091R1.001.001.001.001.001.001.001.001.00R091S0.730.881.170.620.860.890.890.880.72R091T1.200.831.130.950.931.000.930.870.81R091V0.341.201.180.350.941.240.940.980.78R091W0.571.021.090.570.961.150.961.050.82R091Y1.410.860.981.080.941.050.920.940.81E092A0.590.950.820.621.040.981.170.901.02变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCE092C0.661.140.870.461.201.281.501.071.24E092D0.970.920.850.321.011.091.110.880.93E092E1.001.001.001.001.001.001.001.001.00E092F0.610.950.840.500.941.121.050.880.73E092H0.490.910.850.710.921.231.050.840.68E092I0.691.000.820.780.940.971.060.930.83E092K1.481.041.011.430.991.160.951.070.94E092L1.091.040.931.260.951.191.040.970.82E092M0.910.930.981.050.880.930.820.880.75E092N0.721.040.880.440.981.101.160.960.99E092P0.040.040.040.040.040.040.040.040.04E092Q0.860.930.880.680.881.070.970.880.77E092R0.910.970.850.600.921.291.080.960.78E092S0.780.930.870.930.920.960.990.910.80E092T1.110.921.050.980.931.020.950.940.84E092V0.900.980.980.841.001.401.110.950.87E092W0.970.770.940.800.770.920.810.770.66E092Y0.770.910.950.050.901.010.960.920.76R093A0.400.431.470.050.480.120.320.470.28R093C0.380.371.400.050.450.180.290.500.33R093D0.140.101.210.080.310.660.200.320.21R093E0.370.401.380.050.440.120.310.430.20R093F0.200.341.250.050.300.120.310.490.15R093G0.080.151.510.050.290.430.220.500.24R093H0.230.491.120.050.780.700.620.760.47R093I0.110.050.050.150.050.050.050.180.11R093K0.180.311.010.050.561.200.440.620.26R093L0.510.511.370.080.370.100.200.410.22R093M0.630.541.390.050.430.170.210.540.19R093P0.050.050.050.050.050.050.050.050.05R093Q0.160.481.390.050.640.150.290.790.26R093R1.001.001.001.001.001.001.001.001.00R093S0.190.251.470.050.320.170.220.400.20R093T0.150.131.110.050.120.260.080.290.25R093V0.150.131.500.050.110.170.070.270.12R093W0.130.201.290.050.410.160.330.540.24R093Y0.110.050.050.050.050.050.050.160.09E099A0.740.751.020.710.911.170.830.800.80E099C0.050.050.050.050.050.050.050.050.05E099D0.160.260.980.170.811.650.660.760.46E099E1.001.001.001.001.001.001.001.001.00E099F0.480.721.080.560.961.150.970.860.90E099G0.110.050.050.050.110.220.110.270.10E099I0.530.671.100.620.901.240.790.830.77E099K0.590.750.970.720.891.420.790.840.69E099L0.050.050.050.050.050.050.050.050.05变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCE099M0.700.781.020.780.851.230.750.800.70E099N0.700.900.940.860.871.120.840.830.88E099P0.100.050.050.050.100.160.060.290.24E099Q0.110.050.050.050.050.180.050.170.05E099R0.100.050.050.050.050.050.050.170.15E099V0.570.691.000.700.860.980.770.820.79E099W0.510.661.150.490.911.360.890.880.78E099Y0.580.851.030.791.031.311.031.030.85E100A0.601.130.990.051.040.641.011.041.29E100C0.040.040.040.040.040.040.040.040.04E100D0.040.040.040.040.040.040.040.040.04E100E1.001.001.001.001.001.001.001.001.00E100G0.261.140.820.051.190.691.151.361.11E100H0.040.040.040.040.040.040.040.040.04E100I0.311.041.160.050.940.440.941.040.80E100K0.110.261.330.100.540.590.310.380.44E100L0.320.811.020.050.840.410.770.890.82E100M0.301.100.780.150.980.471.011.131.08E100N0.140.640.900.081.050.880.951.091.26E100P0.040.040.040.040.040.040.040.040.04E100Q0.410.990.780.111.010.810.951.060.93E100R0.040.040.040.040.040.040.040.040.04E100S0.521.090.810.061.030.780.971.041.01E100T0.471.170.860.101.040.761.051.150.96E100V0.300.820.770.080.890.400.860.960.75E100W0.040.040.040.040.040.040.040.040.04E100Y0.270.911.270.061.120.341.081.201.00R125A1.290.050.050.050.380.130.050.050.05R125C0.510.050.050.050.710.070.050.050.14R125D1.560.050.050.050.310.190.050.050.06R125E0.650.050.050.050.850.110.050.050.14R125F0.070.050.050.050.050.050.050.050.82R125G0.830.050.050.050.320.380.050.050.07R125I0.170.810.950.150.240.050.730.760.97R125L0.540.050.050.050.720.070.050.050.13R125M0.580.050.050.050.280.070.050.050.10R125P0.150.050.050.050.050.050.050.050.23R125R1.001.001.001.001.001.001.001.001.00R125S0.840.050.050.050.660.080.050.050.05R125T0.930.050.050.050.710.180.050.050.08R125V0.680.050.050.050.050.060.050.050.08R125W0.210.050.050.050.050.050.050.050.21R125Y0.110.050.050.050.370.050.050.050.13K158A1.630.050.050.050.870.180.050.050.05K158C1.160.050.050.050.650.170.050.050.07K158E0.570.050.050.050.050.080.050.050.12变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK158F0.090.050.050.050.670.050.050.050.58K158G1.110.050.050.050.050.120.050.050.05K158H0.210.050.050.101.060.050.050.050.15K158I0.450.050.050.050.410.050.050.050.10K158K1.001.001.001.001.001.001.001.001.00K158L0.820.050.050.050.890.120.050.050.06K158M0.740.050.050.050.350.310.050.050.11K158P0.170.050.050.050.050.050.050.050.49K158Q0.640.050.050.050.050.100.050.050.09K158R0.750.050.050.050.050.130.050.050.09K158S0.670.050.050.050.510.090.050.050.08K158T0.690.050.050.051.330.100.050.050.06K158V0.570.050.050.050.530.100.050.050.10K158Y0.110.050.050.050.310.050.050.050.16H159A1.560.250.980.050.180.470.220.210.23H159C2.770.050.050.050.050.100.050.050.05H159D0.040.040.040.040.040.040.040.040.04H159E1.020.081.530.050.050.290.060.050.12H159F0.540.050.050.050.050.060.050.050.45H159G1.260.091.600.050.060.230.090.060.18H159H1.001.001.001.001.001.001.001.001.00H159I0.420.050.050.050.050.100.050.050.07H159K0.410.050.050.050.050.070.050.050.17H159L0.680.050.050.050.050.050.050.050.05H159M0.350.050.050.050.050.100.050.210.72H159N2.130.050.050.050.050.060.050.050.05H159Q0.400.050.050.050.050.150.050.050.14H159R0.670.050.050.050.050.090.050.050.08H159S1.190.120.620.050.050.140.060.050.11H159T0.040.040.040.040.040.040.040.040.04H159V0.610.050.050.050.050.070.050.380.08H159W1.040.050.050.050.050.060.050.050.05H159Y1.700.050.050.050.050.060.050.050.05N163A1.110.610.330.050.430.580.330.310.33N163C0.880.790.370.050.530.760.580.450.56N163D0.840.990.640.050.720.850.730.620.65N163E0.220.050.050.050.050.110.050.050.05N163F0.320.130.900.050.050.060.050.050.06N163G0.690.860.360.050.480.710.450.510.45N163H1.020.350.230.050.280.340.220.330.14N163I0.260.360.100.050.150.360.220.200.17N163K0.040.040.040.040.040.040.040.040.04N163L0.940.410.350.050.190.300.140.260.21N163M0.740.170.430.050.150.250.080.120.09N163N1.001.001.001.001.001.001.001.001.00N163P0.900.450.310.050.280.370.190.230.21变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN163Q0.930.600.560.050.540.610.440.810.29N163R0.450.050.050.050.050.080.050.050.07N163S1.070.710.380.050.430.540.390.350.41N163T0.630.520.110.050.210.380.250.230.33N163V0.270.480.080.050.130.380.390.250.29N163W0.160.050.050.050.050.100.050.050.15N163Y0.860.560.380.580.400.690.460.320.45E164A0.040.040.040.040.040.040.040.040.04E164C0.650.950.910.050.750.840.930.800.96E164E1.001.001.001.001.001.001.001.001.00E164F0.120.340.700.050.190.300.570.090.22E164G0.200.670.830.050.740.741.130.530.61E164H0.060.050.050.050.050.170.130.050.15E164I0.080.050.050.050.050.050.050.050.07E164K0.100.050.050.050.050.050.050.050.17E164L0.090.050.050.050.050.050.050.050.17E164M0.100.260.680.060.140.210.440.050.17E164N0.110.450.280.050.180.050.700.050.37E164P0.110.320.920.050.140.050.590.050.16E164Q0.150.500.830.050.550.230.960.420.58E164R0.100.050.050.050.050.050.050.050.12E164S0.691.220.930.050.800.651.010.990.87E164T0.430.980.860.670.780.720.900.800.96E164V0.100.450.650.050.180.050.680.100.39E164W0.070.050.050.050.050.050.050.050.12E164Y0.080.050.050.050.070.050.050.050.26Q165C0.531.570.980.050.940.750.901.100.88Q165D0.200.051.030.050.850.630.750.820.85Q165F0.661.180.960.090.820.690.750.950.63Q165G0.220.050.880.050.820.610.761.060.65Q165H0.284.570.920.050.810.590.680.940.63Q165I0.2513.171.030.051.230.790.981.380.54Q165K0.160.051.030.051.080.820.751.010.45Q165L0.2320.391.080.050.820.580.530.810.77Q165M0.373.230.910.051.240.870.961.300.69Q165N0.200.050.950.051.140.730.801.180.51Q165P0.310.780.050.050.160.260.080.070.27Q165Q1.001.001.001.001.001.001.001.001.00Q165R0.267.921.100.050.890.570.640.950.77Q165S0.491.750.910.050.910.640.811.050.98Q165T0.252.320.050.070.190.240.070.100.27Q165V0.2512.111.060.051.440.841.021.540.93Q165W0.511.251.120.050.800.700.710.940.79Q165Y0.871.041.030.090.870.910.850.980.62E166A0.040.040.040.040.040.040.040.040.04E166C0.070.050.830.050.600.750.400.400.45变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCE166D0.393.550.840.051.010.701.131.250.87E166E1.001.001.001.001.001.001.001.001.00E166F0.070.051.180.050.460.530.250.370.48E166G0.180.050.730.050.220.330.150.120.45E166H0.040.040.040.040.040.040.040.040.04E166I0.040.040.040.040.040.040.040.040.04E166K0.2317.911.600.050.160.230.070.100.26E166L0.2311.431.340.050.200.250.070.090.35E166M0.040.040.040.040.040.040.040.040.04E166N0.170.051.290.050.220.290.170.170.28E166P0.245.510.850.050.180.120.080.090.22E166Q0.511.100.470.050.120.140.120.100.10E166R0.281.701.290.050.210.110.050.080.26E166S0.140.051.080.050.280.380.150.210.40E166T0.238.871.230.050.210.180.120.130.38E166V0.200.050.050.050.210.220.110.110.46E166W0.040.040.040.040.040.040.040.040.04E166Y0.320.741.130.050.170.120.060.060.34L167A0.971.060.770.940.930.830.910.840.89L167C0.741.120.720.821.031.121.061.031.02L167D0.761.060.680.050.991.120.990.970.91L167E0.721.050.720.650.971.040.990.940.85L167F0.590.910.730.550.981.020.950.940.77L167G0.681.060.680.330.981.041.000.990.85L167I0.720.940.690.880.900.930.870.820.75L167K0.620.970.630.870.880.910.940.870.90L167L1.001.001.001.001.001.001.001.001.00L167M0.550.940.690.700.831.150.950.790.81L167N0.480.880.690.410.941.001.050.880.87L167P0.120.070.050.050.130.050.050.170.10L167Q0.741.060.680.750.830.800.980.820.87L167R0.801.080.681.230.830.990.970.860.89L167S0.941.210.760.310.911.000.990.880.94L167T0.700.990.730.470.940.970.960.920.91L167V0.601.080.700.740.981.051.041.460.90L167W1.071.070.911.121.021.051.061.081.02L167Y0.941.110.750.660.960.900.971.020.88N168A0.721.040.690.671.011.061.051.020.97N168D0.661.040.670.701.051.211.071.100.98N168E0.541.020.650.661.071.071.101.111.00N168G0.661.090.700.761.091.141.111.080.91N168H0.540.900.620.580.940.951.020.930.99N168I0.560.830.640.240.930.990.890.860.77N168K0.610.850.650.380.900.950.960.870.74N168L0.660.940.700.550.920.900.980.820.82N168M0.520.770.660.470.800.920.870.750.76变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN168N1.001.001.001.001.001.001.001.001.00N168P0.150.050.050.050.050.160.050.050.05N168Q0.540.900.630.450.841.011.010.870.85N168R0.510.870.580.350.840.891.010.920.77N168S0.580.930.660.490.870.940.990.860.83N168T0.580.920.660.550.880.961.080.990.97N168V0.050.050.050.050.050.050.050.050.05N168W0.040.040.040.040.040.040.040.040.04N168Y0.761.090.700.601.051.031.131.211.05R169A1.380.051.810.050.130.190.060.050.09R169C1.190.071.650.050.120.180.080.060.09R169D1.200.050.050.050.150.230.080.050.08R169E1.060.181.590.050.230.320.100.100.11R169F0.420.111.580.050.050.160.050.050.06R169G0.550.050.050.050.050.130.050.050.12R169H0.770.071.540.050.150.270.070.050.15R169I0.690.050.050.050.050.100.050.050.10R169K1.010.050.050.050.190.370.080.060.10R169L0.720.050.050.050.080.200.050.050.05R169M0.400.090.050.050.050.200.050.050.08R169P0.180.050.050.050.050.310.050.050.18R169Q0.560.131.910.050.390.370.210.200.23R169R1.001.001.001.001.001.001.001.001.00R169S0.960.051.830.050.100.180.070.050.08R169T0.670.101.680.050.070.120.050.050.14R169V0.560.080.050.050.050.150.050.050.06R169W0.340.050.050.050.050.120.050.050.13R169Y0.290.120.050.050.050.150.050.050.11E170A1.650.770.930.960.740.950.770.510.62E170D0.930.950.970.711.061.040.900.780.77E170E1.001.001.001.001.001.001.001.001.00E170F0.510.981.120.841.191.131.201.200.84E170G0.050.050.050.050.050.050.050.050.05E170H0.040.040.040.040.040.040.040.040.04E170I0.700.631.030.580.870.850.820.720.70E170K1.090.410.310.590.520.670.580.560.58E170L1.380.821.151.300.780.840.680.600.55E170M0.750.521.050.640.720.790.660.500.61E170N0.050.050.050.050.050.050.050.050.05E170P1.480.790.900.610.750.820.780.560.62E170Q0.710.710.870.700.740.950.720.600.62E170R0.740.560.580.470.530.680.580.470.56E170S0.900.880.820.830.810.940.860.670.86E170T0.100.050.050.050.050.870.050.050.55E170V0.750.661.020.640.850.860.780.670.61E170W1.160.631.110.510.740.610.690.580.60变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCE170Y1.110.531.170.690.820.930.861.030.67P176A1.401.001.001.300.840.891.040.971.05P176C0.040.040.040.040.040.040.040.040.04P176D1.930.951.010.480.820.921.030.930.94P176E0.860.980.900.300.811.120.990.960.78P176F0.731.000.850.060.871.201.041.081.27P176G1.251.060.941.260.861.081.000.930.78P176H0.610.910.890.200.961.121.121.030.92P176I0.260.050.050.120.180.470.140.080.78P176K0.271.080.820.051.330.901.511.381.24P176L0.641.130.910.730.991.241.181.070.92P176M0.790.990.990.270.841.020.930.970.63P176N0.040.040.040.040.040.040.040.040.04P176P1.001.001.001.001.001.001.001.001.00P176Q0.720.930.940.350.851.090.900.920.83P176R0.351.051.070.231.171.121.371.421.15P176S0.540.980.850.530.981.360.980.961.22P176T0.881.020.900.890.841.020.950.970.91P176V0.681.110.910.710.961.361.101.091.00P176W0.201.060.870.051.451.391.611.781.71P176Y0.640.980.920.201.101.101.111.261.08D177A0.990.891.020.520.910.900.840.860.85D177C1.060.910.980.450.940.910.880.890.95D177D1.001.001.001.001.001.001.001.001.00D177E0.530.910.980.711.060.871.031.071.11D177F0.720.890.980.151.051.031.061.041.11D177G1.160.941.050.800.960.990.880.920.99D177H0.440.840.990.541.060.981.071.170.84D177I0.670.870.910.350.970.850.960.980.94D177K1.020.980.980.520.971.020.950.970.94D177L0.580.811.060.541.031.031.021.010.87D177M0.600.941.020.731.131.061.241.211.05D177N1.080.981.020.870.961.050.920.981.02D177Q0.720.891.130.610.971.050.981.020.80D177R0.730.910.960.400.981.061.021.070.92D177S0.130.050.050.050.050.050.050.140.23D177V0.951.010.990.591.011.170.971.051.21D177W0.341.101.010.091.440.051.601.631.20D177Y0.600.870.980.281.050.961.071.100.94D178A1.061.070.931.130.981.040.990.791.01D178C0.831.050.900.891.021.081.060.810.96D178D1.001.001.001.001.001.001.001.001.00D178E1.230.930.940.970.870.850.840.650.81D178G0.870.810.990.660.840.940.780.650.74D178I0.790.820.900.660.840.870.850.670.78D178K0.750.990.910.830.950.971.000.820.84变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCD178L0.920.870.990.780.890.920.880.660.76D178M0.910.840.960.730.850.810.850.680.78D178N0.840.910.970.780.861.040.850.710.82D178P1.210.880.930.900.850.920.810.680.71D178Q0.921.040.980.940.950.900.970.840.86D178R0.731.060.880.840.971.051.030.920.87D178S0.890.811.040.710.880.920.820.710.76D178T1.111.000.941.060.880.830.880.770.77D178V0.650.820.940.570.840.810.800.730.76D178W1.090.900.900.750.860.990.850.700.72D178Y0.220.800.970.051.180.251.601.320.91R179A0.240.561.040.190.991.520.940.840.55R179C0.120.340.960.101.011.350.860.770.38R179D0.040.040.040.040.040.040.040.040.04R179F0.090.050.050.050.100.180.050.050.19R179G0.230.560.900.190.951.280.850.800.53R179H0.090.120.050.070.050.230.050.050.15R179I0.120.420.860.140.841.300.840.780.28R179K0.450.790.990.430.941.281.030.880.72R179L0.050.050.050.050.050.050.050.050.05R179M0.070.400.880.131.281.301.171.180.34R179N0.040.040.040.040.040.040.040.040.04R179P0.110.050.050.060.050.660.050.050.08R179Q0.090.290.890.080.771.350.690.710.28R179R1.001.001.001.001.001.001.001.001.00R179S0.300.860.970.481.041.241.091.120.73R179T0.160.520.960.280.951.230.961.090.46R179V0.190.381.330.180.951.370.990.910.57R179W0.671.010.910.710.910.931.021.100.88R179Y0.040.040.040.040.040.040.040.040.04Q194A1.041.051.001.100.991.021.070.891.04Q194C0.670.950.980.891.041.071.160.941.07Q194E0.620.870.980.810.971.151.030.870.95Q194F0.720.950.980.920.961.181.080.910.89Q194G0.540.800.940.730.961.231.010.880.81Q194H0.730.840.950.860.891.280.940.790.90Q194I0.540.730.890.150.890.990.950.830.78Q194K0.901.050.921.080.901.241.010.910.87Q194L0.120.320.890.241.001.111.101.010.56Q194M0.660.910.890.910.921.130.920.870.98Q194N0.040.040.040.040.040.040.040.040.04Q194P0.130.070.050.120.050.200.050.050.10Q194Q1.001.001.001.001.001.001.001.001.00Q194R0.840.930.931.010.861.210.900.910.75Q194S0.770.910.950.860.870.940.880.850.80Q194T0.560.790.980.720.881.080.930.930.84变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ194W0.830.890.980.700.891.210.880.890.83Q194Y1.131.070.920.990.921.000.960.980.94N196A0.250.430.930.220.810.950.850.690.61N196E0.591.011.030.691.031.121.170.881.10N196F0.040.040.040.040.040.040.040.040.04N196G0.700.891.020.660.910.890.890.810.80N196H0.650.950.980.690.971.050.990.880.88N196I0.150.050.050.050.050.070.070.050.13N196K0.140.050.050.050.050.050.060.050.14N196L0.330.950.970.551.171.251.351.121.17N196M0.480.761.010.450.890.990.920.760.77N196N1.001.001.001.001.001.001.001.001.00N196P0.170.191.040.070.410.690.500.390.29N196Q0.570.980.980.661.010.961.110.970.98N196R0.420.790.970.461.001.180.991.000.96N196T0.150.490.970.221.101.291.311.150.75N196V0.040.040.040.040.040.040.040.040.04N196Y0.400.500.990.250.730.890.710.740.58S199A1.730.881.071.981.010.920.880.910.94S199C0.920.960.950.800.900.900.850.950.99S199F0.060.050.050.050.050.380.050.130.26S199G0.561.000.890.520.981.040.940.960.99S199H0.100.050.050.050.080.190.050.050.14S199I0.150.420.630.050.470.550.520.750.57S199K0.120.050.050.050.050.170.050.050.13S199L0.070.100.570.050.140.190.170.180.19S199M0.070.050.050.050.060.380.110.110.36S199N0.170.400.970.070.611.260.580.880.68S199Q0.150.100.050.050.050.340.150.170.22S199R0.090.050.050.050.050.080.050.050.15S199T0.470.860.930.560.961.460.900.990.86S199V0.300.690.870.260.681.060.780.800.53S199W0.080.050.050.050.050.110.050.050.05S199Y0.160.050.050.050.050.480.050.050.09Y204A1.390.050.050.050.050.120.100.050.06Y204C0.270.050.050.080.050.190.320.050.42Y204E1.060.050.050.210.060.210.150.050.23Y204F0.591.190.560.400.650.770.620.500.53Y204G1.720.050.050.050.050.130.090.050.20Y204H1.520.050.050.170.050.080.080.050.06Y204I1.000.080.050.110.050.130.130.050.09Y204K1.630.050.050.080.050.140.100.050.10Y204L0.740.600.550.460.310.530.440.270.32Y204M0.800.161.220.150.210.330.310.140.23Y204P1.430.050.050.060.050.090.080.050.05Y204Q1.090.050.050.050.050.140.130.050.22变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCY204R1.780.050.050.050.050.070.070.050.05Y204S1.150.060.050.170.050.060.100.050.17Y204T1.250.050.050.050.070.220.150.160.20Y204V1.440.070.050.050.130.250.160.200.17Y204W1.410.060.500.070.050.140.110.050.13Y204Y1.001.001.001.001.001.001.001.001.00N208A0.040.040.040.040.040.040.040.040.04N208C0.330.110.050.050.240.190.080.540.34N208D0.300.250.960.170.610.420.390.890.87N208E0.270.110.050.110.290.140.080.600.33N208F0.290.250.950.110.510.340.300.820.53N208G0.040.040.040.040.040.040.040.040.04N208H0.310.330.050.290.670.530.440.920.64N208K0.250.340.860.150.840.630.531.120.68N208N1.001.001.001.001.001.001.001.001.00N208R0.300.251.020.070.560.520.360.830.86N208T0.040.040.040.040.040.040.040.040.04N208V0.250.270.900.110.650.470.410.980.74N208W0.040.040.040.040.040.040.040.040.04T209A0.040.040.040.040.040.040.040.040.04T209C0.651.520.920.861.111.151.281.361.36T209D0.731.150.870.730.981.051.041.091.06T209E0.631.220.910.790.991.021.061.141.26T209G0.701.080.970.731.041.121.061.061.18T209H0.421.290.970.481.051.131.101.210.81T209I0.451.330.920.481.091.551.151.190.97T209K0.351.071.010.330.971.070.981.010.39T209L0.441.061.000.451.121.001.061.061.08T209M0.471.271.030.581.041.051.161.170.98T209P0.810.940.950.050.840.750.790.820.73T209Q0.631.240.950.631.051.121.101.060.86T209R0.691.071.060.610.991.001.000.980.92T209S0.721.140.970.821.080.971.091.071.08T209T1.001.001.001.001.001.001.001.001.00T209V0.731.220.960.771.091.041.211.191.21T209W0.601.080.950.410.951.050.950.991.04T209Y0.252.560.990.632.121.882.432.671.79E214A0.581.451.010.050.860.560.901.050.92E214C0.2415.690.820.050.700.640.690.840.92E214D0.581.610.830.360.991.060.991.101.07E214E1.001.001.001.001.001.001.001.001.00E214F0.200.050.840.050.580.310.440.490.46E214G0.352.980.890.050.880.720.871.060.94E214H0.363.050.850.050.780.420.800.960.88E214I0.200.050.850.050.680.430.730.840.61E214K0.210.051.020.050.760.410.620.740.62变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCE214L0.314.720.790.050.850.310.870.950.74E214M0.402.710.690.050.820.630.870.970.63E214N0.343.570.740.050.780.660.790.920.66E214P0.170.051.030.050.350.180.310.380.44E214Q0.521.620.890.300.910.880.830.970.57E214R0.266.731.070.050.740.320.761.010.61E214S0.402.390.870.050.870.620.861.010.82E214T0.314.470.630.050.800.650.780.930.64E214V0.210.050.810.050.750.310.730.861.02E214W0.080.051.020.051.240.691.201.420.59E214Y0.312.481.010.050.470.360.400.460.66D215A0.480.981.000.510.990.970.961.000.97D215C0.500.961.040.591.031.031.110.981.05D215D1.001.001.001.001.001.001.001.001.00D215E0.391.170.970.651.060.861.161.121.01D215F0.160.491.020.190.630.670.460.630.45D215G0.440.961.070.600.990.931.000.950.93D215H0.340.941.120.500.980.900.971.030.65D215I0.100.180.050.170.120.260.050.270.05D215K0.250.790.900.340.820.810.670.850.46D215L0.141.100.870.401.201.301.191.190.60D215M0.250.981.140.470.901.061.040.980.66D215N0.461.401.000.971.090.991.301.131.22D215Q0.361.121.100.611.000.911.120.930.72D215R0.180.660.990.280.550.810.580.620.27D215S0.421.671.081.121.381.141.841.401.19D215V0.060.341.020.120.380.640.050.760.38D215W0.160.651.040.280.831.040.611.070.36Q216A0.930.971.100.731.061.141.031.101.08Q216C0.530.831.060.311.001.061.050.910.81Q216D1.600.791.120.760.991.250.941.010.92Q216E1.001.001.051.011.001.090.990.990.95Q216F0.720.971.080.660.981.151.061.030.98Q216G0.831.220.990.851.161.131.131.071.00Q216H0.810.971.010.621.041.160.991.031.05Q216I0.611.021.050.761.111.291.201.231.09Q216K0.950.921.070.890.971.101.001.030.96Q216L0.771.021.071.041.000.991.061.131.08Q216M0.510.941.020.921.030.981.161.110.93Q216N1.131.050.991.211.001.140.961.060.96Q216P0.530.821.030.520.941.041.000.960.85Q216Q1.001.001.001.001.001.001.001.001.00Q216R1.000.901.050.650.961.090.960.940.94Q216S0.461.030.960.871.061.231.211.131.04Q216T0.770.991.110.920.991.011.071.110.89Q216W0.580.971.100.791.121.371.301.191.15变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ216Y0.731.131.070.861.161.161.271.291.22K224A0.080.050.050.050.140.060.050.050.37K224C0.110.220.050.050.180.290.080.050.30K224D0.090.050.050.050.050.260.050.050.41K224E0.080.190.050.050.360.370.370.160.39K224F0.100.200.050.050.530.180.370.200.46K224G0.090.220.050.050.630.490.490.230.61K224H0.210.660.810.071.040.980.971.020.60K224I0.100.270.900.050.540.440.470.220.45K224K1.001.001.001.001.001.001.001.001.00K224L0.110.230.690.110.530.290.420.230.36K224M0.070.050.050.050.250.180.200.050.33K224N0.100.260.680.050.600.700.530.240.52K224P0.090.050.050.050.050.070.050.050.23K224R0.521.190.730.331.131.181.141.020.93K224S0.080.050.050.050.290.180.260.050.47K224T0.070.050.050.050.280.230.310.050.30K224V0.431.110.820.551.100.981.121.070.86K224W0.100.050.050.050.140.130.180.050.29K224Y0.120.340.720.050.640.700.540.350.53D225A0.940.761.110.940.891.070.930.900.96D225C1.350.851.091.310.911.100.960.990.90D225D1.001.001.001.001.001.001.001.001.00D225E0.250.491.010.460.910.810.911.031.02D225F0.530.811.040.860.951.121.081.270.94D225G0.630.951.001.141.061.111.131.131.04D225H0.400.731.030.761.061.151.081.151.04D225I0.470.771.060.621.011.141.121.101.16D225L0.670.881.091.251.041.151.061.281.12D225M0.470.810.960.841.021.071.061.090.99D225P0.050.050.050.050.100.500.050.050.18D225Q0.640.811.031.020.921.110.980.990.90D225R0.040.040.040.040.040.040.040.040.04D225S0.720.830.981.060.911.070.900.920.92D225T0.570.941.071.091.061.091.241.261.09D225V0.711.021.011.061.041.181.111.210.97D225W0.480.821.130.731.181.131.261.301.27D225Y0.520.881.010.921.091.221.161.221.11Q226A1.101.041.031.010.940.951.041.070.98Q226C0.630.961.110.630.991.161.051.151.42Q226D0.520.891.040.451.001.110.981.061.06Q226E0.690.921.020.580.881.060.951.000.88Q226F0.531.030.920.511.151.201.181.141.17Q226H0.650.860.950.410.891.100.930.870.69Q226I0.410.891.060.451.101.541.101.040.79Q226K0.480.781.010.420.931.370.970.900.65变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ226L0.521.000.910.481.051.251.081.000.88Q226M0.470.900.990.451.101.561.100.990.85Q226N0.330.850.970.290.981.311.010.911.03Q226Q1.001.001.001.001.001.001.001.001.00Q226R0.440.860.900.531.021.361.040.891.15Q226S0.220.050.050.090.260.500.150.131.06Q226T0.590.980.960.550.991.120.980.980.99Q226V0.310.850.960.371.141.631.030.940.98Q226W0.371.081.060.541.501.611.491.511.47Q226Y0.421.230.990.511.441.551.461.541.17D236A1.300.050.050.050.050.070.050.050.07D236C0.570.050.050.050.050.060.050.050.21D236D1.001.001.001.001.001.001.001.001.00D236E0.850.050.050.050.050.050.050.050.13D236F0.190.050.050.050.050.140.050.050.26D236G1.450.050.050.050.050.080.050.050.10D236H0.040.040.040.040.040.040.040.040.04D236I0.260.050.050.050.050.140.050.050.18D236K0.040.040.040.040.040.040.040.040.04D236L0.150.050.050.120.050.230.050.240.23D236M0.860.050.050.050.050.120.050.050.10D236P1.140.050.050.050.050.070.050.050.14D236Q1.830.050.050.050.050.070.050.050.05D236R0.060.050.050.050.050.640.050.070.33D236S1.290.050.050.050.050.050.050.050.08D236T1.400.050.050.050.050.050.050.050.06D236V0.210.050.050.050.050.200.050.060.45D236W0.210.050.050.050.050.090.050.050.30D236Y0.160.050.050.050.050.190.050.060.23W237A1.000.060.050.050.060.130.050.050.14W237C0.430.050.050.050.050.060.050.050.12W237D0.580.050.050.050.050.050.050.050.16W237E0.780.050.050.050.050.050.050.050.07W237G0.460.050.050.050.050.050.050.050.30W237H1.280.050.050.050.050.050.050.050.10W237I1.890.050.050.050.050.050.050.050.05W237K1.680.050.050.050.050.050.050.050.21W237M1.310.050.050.050.050.060.050.050.05W237N0.930.050.050.050.050.050.050.050.09W237P1.870.050.050.050.050.050.050.050.05W237Q0.930.050.050.050.050.050.050.050.10W237R1.420.050.050.050.050.050.050.050.10W237S1.320.050.050.050.050.080.050.050.15W237T1.370.050.050.050.050.050.050.050.08W237V0.980.050.050.050.050.050.050.050.09W237W1.001.001.001.001.001.001.001.001.00变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCW237Y0.740.391.510.050.220.170.210.280.30N238A0.781.361.021.201.221.231.231.061.09N238C0.380.921.000.481.311.031.431.270.96N238D0.890.881.050.690.950.950.920.930.79N238E0.441.160.910.451.120.921.191.050.83N238F0.490.201.330.270.490.500.330.360.31N238G0.431.091.020.711.041.310.930.890.61N238H0.490.800.780.200.660.700.800.690.59N238I0.390.860.670.060.460.510.680.610.45N238K0.330.790.730.050.340.400.680.510.32N238L0.600.820.900.420.450.780.760.740.54N238M0.321.010.770.540.581.030.920.870.60N238P0.110.421.180.050.200.250.160.320.24N238R0.390.520.570.100.270.270.570.420.24N238S0.561.091.050.730.851.000.880.890.76N238T0.551.280.870.470.650.960.940.840.72N238V0.370.820.970.330.580.850.840.930.61N238W0.440.301.400.160.940.630.590.750.44N238Y0.530.830.870.480.750.770.770.890.59T242A0.751.210.820.091.071.001.020.950.92T242C0.621.390.940.471.291.191.361.281.17T242E0.961.280.890.461.101.101.091.021.03T242F0.501.140.840.171.100.701.030.920.84T242G0.751.180.860.271.080.891.000.890.87T242H0.941.311.061.231.190.931.271.241.17T242I0.381.100.660.050.960.050.940.810.73T242K0.481.190.810.361.040.721.040.990.82T242L0.801.390.870.051.120.461.100.991.05T242M0.521.220.850.071.170.581.140.980.94T242N0.821.160.900.931.040.860.950.910.90T242P0.090.050.050.050.090.050.050.050.39T242Q0.501.280.770.491.150.861.151.090.88T242R0.721.180.800.381.020.650.930.940.79T242S1.560.931.232.001.021.021.000.951.01T242T1.001.001.001.001.001.001.001.001.00T242V0.401.180.860.051.150.271.130.970.88T242W0.541.500.860.191.240.841.301.191.13T242Y0.521.360.820.211.230.951.221.181.03N248A1.071.020.981.101.010.800.950.950.99N248C1.221.020.991.350.950.890.950.931.03N248F0.581.010.880.791.020.720.940.990.96N248G0.250.900.840.451.210.921.181.270.99N248H0.700.880.890.760.940.680.860.820.77N248K0.510.840.870.590.960.710.870.830.78N248L0.930.980.931.100.960.750.860.840.82N248M0.610.890.830.700.850.690.770.790.75变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN248P0.130.340.920.080.720.300.700.740.58N248Q0.560.880.930.700.940.800.890.880.78N248R0.440.890.880.540.990.810.940.950.82N248S0.750.850.870.730.860.770.760.760.75N248T0.841.120.871.121.031.010.981.000.96N248V0.580.910.890.740.920.850.870.920.76N248W0.641.200.911.001.090.961.191.151.14N248Y0.510.980.870.741.000.931.031.000.89S249A1.040.050.050.320.891.110.910.970.75S249C0.930.430.900.500.840.960.880.930.81S249D0.510.290.050.670.730.800.630.640.55S249E0.240.050.050.360.170.330.090.050.26S249G0.580.050.050.570.841.050.790.850.67S249H0.690.050.050.480.880.830.790.750.53S249I0.730.700.881.040.900.880.870.780.67S249K0.430.270.050.680.880.940.740.700.40S249L0.480.280.900.970.980.850.870.870.72S249M0.660.691.050.841.070.920.840.710.61S249N0.650.450.900.730.830.800.750.610.50S249P0.270.050.050.570.170.160.100.050.20S249Q0.810.050.050.440.920.970.900.720.62S249R0.740.400.870.630.790.810.770.570.56S249S1.001.001.001.001.001.001.001.001.00S249T0.810.280.050.660.870.990.830.730.82S249V0.550.530.760.361.030.760.970.710.74S249W0.520.050.050.050.860.850.790.810.62S249Y0.630.720.970.050.950.860.860.840.57N263A0.961.031.000.960.991.070.960.961.08N263C0.611.291.060.911.181.221.581.481.54N263D0.930.961.010.931.001.220.950.971.07N263E0.920.931.080.870.940.971.040.891.01N263F0.590.971.010.710.980.681.190.990.97N263G0.761.111.000.931.010.991.051.091.16N263H0.841.031.020.940.970.820.890.911.02N263I0.500.781.000.500.910.850.980.870.91N263K0.570.831.010.590.890.800.940.920.93N263L0.680.931.060.780.960.990.980.921.06N263M0.040.040.040.040.040.040.040.040.04N263N1.001.001.001.001.001.001.001.001.00N263P0.620.771.040.530.660.420.770.730.75N263Q0.610.951.050.670.961.011.050.971.02N263R0.680.930.970.680.920.820.920.871.02N263S0.471.301.100.861.281.451.471.501.56N263T0.511.011.110.711.091.161.381.181.25N263V0.470.831.110.520.940.851.070.940.88N263W0.040.040.040.040.040.040.040.040.04变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN263Y0.600.910.950.650.880.861.070.890.90N264A0.370.911.120.060.930.960.960.710.95N264C0.711.291.040.901.101.181.251.011.26N264D1.080.861.030.710.900.890.870.710.75N264E1.030.771.100.700.840.860.820.620.70N264G0.970.881.130.680.860.780.880.660.70N264H1.100.941.020.940.900.870.910.710.71N264K0.780.851.140.680.800.790.870.670.70N264L1.160.661.150.580.790.830.750.610.65N264M0.810.741.220.610.800.850.810.710.66N264N1.001.001.001.001.001.001.001.001.00N264P0.850.810.760.050.570.440.740.510.58N264Q0.780.911.090.660.911.000.960.800.91N264R1.010.851.040.910.770.780.830.720.76N264S1.170.871.120.890.900.870.880.750.82N264T0.860.811.100.610.810.950.810.710.70N264V1.030.841.090.700.830.760.840.690.73N264W0.040.040.040.040.040.040.040.040.04N264Y1.200.761.020.730.790.750.760.700.71R265A1.180.891.010.720.921.100.870.730.85R265E0.871.031.110.610.971.051.020.830.89R265F0.980.901.130.410.890.900.950.780.82R265G1.020.961.040.160.860.830.880.730.79R265I0.840.961.150.290.991.010.960.920.92R265K0.921.150.960.950.980.991.030.900.86R265L0.921.050.920.520.921.010.920.800.82R265M0.890.921.220.610.951.030.940.840.82R265N0.901.140.950.350.960.940.910.840.91R265P0.750.771.310.360.780.800.790.700.68R265Q0.981.041.010.870.930.990.930.860.95R265R1.001.001.001.001.001.001.001.001.00R265S0.840.941.120.260.820.900.860.850.64R265T0.890.761.220.200.820.840.860.850.77R265V0.740.841.220.270.910.870.860.950.76R265W0.870.970.950.370.830.970.900.900.88R265Y1.220.910.960.500.851.000.900.910.72N276A0.280.511.020.271.121.070.951.331.44N276C0.090.340.980.171.781.691.302.731.75N276E0.270.260.900.140.620.440.390.870.45N276F0.190.390.980.211.180.870.951.681.15N276H0.300.280.850.110.490.400.270.770.72N276K0.250.350.820.050.680.520.480.981.01N276M0.240.290.860.150.700.490.481.010.50N276N1.001.001.001.001.001.001.001.001.00N276P0.200.190.730.120.410.210.130.740.28N276Q0.300.390.900.240.800.660.521.031.00变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN276R0.320.310.940.150.590.480.410.850.80N276T0.340.350.870.160.680.630.460.910.86N276V0.040.040.040.040.040.040.040.040.04N276W0.040.040.040.040.040.040.040.040.04S277A1.030.961.010.910.951.091.030.860.81S277C0.530.941.090.601.201.251.411.151.05S277D1.290.881.070.890.941.030.990.840.76S277E0.860.841.000.860.871.320.900.790.76S277F0.560.821.040.601.001.461.080.930.73S277G0.740.831.050.700.901.470.890.860.73S277H0.620.750.990.610.901.270.920.870.80S277I0.540.771.000.540.921.161.020.900.82S277K0.740.770.960.510.790.950.840.610.68S277L0.560.830.940.700.920.910.980.560.76S277M0.540.901.020.630.981.131.080.650.82S277N0.700.781.110.650.891.000.890.800.71S277P0.260.590.920.180.941.211.050.720.93S277Q0.690.871.020.730.951.040.990.880.87S277R0.730.891.030.680.920.971.060.710.74S277S1.001.001.001.001.001.001.001.001.00S277T0.490.760.930.050.890.960.960.920.91S277V0.620.640.490.050.410.550.460.400.47S277W0.061.471.010.606.076.639.149.253.36S277Y0.560.970.940.791.081.181.241.220.93N278A1.010.971.000.791.001.411.080.930.90N278C0.901.001.040.721.051.251.191.020.98N278D1.590.881.031.170.971.130.940.880.80N278F0.501.091.000.691.252.261.571.321.33N278G0.800.921.010.791.041.141.080.920.88N278H0.150.410.730.050.610.121.210.640.59N278I0.590.840.980.570.941.501.120.950.88N278L0.670.890.970.740.910.991.060.780.87N278M0.720.930.920.650.911.211.050.900.76N278N1.001.001.001.001.001.001.001.001.00N278P0.220.340.900.050.700.440.850.690.52N278Q0.740.791.050.670.881.180.990.870.78N278R0.940.891.050.820.921.121.050.980.78N278S0.600.900.990.660.971.101.090.860.89N278T0.790.980.950.650.921.141.060.980.88N278V0.800.981.020.811.041.241.131.070.94N278W0.700.930.950.550.911.200.871.081.00N278Y0.600.820.990.740.981.291.100.920.95Q279A0.050.050.050.050.050.050.050.050.05Q279C0.631.361.020.811.311.351.381.471.50Q279D0.870.971.030.850.991.130.961.021.00Q279E0.680.780.950.600.811.040.780.810.82变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ279G0.731.000.940.840.951.020.950.960.92Q279H0.520.890.930.580.911.110.850.950.75Q279I0.390.760.990.370.861.250.870.970.83Q279K0.280.790.890.370.881.320.991.090.70Q279L0.770.740.990.650.780.790.670.720.60Q279M0.730.740.970.590.730.940.630.690.70Q279N0.270.410.950.200.581.080.550.660.51Q279P0.120.180.960.050.350.470.510.460.32Q279S0.560.850.940.560.821.200.860.920.83Q279T0.610.870.930.640.851.060.820.860.80Q279V0.490.901.000.580.951.201.011.010.83Q279Y0.450.900.980.550.951.101.061.090.86T282C0.921.080.991.141.040.951.051.141.15T282D1.090.941.011.040.820.960.921.020.99T282E0.720.960.970.840.920.810.870.940.92T282F0.790.990.910.750.830.840.850.930.79T282G0.990.990.981.050.970.970.870.970.87T282H0.100.131.330.050.050.050.050.050.65T282I0.870.830.970.820.810.840.790.840.73T282K0.910.861.030.970.871.020.830.930.79T282L0.610.940.920.780.870.880.840.931.03T282M0.751.000.930.850.890.920.900.990.96T282N1.190.960.971.190.860.940.820.870.79T282P0.580.910.980.780.940.840.941.081.03T282Q0.880.870.980.840.820.790.800.850.78T282R1.240.960.971.100.890.960.910.960.84T282S0.470.980.910.710.980.940.971.190.99T282T1.001.001.001.001.001.001.001.001.00T282V1.100.990.971.100.900.950.900.970.87T282Y0.700.820.970.740.850.870.890.930.85R284A0.190.130.680.050.350.640.140.920.52R284C0.390.160.050.050.260.200.140.510.30R284E0.310.150.050.080.270.160.110.570.76R284F0.350.130.050.050.230.210.110.490.28R284G0.040.040.040.040.040.040.040.040.04R284H0.140.150.050.060.530.360.231.180.74R284I0.360.150.050.080.230.200.100.500.50R284K0.040.040.040.040.040.040.040.040.04R284M0.100.200.050.120.830.730.451.841.02R284N0.360.121.000.050.290.220.170.540.41R284P0.290.140.920.050.260.150.100.540.43R284R1.001.001.001.001.001.001.001.001.00R284S0.240.140.050.150.290.150.110.680.59R284T0.360.190.890.140.330.270.200.590.86R284V0.310.640.250.050.470.430.270.730.43R284W0.040.040.040.040.040.040.040.040.04变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR284Y0.040.040.040.040.040.040.040.040.04D287C0.961.171.150.761.051.151.181.181.12D287D1.001.001.001.001.001.001.001.001.00D287E0.721.070.940.610.990.990.990.980.91D287F0.310.291.330.070.330.410.270.190.66D287G0.710.980.920.600.971.160.960.940.93D287H0.220.781.320.360.851.080.800.740.73D287I0.310.631.540.180.621.030.610.570.55D287K0.350.921.130.210.941.250.930.941.04D287L0.280.471.260.310.480.590.440.370.47D287M0.300.851.150.220.831.150.830.820.63D287N0.651.040.890.620.911.130.920.930.90D287P0.140.050.050.080.390.440.280.170.27D287R0.340.200.050.050.230.330.160.140.47D287S0.461.090.910.521.001.351.061.030.97D287V0.360.561.160.120.660.800.590.540.93D287W0.230.521.560.080.610.750.520.470.62D287Y0.290.322.510.050.370.440.290.220.08R291A0.080.300.910.050.290.830.350.410.30R291C0.080.050.050.050.050.480.050.110.23R291D0.070.050.050.050.050.340.050.050.26R291E0.070.050.050.050.050.500.050.050.21R291G0.060.050.050.050.050.870.050.180.25R291H0.050.050.050.050.050.050.050.050.05R291I0.050.050.050.050.050.250.050.050.31R291K0.080.270.050.050.200.980.310.470.37R291L0.050.050.050.050.050.050.050.050.05R291M0.050.050.050.050.050.050.050.050.05R291N0.060.050.050.050.050.330.050.090.31R291Q0.060.050.050.050.050.320.050.090.29R291S0.070.310.980.050.280.750.280.390.22R291T0.050.050.050.050.050.400.050.180.43R291V0.050.050.050.050.050.050.050.050.05R291W0.060.050.050.050.050.680.050.050.09R291Y0.060.050.050.050.050.320.050.130.22Q301A0.671.200.880.060.931.031.000.840.93Q301E0.420.841.090.300.891.020.910.760.78Q301F0.220.750.950.050.610.680.640.530.34Q301G0.120.990.970.051.011.221.250.850.47Q301H0.200.960.960.050.820.900.860.750.47Q301K0.081.080.910.081.141.321.331.130.48Q301L0.331.091.050.051.010.980.950.950.79Q301N0.221.220.950.140.980.980.980.980.60Q301Q1.001.001.001.001.001.001.001.001.00Q301R0.221.190.850.050.881.090.880.970.57Q301S0.351.120.960.050.870.760.900.880.78变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ301T0.211.031.050.050.890.830.950.900.58Q301V0.261.121.020.050.840.850.880.950.77Q301Y0.240.950.940.050.780.670.860.900.59D302A0.381.060.930.551.151.481.171.261.20D302C0.231.010.990.361.341.661.361.491.32D302D1.001.001.001.001.001.001.001.001.00D302E0.350.830.890.511.031.300.971.081.05D302F0.330.880.810.481.031.331.011.151.41D302G0.390.980.860.491.091.351.121.281.22D302I0.050.050.050.050.050.050.050.050.05D302K0.290.740.940.381.201.190.880.980.93D302L0.290.720.910.090.900.860.831.110.81D302M0.270.770.900.271.021.260.951.030.79D302N0.510.870.860.710.931.050.941.010.65D302P0.240.750.880.311.001.050.850.950.67D302S0.480.940.860.680.961.180.930.990.87D302T0.410.830.930.391.031.020.900.930.64D302V0.230.300.820.190.550.840.340.290.16D302W0.591.020.990.580.871.020.850.920.85D302Y0.500.930.850.480.971.370.830.940.83Q303A1.021.080.851.401.010.921.010.820.90Q303C0.561.280.910.951.311.091.511.101.28Q303D0.950.980.931.220.981.011.070.860.97Q303E1.061.040.961.370.980.910.940.790.89Q303F0.880.910.911.190.920.950.860.710.85Q303G0.800.990.921.100.980.900.950.800.84Q303H0.741.060.940.930.980.920.990.850.86Q303I0.821.060.961.360.950.890.950.860.83Q303K0.741.090.881.311.040.930.990.880.94Q303L0.791.110.901.410.980.900.920.880.93Q303M0.871.030.911.341.010.950.870.800.87Q303N1.231.050.971.880.990.890.810.880.84Q303P0.370.970.850.661.050.851.000.870.91Q303Q1.001.001.001.001.001.001.001.001.00Q303R0.941.050.911.310.911.010.930.860.80Q303S0.921.070.911.430.930.920.900.900.80Q303T0.871.020.941.510.940.980.900.880.91Q303V0.851.110.911.170.860.840.950.940.86Q303W0.710.950.920.850.941.040.990.890.81Q303Y0.691.040.901.220.940.880.940.920.93Y306A1.280.840.880.440.810.830.770.770.86Y306C0.651.011.760.240.890.870.850.891.05Y306D0.110.050.050.050.360.440.300.220.81Y306E1.060.960.840.350.850.790.840.860.93Y306F1.030.950.901.050.920.790.860.880.91Y306G0.851.130.830.621.050.930.940.941.00变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCY306I1.001.080.871.020.880.970.840.860.90Y306K0.441.380.990.121.120.991.051.130.94Y306L1.191.120.891.210.920.830.940.940.78Y306M0.611.230.780.781.081.040.981.081.05Y306N0.571.200.860.480.980.940.930.921.00Y306P0.851.050.790.560.870.860.860.870.84Y306Q0.171.640.870.241.520.101.361.391.28Y306R0.441.440.051.031.191.391.201.200.98Y306S0.891.010.880.390.880.910.840.870.84Y306T0.791.030.810.520.860.910.830.850.94Y306V0.421.280.830.901.191.040.950.981.17Y306W1.330.950.921.400.880.890.840.870.88Y306Y1.001.001.001.001.001.001.001.001.00S312A1.090.871.030.900.921.000.840.870.68S312C0.971.311.161.041.241.261.381.351.50S312D1.220.891.080.881.051.100.990.671.02S312F0.040.040.040.040.040.040.040.040.04S312G1.020.891.080.840.961.060.910.880.89S312I1.020.871.010.560.880.920.850.870.71S312K0.771.001.080.730.980.940.971.000.75S312L0.730.881.030.560.760.810.820.830.55S312M0.640.981.020.600.880.970.900.930.69S312N1.530.851.021.330.931.040.880.710.70S312P0.260.850.870.050.510.150.670.970.20S312Q0.940.961.110.900.921.030.960.990.67S312R1.040.811.040.680.851.080.830.910.70S312S1.001.001.001.001.001.001.001.001.00S312T0.861.080.980.940.921.020.960.970.72S312V0.851.070.990.680.981.190.970.990.86S312W0.136.541.020.883.600.065.395.542.47S312Y0.511.571.090.781.221.291.411.461.21R313A0.780.860.950.470.921.010.880.760.93R313C0.670.830.970.350.921.050.970.780.91R313D1.010.940.970.050.911.060.890.780.90R313E1.050.951.000.460.880.850.690.710.83R313F0.510.680.990.260.800.930.750.660.71R313G0.830.921.000.520.861.000.840.730.79R313H0.930.900.910.730.840.880.820.720.76R313I0.850.840.980.620.840.990.800.670.69R313K0.670.880.920.750.951.010.950.820.75R313L0.970.981.000.830.860.970.770.740.78R313M0.890.870.990.630.850.890.750.700.68R313N0.840.860.970.570.831.080.780.710.70R313P0.230.490.870.050.760.420.800.720.55R313R1.001.001.001.001.001.001.001.001.00R313S1.000.891.040.540.830.930.730.770.72变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR313V0.630.801.020.500.850.880.810.820.75R313W0.700.831.010.340.860.920.850.830.73Q316A1.470.971.140.580.860.990.940.990.92Q316C0.891.011.060.330.950.971.031.151.04Q316D1.030.931.020.270.880.860.951.010.98Q316E0.870.931.050.750.890.920.880.890.79Q316F1.050.911.040.240.910.930.870.950.90Q316G0.660.781.060.240.920.990.711.040.82Q316H0.270.421.080.060.900.880.821.280.69Q316I0.740.911.010.050.950.910.921.050.92Q316K0.530.811.020.201.051.140.991.200.93Q316L0.840.920.970.150.871.030.770.830.79Q316M0.230.420.890.250.971.140.760.970.63Q316N0.490.661.070.060.920.940.800.950.73Q316P0.471.091.010.051.180.781.141.411.07Q316Q1.001.001.001.001.001.001.001.001.00Q316R0.370.611.040.161.031.020.901.050.79Q316S0.380.821.010.131.170.851.091.391.01Q316T0.501.261.010.521.401.581.541.541.31Q316V0.720.941.040.200.970.920.920.920.89Q316W0.460.541.060.140.840.910.680.850.62Q316Y0.781.001.090.141.011.121.111.111.05K320A1.680.730.970.770.770.890.820.700.69K320C0.861.271.001.351.231.211.411.661.23K320E0.940.931.020.300.810.910.961.210.95K320G0.980.821.050.800.841.050.870.780.93K320H1.550.800.981.480.780.930.871.290.66K320K1.001.001.001.001.001.001.001.001.00K320L0.520.740.990.400.570.860.661.880.77K320M0.870.841.020.800.750.980.791.310.91K320N1.620.951.051.780.841.061.010.891.02K320P0.820.930.940.650.841.020.901.900.87K320Q1.000.770.930.800.730.950.771.840.63K320R0.980.860.920.490.780.990.902.990.80K320S1.300.980.971.570.901.011.061.780.78K320T0.810.921.010.600.890.911.041.890.90K320V0.040.040.040.040.040.040.040.040.04K320W0.850.880.950.710.590.790.920.640.90K320Y1.340.880.971.650.821.091.210.940.91R324C0.650.941.090.570.981.261.091.131.10R324D0.610.801.000.260.901.130.940.980.90R324E0.560.960.950.630.871.120.950.980.77R324F0.680.940.930.680.891.190.940.960.90R324H0.560.870.970.600.841.190.870.920.89R324I0.260.490.920.210.721.330.850.780.64R324K0.380.680.990.400.751.230.950.940.81变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR324L0.570.870.920.570.831.180.880.890.77R324M0.550.880.950.540.871.060.960.980.83R324N0.250.050.050.050.170.130.050.050.15R324P0.070.050.050.050.050.110.080.090.36R324Q0.460.770.910.410.791.060.910.900.75R324S0.140.050.050.050.050.160.050.060.11R324V0.280.530.920.220.731.250.890.890.63R324W0.360.670.970.350.761.370.910.920.79R324Y0.770.980.990.840.951.051.031.040.93R328C0.391.001.130.360.971.050.941.090.54R328D0.140.050.050.050.080.060.050.490.05R328E0.351.121.010.440.890.820.931.050.67R328F0.060.050.050.050.090.530.051.100.15R328G0.361.111.000.450.900.970.891.160.42R328I0.140.621.040.050.480.590.541.030.05R328K0.501.090.940.510.851.170.961.040.70R328L0.131.121.030.130.700.730.891.400.32R328M0.131.330.990.160.860.751.141.600.40R328N0.230.890.880.120.520.770.730.990.32R328P0.050.050.050.050.050.050.050.050.05R328Q0.391.061.040.430.820.990.971.070.57R328R1.001.001.001.001.001.001.001.001.00R328S0.124.020.970.052.591.673.383.431.34R328T0.831.060.940.910.930.880.980.970.79R328V0.411.130.910.300.990.970.991.090.61R328W0.120.050.050.050.060.210.050.510.39R328Y0.060.050.050.050.200.440.092.090.16D329A0.931.241.001.341.051.081.101.001.16D329D1.001.001.001.001.001.001.001.001.00D329E0.691.170.970.910.940.850.920.910.72D329F0.120.601.180.180.400.440.370.850.53D329G0.671.200.990.800.940.981.000.940.73D329H0.241.180.950.260.771.050.930.970.41D329I0.070.050.050.050.050.050.050.730.05D329K0.090.480.050.050.220.280.270.900.34D329L0.050.050.050.050.050.050.050.050.05D329M0.211.321.000.290.690.870.921.130.38D329N0.601.340.950.850.770.891.040.990.72D329P0.050.050.050.050.050.050.050.050.05D329Q0.451.171.000.590.740.980.930.950.62D329R0.050.050.050.050.050.050.050.050.05D329S0.661.390.960.970.971.071.011.001.02D329T0.151.261.000.240.850.940.901.310.46D329V0.050.050.050.050.050.050.050.050.05D329W0.050.050.050.050.050.050.050.050.05D329Y0.220.951.030.200.761.000.721.060.39变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCL334A0.120.330.900.191.221.841.321.140.70L334C0.490.670.970.470.991.401.040.890.89L334E0.090.070.050.050.050.100.050.050.14L334F0.130.310.870.150.931.371.060.950.97L334G0.040.040.040.040.040.040.040.040.04L334H0.050.050.050.050.050.050.050.050.05L334I0.200.380.980.230.910.990.980.890.44L334K0.090.060.050.080.070.430.050.050.16L334L1.001.001.001.001.001.001.001.001.00L334M0.440.721.000.530.931.201.040.950.66L334N0.120.080.050.090.050.180.060.060.10L334P0.100.130.050.110.170.370.240.310.22L334R0.040.040.040.040.040.040.040.040.04L334S0.110.070.050.100.120.310.140.150.07L334T0.160.350.930.240.961.281.091.080.70L334V0.530.701.040.671.011.111.080.980.88L334W0.900.920.930.840.861.040.940.960.86L334Y0.140.090.050.090.150.280.150.160.18K335A0.991.051.001.591.011.100.880.190.98K335D0.471.380.980.771.091.061.030.491.05K335F0.591.261.000.870.991.000.940.630.83K335G0.120.751.160.150.560.620.470.940.37K335H0.771.160.981.001.031.200.960.830.80K335I0.411.150.950.540.870.910.850.890.79K335K1.001.001.001.001.001.001.001.001.00K335L1.031.090.951.440.951.060.890.800.89K335M0.681.280.930.910.990.910.920.890.84K335N0.201.250.900.310.790.870.851.070.59K335P0.040.040.040.040.040.040.040.040.04K335R0.731.300.981.090.961.031.000.960.87K335S1.031.001.011.100.931.120.860.810.88K335T1.141.141.041.600.970.880.940.870.85K335V0.501.531.000.871.081.101.071.070.90K335W0.691.301.021.021.041.161.050.820.96N336A0.480.720.990.641.041.110.950.771.12N336C0.710.850.970.790.951.040.940.771.02N336F0.140.160.830.150.420.640.420.330.51N336G0.210.470.940.361.031.210.950.790.66N336H0.460.741.000.590.951.190.930.771.01N336I0.040.040.040.040.040.040.040.040.04N336K0.120.170.760.110.400.810.450.340.41N336L0.240.510.970.310.941.250.940.790.82N336M0.300.550.980.450.921.210.690.760.78N336N1.001.001.001.001.001.001.001.001.00N336P0.130.070.050.050.060.070.050.050.33N336Q0.210.430.960.300.891.140.930.820.77变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN336R0.100.300.860.190.971.451.030.920.40N336S0.560.721.040.590.930.900.860.780.88N336T0.270.511.000.370.901.170.900.820.74N336V0.120.320.970.170.961.220.920.920.63N336W0.040.040.040.040.040.040.040.040.04N336Y0.320.551.000.420.911.090.870.810.84D337A0.831.081.030.900.531.070.911.011.13D337C0.711.171.030.872.280.830.971.111.18D337E0.571.070.970.660.210.520.930.930.90D337F0.480.990.980.510.050.460.830.890.94D337G0.921.160.950.930.910.970.930.980.81D337H0.361.090.960.450.570.370.920.930.98D337K0.531.090.950.541.080.560.810.881.07D337L0.351.070.890.400.300.330.870.940.93D337M0.371.100.990.470.610.380.960.921.03D337N0.681.140.950.750.840.660.940.980.97D337P0.240.670.960.200.530.150.530.650.66D337Q0.560.990.990.620.440.660.880.860.94D337R0.571.020.960.670.890.610.870.970.97D337S0.561.040.990.650.050.590.920.880.81D337T0.461.341.000.670.770.451.181.211.15D337V0.841.130.930.840.400.951.011.030.98D337W0.420.961.020.461.390.420.910.900.90D337Y0.541.030.960.560.570.460.880.880.97A338A1.001.001.001.001.001.001.001.001.00A338C0.921.210.971.091.031.071.191.241.55A338D1.710.931.001.690.951.241.031.031.18A338E1.050.970.980.950.831.180.940.961.27A338F0.811.001.040.850.871.140.951.021.05A338G1.041.071.001.250.951.121.031.051.24A338H0.691.020.990.650.841.230.900.970.92A338I0.891.170.991.030.921.221.061.100.98A338K0.840.960.990.860.811.270.900.941.06A338L0.851.110.960.910.871.071.001.001.12A338M0.881.110.910.740.840.940.971.001.11A338N1.041.060.931.060.811.140.910.930.97A338P0.561.161.010.570.911.290.981.011.03A338Q0.810.961.030.730.871.080.920.950.92A338R1.091.001.000.950.861.080.930.950.98A338S0.040.040.040.040.040.040.040.040.04A338T0.040.040.040.040.040.040.040.040.04A338V0.861.121.031.160.981.271.071.121.07A338W0.661.901.001.011.201.291.621.631.61A338Y0.960.921.040.790.841.080.890.921.04N339A0.310.630.970.320.750.920.730.630.76N339C0.260.670.980.270.860.650.850.820.90变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN339D0.220.591.090.220.780.710.750.660.66N339E0.160.720.950.252.340.911.030.900.89N339F0.050.050.050.050.050.050.050.050.05N339G0.150.481.000.151.620.670.670.520.63N339H0.210.560.990.241.200.830.700.640.67N339I0.160.361.140.150.910.410.490.450.68N339K0.140.420.050.161.130.680.660.450.14N339L0.230.521.210.231.040.660.600.590.72N339N1.001.001.001.001.001.001.001.001.00N339P0.200.381.250.130.530.580.510.410.39N339Q0.140.391.130.140.640.560.560.500.42N339R0.180.461.880.160.650.810.640.460.75N339S0.260.650.890.250.800.730.710.680.63N339T0.200.480.950.190.710.670.640.560.49N339V0.190.411.400.140.570.530.580.430.61N339W0.290.460.970.180.580.620.480.440.73N339Y0.210.591.010.220.750.680.710.650.58K344D0.851.041.020.881.031.091.051.011.07K344E0.581.090.940.750.990.980.990.860.98K344F0.511.121.000.651.091.141.071.011.11K344G0.601.040.930.680.951.000.940.871.08K344I0.421.090.930.591.050.971.041.030.95K344K1.001.001.001.001.001.001.001.001.00K344L0.471.210.920.761.041.121.010.891.08K344M0.521.200.980.781.061.040.990.980.80K344N0.561.110.930.740.951.010.950.910.84K344P0.260.930.940.361.071.291.060.890.78K344Q0.370.970.930.510.980.951.000.921.01K344R0.691.060.940.790.970.990.920.941.02K344S0.801.270.951.211.051.000.990.980.98K344T0.451.090.980.671.031.170.980.950.81K344V0.481.320.960.721.081.061.131.011.06K344W0.050.050.050.050.050.050.050.050.05K345A0.941.031.060.961.021.011.071.071.15K345D0.510.861.090.491.011.201.090.940.95K345E0.431.381.010.801.301.211.441.341.26K345F0.122.990.990.893.983.645.335.282.82K345G0.230.911.030.331.181.411.261.120.76K345H0.681.061.020.760.991.131.060.940.94K345I0.050.050.050.050.050.050.050.050.05K345K1.001.001.001.001.001.001.001.001.00K345L0.050.050.050.050.050.050.050.050.05K345M0.050.050.050.050.050.050.050.050.05K345N0.250.780.990.340.941.280.920.800.41K345P0.481.000.980.620.981.161.000.910.93K345Q0.661.011.010.810.980.931.001.010.73变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK345R0.701.010.960.720.951.270.780.970.98K345S0.411.100.890.581.021.221.031.081.09K345T0.511.110.910.710.990.971.051.010.98K345V0.371.040.940.541.081.421.131.150.91K345W0.440.930.990.440.971.210.991.010.94K345Y0.111.981.020.583.273.123.914.231.59A347D1.001.031.051.110.961.041.010.960.98A347F0.991.101.031.120.961.050.970.920.96A347H0.810.940.990.850.911.140.920.840.95A347I0.660.931.040.740.921.090.950.900.79A347K0.680.881.070.770.911.090.970.880.81A347L0.610.911.100.760.991.140.930.930.94A347M0.770.981.100.940.981.030.950.870.84A347P0.721.021.020.960.991.100.990.860.96A347Q0.610.881.080.090.971.090.930.860.75A347R0.730.991.000.980.961.180.960.920.73A347S0.650.941.030.731.001.100.940.870.84A347T0.210.400.910.130.600.830.240.370.50A347V0.200.050.050.050.320.340.100.160.60A347Y1.171.341.071.641.071.101.141.071.10H361A0.890.921.020.051.011.081.050.990.97H361C0.830.931.010.051.020.451.081.030.93H361D0.940.881.000.050.960.610.900.741.01H361E0.560.881.030.050.890.731.010.770.78H361F0.040.040.040.040.040.040.040.040.04H361G0.921.111.060.051.101.141.151.201.08H361H1.001.001.001.001.001.001.001.001.00H361I0.150.120.920.050.050.120.130.050.21H361K0.160.471.030.050.150.320.890.150.39H361L0.170.631.000.050.770.071.210.730.48H361M0.400.821.040.050.950.181.111.010.68H361N0.980.950.970.050.921.030.990.990.92H361P0.430.661.090.050.830.250.970.850.58H361R0.110.420.970.050.210.410.950.220.36H361S0.930.841.120.050.890.950.940.960.82H361T0.280.760.950.050.720.171.020.730.40H361V0.180.110.050.050.050.200.110.050.19H361Y0.190.131.060.050.050.150.190.050.07R363A1.220.921.100.620.990.960.990.991.11R363C0.451.061.010.161.191.311.221.251.29R363E0.380.901.060.860.920.051.031.041.08R363F0.040.040.040.040.040.040.040.040.04R363G1.190.971.050.051.030.930.971.011.19R363K1.051.061.010.801.051.021.111.091.14R363L0.811.001.031.060.990.911.030.980.80R363M1.100.980.980.980.961.031.030.981.16变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR363N0.220.511.060.100.600.930.410.720.45R363P0.050.050.050.050.050.050.050.050.05R363Q0.801.011.010.281.011.241.051.091.02R363R1.001.001.001.001.001.001.001.001.00R363S0.210.811.080.061.021.010.831.190.78R363T0.681.011.060.301.001.020.831.071.07R363V1.750.771.150.090.971.060.910.931.15R363W0.141.621.040.052.221.972.663.181.37R363Y0.132.650.930.053.320.104.394.921.47N369A0.110.381.100.240.620.800.300.720.27N369C0.301.881.051.091.771.282.222.091.45N369D0.961.061.080.321.081.201.111.081.00N369E0.741.311.051.831.110.861.201.071.06N369F0.501.330.981.331.090.741.161.100.95N369I0.460.901.331.030.850.690.830.760.54N369K0.050.050.050.050.050.050.050.050.05N369L0.631.151.081.540.910.840.970.940.77N369M0.701.281.011.870.970.851.140.960.84N369N1.001.001.001.001.001.001.001.001.00N369R0.940.881.061.560.760.800.820.690.72N369S0.471.200.960.930.971.071.150.930.94N369T0.761.141.121.440.940.961.120.931.08N369V0.911.121.051.950.890.681.010.910.83N369W0.262.121.011.381.820.982.222.131.08N369Y0.341.541.061.211.340.961.481.510.91D370D1.001.001.001.001.001.001.001.001.00D370E1.141.071.080.050.410.430.470.500.70D370F0.721.481.110.050.420.170.570.370.66D370G0.541.050.870.050.590.310.640.520.80D370I0.390.810.820.050.050.050.110.070.38D370K0.590.280.570.050.050.080.050.050.27D370L0.580.640.840.050.050.090.050.050.46D370M0.660.770.920.050.140.170.250.110.43D370N0.990.990.830.050.300.430.400.270.48D370P0.180.380.980.050.050.180.130.050.55D370Q1.080.780.780.050.230.370.320.210.48D370R0.360.190.640.050.050.090.050.050.21D370S0.751.080.820.050.490.550.560.450.70D370T0.360.940.830.050.130.060.290.130.66D370V0.620.990.850.050.120.050.200.120.42D370W0.751.141.450.050.250.070.320.210.52D370Y0.751.051.000.050.330.180.430.330.57K371A1.131.011.080.051.040.940.930.951.17K371C0.470.920.970.050.900.940.890.930.98K371D0.940.951.080.050.850.730.890.850.86K371F0.381.071.040.050.750.050.820.800.87变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK371G0.651.041.080.051.011.050.670.860.92K371H1.060.970.950.300.850.860.850.771.00K371K1.001.001.001.001.001.001.001.001.00K371L0.881.131.080.051.040.900.950.881.14K371N0.411.060.910.050.810.560.840.730.98K371P0.370.840.720.050.140.070.210.100.36K371Q0.491.030.980.050.780.460.810.760.60K371R0.581.180.850.050.890.870.980.880.85K371S0.721.151.060.050.970.480.920.890.98K371T0.191.861.030.051.370.101.741.581.57K371V0.271.030.940.050.740.120.780.790.61K371W0.250.621.040.050.360.080.420.150.47K371Y0.320.580.980.050.270.060.310.160.32G372A0.651.441.101.191.211.191.471.241.21G372C0.511.031.050.110.980.981.080.890.77G372D0.660.971.080.050.930.941.070.920.86G372E0.591.100.910.050.700.480.860.790.73G372F0.140.690.610.050.420.050.740.480.38G372G1.001.001.001.001.001.001.001.001.00G372H0.200.880.680.050.550.120.940.730.48G372I0.100.140.050.060.050.280.050.050.26G372K0.090.670.960.071.030.651.661.470.73G372L0.141.000.750.050.390.080.630.520.43G372M0.281.140.850.050.630.411.131.060.73G372N0.350.961.040.120.610.531.020.980.71G372Q0.390.900.990.060.620.710.980.960.73G372R0.420.930.950.050.530.540.840.760.66G372S0.410.811.050.050.730.950.870.780.71G372T0.461.310.820.050.540.540.930.770.74G372V0.181.190.740.110.540.121.220.960.57G372W0.060.740.840.051.350.332.021.730.57G372Y0.170.970.740.050.840.201.231.100.54D374A1.140.761.080.050.890.820.820.740.79D374C0.701.280.970.151.101.131.131.201.03D374F0.161.110.950.090.901.091.211.190.78D374G0.211.230.930.050.980.351.221.330.93D374I0.080.811.100.050.980.760.961.520.62D374K0.050.050.050.050.050.050.050.050.05D374L0.321.370.900.051.120.801.361.371.09D374M0.191.370.930.051.160.281.201.621.03D374N0.591.110.900.900.861.050.830.930.84D374P0.110.050.050.050.050.450.050.050.13D374Q0.201.160.920.051.030.441.411.480.68D374R0.060.361.020.050.090.780.210.370.43D374S0.501.240.930.051.031.111.041.201.02D374T0.210.880.920.050.760.240.821.000.65变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCD374V0.211.030.930.051.210.561.111.300.78D374W0.070.391.050.050.310.590.360.670.44D374Y0.260.980.930.220.750.900.991.000.61D375A0.781.020.870.050.980.900.980.880.95D375C0.661.110.890.051.110.991.211.321.04D375D1.001.001.001.001.001.001.001.001.00D375E0.651.160.980.051.090.991.221.140.89D375F0.560.850.890.050.910.550.920.900.81D375G0.540.850.930.050.910.610.930.960.85D375H0.560.890.880.050.930.860.951.000.87D375I0.731.010.950.050.900.820.931.050.85D375K0.470.810.870.050.890.680.910.980.85D375L0.540.890.930.050.920.740.870.930.76D375M0.570.890.930.050.890.770.920.970.76D375N0.650.940.930.050.920.790.920.920.85D375P0.090.240.860.050.070.260.050.260.35D375Q0.310.620.900.050.880.960.860.910.56D375R0.450.800.850.050.880.760.851.130.76D375S0.580.900.910.050.880.920.860.900.69D375T0.550.910.960.050.960.800.950.970.80D375V0.601.070.920.050.970.681.031.060.83D375W0.410.930.910.051.080.661.091.200.96D375Y0.620.900.880.050.890.560.870.930.73M380A0.180.050.050.050.050.070.050.050.09M380E0.790.981.170.050.890.840.950.850.84M380F0.520.971.070.050.970.770.950.990.94M380G0.140.471.050.050.790.220.840.950.62M380I0.501.160.940.051.000.690.971.120.98M380K0.060.050.050.050.150.360.130.400.35M380L0.561.181.050.051.000.770.910.980.83M380M1.001.001.001.001.001.001.001.001.00M380N0.120.750.950.051.170.131.151.250.53M380Q0.591.151.020.050.920.810.920.991.00M380R0.050.050.050.050.050.050.050.050.05M380S0.601.110.990.050.970.600.900.940.85M380T0.591.041.010.051.000.930.901.031.06M380V0.591.121.130.051.060.690.931.001.33M380W0.040.040.040.040.040.040.040.040.04M380Y0.120.551.030.050.790.400.961.340.42G381A0.210.050.050.050.050.080.050.050.16G381C0.180.050.050.050.050.070.050.050.07G381D0.110.050.050.050.050.070.050.060.16G381E0.180.050.050.050.050.110.050.050.07G381F0.170.050.050.050.050.050.050.080.08G381G1.001.001.001.001.001.001.001.001.00G381H0.060.050.050.050.050.190.050.051.18变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCG381I0.050.050.050.050.050.240.050.060.13G381K0.070.050.050.050.050.170.050.050.22G381L0.050.050.050.050.050.050.050.050.05G381N0.130.050.050.060.050.100.050.050.05G381P0.050.050.050.050.050.050.050.050.05G381Q0.060.050.050.050.050.070.050.050.05G381R0.050.050.050.050.050.050.050.050.05G381S0.050.050.050.050.050.050.050.050.05G381T0.050.050.050.050.050.260.050.050.18G381V0.060.050.050.050.050.420.050.050.32G381W0.040.040.040.040.040.040.040.040.04G381Y0.040.040.040.040.040.040.040.040.04W382A0.040.040.040.040.040.040.040.040.04W382C0.040.040.040.040.040.040.040.040.04W382D0.040.040.040.040.040.040.040.040.04W382E0.040.040.040.040.040.040.040.040.04W382F0.250.740.760.061.030.450.961.131.68W382G0.040.040.040.040.040.040.040.040.04W382H0.180.160.050.230.290.260.080.050.31W382I0.040.040.040.040.040.040.040.040.04W382K0.170.220.050.190.240.200.110.090.44W382L0.040.040.040.040.040.040.040.040.04W382M0.180.170.050.250.250.350.140.080.83W382N0.180.251.110.170.580.920.370.340.77W382P0.190.380.050.150.280.300.090.060.72W382R0.040.040.040.040.040.040.040.040.04W382S0.260.190.050.240.240.230.140.090.63W382T0.040.040.040.040.040.040.040.040.04W382V0.280.150.050.250.180.160.090.050.58W382Y0.490.911.020.141.000.260.950.990.87Y396A1.081.060.920.481.010.971.000.991.00Y396C0.861.070.980.411.010.971.021.060.92Y396D0.781.060.900.330.991.180.991.050.94Y396E0.791.010.910.391.001.010.991.040.86Y396F0.671.080.970.881.031.100.971.120.91Y396G0.741.060.940.121.001.001.061.130.92Y396H0.850.920.940.800.961.020.971.090.85Y396I0.761.020.940.750.920.950.951.030.84Y396K0.621.120.920.251.011.061.041.170.91Y396L0.670.980.930.660.931.060.961.070.75Y396M0.510.940.940.560.901.040.921.080.64Y396N0.560.940.950.480.991.120.981.120.82Y396P0.150.050.050.050.050.100.050.100.05Y396Q0.600.970.950.220.941.041.001.080.76Y396R0.611.000.960.060.971.060.971.090.67Y396S0.861.000.900.380.920.930.951.040.79变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCY396T0.771.021.000.100.940.930.920.990.72Y396V0.540.930.960.381.040.970.931.060.71Y396W0.820.930.920.810.960.990.911.040.80Y396Y1.001.001.001.001.001.001.001.001.00D397A0.670.970.940.110.970.991.051.030.99D397C0.611.291.000.241.221.321.501.481.30D397D1.001.001.001.001.001.001.001.001.00D397E0.701.210.890.441.121.091.151.110.93D397F0.180.050.050.050.050.051.130.050.25D397H0.501.230.770.091.121.111.111.100.93D397I0.601.230.740.051.071.231.071.010.90D397K0.871.130.880.051.051.111.091.051.00D397L0.561.080.790.051.050.981.050.940.92D397M0.571.120.840.071.071.071.051.060.91D397N1.131.151.060.771.051.011.121.311.10D397P0.731.270.720.330.931.061.051.010.87D397Q0.641.100.950.291.091.221.171.110.98D397R0.691.230.900.141.131.261.261.201.19D397S0.611.170.830.231.051.021.151.190.90D397T0.831.190.940.171.071.131.121.201.05D397V0.611.320.950.241.211.361.281.241.09D397Y0.581.130.910.051.111.151.161.141.02A398C0.871.071.100.720.981.091.110.740.72A398D0.591.271.010.460.991.451.010.990.77A398E0.651.341.030.771.011.271.120.970.74A398F0.461.601.040.360.891.481.050.840.82A398G0.491.331.020.280.841.530.910.800.76A398H0.631.301.030.581.001.350.990.930.93A398I0.551.330.970.630.861.631.010.800.82A398K0.392.160.970.450.991.701.151.070.90A398L0.581.361.010.610.971.460.970.900.91A398M0.391.680.980.410.721.700.960.750.62A398N0.332.681.000.420.972.751.170.930.84A398P0.691.261.040.660.981.641.010.920.94A398Q0.481.600.980.580.941.651.181.140.90A398R0.362.720.910.511.031.991.281.091.02A398S0.421.681.090.470.901.371.010.820.77A398T0.641.351.000.860.971.601.150.910.93A398V0.541.620.990.761.001.711.150.930.92A398W0.304.140.970.431.202.971.551.331.16A398Y0.441.681.020.370.891.461.140.940.81I399A0.410.780.940.660.991.051.031.151.00I399C0.190.460.850.331.071.261.131.280.78I399D0.140.360.860.240.901.101.151.330.68I399E0.300.620.880.410.971.081.001.100.87I399F0.600.970.940.680.950.911.020.960.95变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCI399G0.110.310.760.180.891.250.691.370.55I399H0.040.040.040.040.040.040.040.040.04I399I1.001.001.001.001.001.001.001.001.00I399K0.050.050.050.050.050.050.050.050.05I399L1.011.101.021.160.970.980.950.921.04I399M0.550.890.890.660.931.020.900.930.86I399N0.130.050.050.050.060.150.060.220.26I399P0.170.050.050.050.050.140.060.180.09I399Q0.140.390.800.180.941.121.221.410.49I399R0.140.050.050.050.050.100.080.070.17I399S0.220.520.860.330.901.121.081.090.74I399T0.240.630.830.391.001.021.181.290.74I399V0.581.010.900.780.990.941.071.041.01I399W0.430.730.890.340.931.060.960.850.74I399Y0.220.530.840.261.021.041.031.310.65R402A0.621.270.911.011.221.181.271.291.22R402C0.570.811.020.640.951.010.960.930.78R402D0.220.330.890.150.740.870.700.790.50R402E0.310.611.090.551.000.891.001.020.66R402F0.590.840.960.921.000.901.010.990.81R402G0.250.631.020.411.091.021.141.180.78R402I0.540.891.100.800.980.830.980.981.11R402K0.070.150.050.130.320.460.280.620.50R402L0.410.851.020.921.070.991.071.080.77R402N0.050.050.050.050.050.050.050.050.05R402P0.160.362.840.270.820.970.770.960.49R402Q0.320.651.050.491.020.900.961.030.46R402R1.001.001.001.001.001.001.001.001.00R402S0.180.761.100.341.391.041.451.500.87R402T0.120.050.050.110.050.050.050.070.26R402V0.520.801.210.770.950.830.950.950.89R402W0.470.940.980.851.040.981.021.160.94R402Y0.600.861.030.800.951.000.941.000.80Q409C0.180.481.340.140.760.790.680.630.52Q409D0.180.631.160.180.971.000.980.750.24Q409E0.130.430.860.310.920.840.630.590.35Q409G0.150.571.100.191.060.910.830.710.65Q409H0.150.511.730.160.740.720.750.660.53Q409I0.200.431.040.160.590.510.540.410.22Q409L0.200.350.960.150.520.550.330.330.48Q409M0.150.390.710.260.690.660.470.480.43Q409Q1.001.001.001.001.001.001.001.001.00Q409R0.040.040.040.040.040.040.040.040.04Q409S0.180.470.840.200.670.540.660.500.82Q409T0.230.580.890.260.810.740.770.700.52Q409V0.170.511.210.120.730.750.740.580.44变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ409W0.040.040.040.040.040.040.040.040.04V410A0.960.970.990.650.971.130.981.000.96V410C0.951.010.970.930.931.070.951.000.96V410D0.140.140.970.050.420.610.290.660.38V410F0.670.861.000.530.980.920.881.091.00V410G0.220.251.020.160.600.730.490.820.49V410H0.220.390.960.180.910.950.721.200.64V410I0.630.820.990.790.921.070.951.040.87V410L0.950.971.051.070.921.040.860.910.89V410M0.680.870.980.890.960.980.900.860.84V410N0.220.460.910.320.991.360.870.960.75V410P0.270.410.980.240.850.970.680.890.61V410Q0.170.300.920.110.810.920.620.930.47V410R0.080.200.920.051.001.120.781.311.41V410S0.440.690.980.500.981.110.961.061.04V410T0.830.950.970.880.901.120.870.890.81V410V1.001.001.001.001.001.001.001.001.00V410W0.350.530.990.280.911.050.821.120.64V410Y0.510.710.970.330.950.920.891.070.82T411D0.761.150.880.911.081.181.041.070.96T411E0.551.330.920.731.181.001.241.251.07T411F0.761.200.921.121.071.001.111.040.94T411G0.471.210.840.681.171.041.141.040.91T411H0.681.290.851.091.110.871.131.121.02T411I0.681.190.800.871.040.940.971.030.80T411K0.551.130.790.701.030.901.020.970.84T411L0.841.210.911.141.030.901.030.930.86T411M0.050.050.050.050.050.050.050.050.26T411N0.571.310.900.901.181.131.291.221.07T411P0.170.700.750.250.920.740.830.680.47T411Q0.741.250.921.061.111.171.131.160.95T411R0.771.180.850.861.030.981.041.030.89T411S0.601.070.920.861.011.091.051.020.92T411T1.001.001.001.001.001.001.001.001.00T411V0.821.200.900.981.040.991.051.020.97T411W0.040.040.040.040.040.040.040.040.04T411Y0.981.090.971.220.981.170.951.030.91S420A1.020.950.921.010.960.860.880.900.86S420C0.411.480.940.851.541.241.751.791.71S420D1.151.030.881.100.990.940.930.951.01S420F0.711.000.920.800.950.850.870.910.96S420G0.511.400.910.881.270.921.281.401.35S420H0.580.950.940.711.020.790.930.920.84S420I0.040.040.040.040.040.040.040.040.04S420K0.661.060.860.741.020.820.940.970.98S420L0.560.930.860.690.900.750.820.870.81变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCS420M0.150.350.900.160.590.700.540.670.58S420N0.461.020.950.731.060.831.021.121.09S420Q0.411.090.840.681.110.961.111.210.97S420S1.001.001.001.001.001.001.001.001.00S420T0.481.050.930.741.100.841.071.121.03S420V0.050.900.970.293.230.193.774.551.39S420W0.040.040.040.040.040.040.040.040.04S420Y0.701.110.860.921.000.961.051.040.96R426A0.062.431.030.849.135.6011.4212.313.54R426E0.900.951.030.890.951.020.990.980.89R426F0.870.961.091.030.981.101.080.990.93R426G0.050.050.050.050.050.050.050.050.05R426I0.890.801.120.760.881.230.960.900.91R426K0.900.851.110.970.921.070.950.920.80R426L0.770.991.010.970.930.931.001.080.85R426M0.680.801.030.610.880.980.920.950.78R426N0.970.941.101.020.951.171.050.970.86R426P0.240.520.920.190.981.120.990.930.48R426Q0.810.861.150.780.961.091.010.990.79R426R1.001.001.001.001.001.001.001.001.00R426S0.840.821.000.800.901.040.680.870.72R426T0.931.001.070.940.980.981.031.100.99R426V0.050.050.050.050.050.050.050.050.05R426W0.730.950.970.780.961.061.030.990.95R426Y0.920.901.100.920.961.021.001.060.74G427C0.381.390.990.681.491.241.571.541.25G427D0.950.971.060.881.010.900.970.980.95G427E1.271.001.081.241.021.000.950.941.12G427F0.531.351.040.941.331.071.281.191.48G427G1.001.001.001.001.001.001.001.001.00G427H0.931.070.950.981.011.060.930.841.06G427I0.420.900.930.481.001.000.870.820.78G427K0.610.930.960.690.881.060.890.860.82G427L0.891.051.060.980.990.930.940.911.03G427M0.591.040.980.740.970.880.900.930.92G427N0.570.941.030.650.891.030.770.910.81G427P0.190.650.970.131.140.970.941.210.73G427Q0.720.981.000.870.941.050.900.930.85G427R0.580.950.980.640.841.060.890.890.86G427S1.051.001.051.070.991.000.800.830.97G427T0.600.961.000.700.961.070.850.910.76G427V0.320.941.000.451.031.020.961.040.76G427W0.940.891.030.890.930.880.800.680.82G427Y0.621.160.980.821.070.941.010.981.01K428A1.181.161.051.541.120.831.071.120.91K428C0.950.970.980.990.941.150.880.910.92变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK428D0.310.470.900.300.771.310.630.750.36K428E0.620.780.980.670.911.270.780.850.62K428F0.420.520.890.310.711.490.570.620.48K428G0.500.790.880.600.932.700.800.850.65K428H0.810.840.940.820.881.430.770.800.77K428I0.430.700.970.550.921.620.790.850.77K428K1.001.001.001.001.001.001.001.001.00K428L0.630.760.940.600.851.120.730.770.70K428M0.570.900.910.670.911.790.850.880.84K428N0.270.670.890.401.012.120.910.990.82K428P0.700.781.010.770.861.060.790.740.76K428Q0.470.740.930.560.871.960.760.830.83K428R0.520.860.920.620.921.320.910.910.85K428S0.960.900.921.060.901.080.770.780.80K428T0.660.820.930.730.871.230.760.800.77K428V0.520.810.960.620.911.500.840.850.78K428W0.430.500.970.310.701.710.580.680.53K428Y0.550.780.940.530.871.300.820.840.75E441A1.950.050.050.050.050.050.050.050.05E441C1.380.050.050.050.050.050.050.050.08E441D1.960.050.050.050.050.050.050.050.05E441E1.001.001.001.001.001.001.001.001.00E441F0.040.040.040.040.040.040.040.040.04E441G1.200.050.050.050.050.050.050.050.05E441H0.170.050.050.050.050.100.050.050.08E441I0.700.050.050.050.050.050.050.050.05E441K0.350.050.050.050.050.050.050.050.07E441L0.310.050.050.050.050.050.050.050.08E441M0.540.050.050.050.050.050.050.050.05E441N0.870.050.050.050.050.050.050.050.12E441P0.280.050.050.050.050.090.050.050.05E441Q0.690.050.050.050.050.050.050.050.08E441R0.080.050.050.100.050.200.050.050.05E441S0.980.050.050.050.050.050.050.050.05E441T0.670.050.050.050.050.070.050.050.15E441V0.740.050.050.050.050.050.050.050.05E441Y0.270.080.050.050.050.050.050.050.05T445A0.881.011.080.911.031.111.111.011.06T445C0.321.151.030.601.311.641.791.651.54T445D0.740.960.981.490.971.150.950.971.02T445E0.611.011.040.741.060.911.080.941.06T445F0.551.071.040.781.040.991.191.241.12T445G0.611.011.231.141.050.891.181.191.16T445H0.040.040.040.040.040.040.040.040.04T445I0.571.001.000.680.950.951.100.981.09T445K0.480.850.960.530.861.031.120.970.99变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCT445L0.570.980.970.650.940.851.110.890.88T445M0.461.071.060.721.111.011.221.161.13T445N0.270.750.930.360.971.001.111.110.96T445P0.430.991.110.421.020.601.211.081.04T445Q0.420.811.030.470.881.091.070.920.94T445R0.650.920.920.670.880.911.040.950.86T445S0.430.831.060.520.961.091.110.990.97T445T1.001.001.001.001.001.001.001.001.00T445V0.491.051.020.701.030.891.171.141.11T445W0.050.050.050.050.050.050.050.050.05T445Y0.611.031.070.751.020.781.181.011.01V446A1.160.990.940.050.920.900.960.961.13V446C0.820.870.890.050.951.080.960.901.06V446E0.260.540.810.050.850.180.880.940.60V446F0.170.290.870.050.670.330.720.790.70V446G0.230.390.910.050.840.180.840.780.79V446I0.700.830.850.050.860.860.890.840.86V446K0.110.340.720.050.920.361.041.020.48V446L0.230.520.890.050.920.540.790.840.70V446M0.050.050.050.050.050.050.050.050.05V446N0.290.550.900.090.810.320.820.810.71V446P0.120.170.710.050.390.130.390.500.34V446Q0.260.730.860.111.120.921.251.160.82V446R0.120.430.780.051.090.541.301.300.50V446S0.350.710.910.050.960.761.000.940.86V446V1.001.001.001.001.001.001.001.001.00V446W0.120.290.880.050.750.230.790.940.36E447A1.000.961.020.050.870.820.830.870.81E447C0.080.080.050.050.090.050.050.050.14E447E1.001.001.001.001.001.001.001.001.00E447F0.400.600.920.050.920.250.860.960.52E447G0.070.050.050.050.050.390.050.050.28E447I0.480.560.960.050.810.200.780.910.69E447K0.420.620.960.050.870.340.851.020.66E447L0.420.661.010.050.920.560.891.030.80E447M0.050.050.050.050.050.110.050.050.05E447N1.010.860.950.050.820.700.750.880.74E447P0.070.110.980.050.130.150.060.050.25E447R0.560.560.950.060.690.280.590.680.59E447S0.530.760.930.050.920.600.891.150.92E447T0.050.050.050.050.050.050.050.050.17E447V0.350.630.930.051.030.230.991.130.85E447W0.450.481.080.100.750.200.610.720.59E447Y0.250.440.870.050.960.300.901.070.56G448A1.011.070.970.050.981.051.020.900.95G448C0.821.330.920.071.091.121.221.021.04变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCG448D1.141.021.040.061.011.261.010.900.92G448E0.851.051.130.051.021.201.040.900.98G448F1.320.991.120.051.041.021.100.901.05G448G1.001.001.001.001.001.001.001.001.00G448H0.871.011.090.050.971.000.960.991.02G448K0.850.941.010.050.930.930.920.900.88G448L0.690.991.040.050.970.890.920.880.74G448M1.021.001.020.050.950.980.940.880.85G448N1.360.971.120.211.021.100.960.950.98G448P0.180.870.990.050.610.110.970.760.44G448Q0.820.941.110.050.970.981.000.980.82G448R1.300.940.970.050.950.900.920.990.88G448S1.000.991.080.050.931.000.930.990.91G448T1.051.041.000.050.981.020.990.980.89G448V0.960.851.100.050.930.630.830.850.81G448W0.980.920.990.050.880.810.860.910.73G448Y1.080.891.060.050.941.030.941.040.93N449A0.831.040.990.051.001.041.041.001.13N449C0.661.071.090.231.021.101.061.041.10N449E0.731.061.120.051.000.880.950.940.93N449F0.471.030.970.050.920.470.970.930.93N449G0.220.641.020.051.010.111.150.870.69N449H0.410.880.950.050.980.941.021.070.73N449K0.160.461.220.051.010.141.081.010.79N449L0.340.851.170.050.870.270.810.890.66N449M0.731.120.940.050.940.780.820.850.91N449N1.001.001.001.001.001.001.001.001.00N449P0.121.060.600.050.690.101.240.540.36N449Q0.680.980.980.050.860.930.810.800.79N449R0.740.871.120.050.910.760.840.810.85N449S0.661.000.920.050.930.920.840.870.77N449T0.761.130.990.050.940.900.870.860.66N449V0.791.091.110.050.920.810.850.860.75N449W0.430.921.090.050.870.260.820.850.72N449Y0.040.040.040.040.040.040.040.040.04D452A0.050.050.050.050.050.050.050.050.05D452C0.040.040.040.040.040.040.040.040.04D452D1.001.001.001.001.001.001.001.001.00D452E0.140.050.050.050.050.170.050.050.29D452F0.180.050.050.050.050.050.070.050.39D452G0.050.050.050.050.050.050.050.050.05D452H0.040.040.040.040.040.040.040.040.04D452I0.130.050.050.050.070.170.130.050.30D452K0.140.050.050.050.050.050.050.050.12D452L0.140.050.050.050.050.050.070.050.20D452M0.140.050.050.050.050.050.110.050.26变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCD452N0.260.201.070.050.200.130.240.230.29D452P0.130.050.050.050.050.060.050.050.25D452Q0.040.040.040.040.040.040.040.040.04D452R0.140.050.050.050.110.100.050.050.16D452S0.040.040.040.040.040.040.040.040.04D452T0.140.080.050.050.050.050.140.050.24D452V0.150.050.050.050.050.050.050.050.28D452W0.130.050.050.050.050.070.170.050.24D452Y0.140.050.050.050.050.050.170.050.28R453A0.190.081.360.050.050.100.180.050.24R453C0.200.050.050.050.050.110.080.050.54R453D0.200.050.050.050.050.050.100.050.14R453E0.080.121.600.050.180.250.740.160.38R453F0.080.050.050.080.050.150.090.050.14R453I0.040.040.040.040.040.040.040.040.04R453K0.090.171.000.070.250.140.700.410.34R453L0.110.121.600.050.220.250.520.370.49R453M0.170.301.330.050.590.250.700.680.52R453N0.090.090.050.050.090.090.420.200.23R453P0.110.050.050.050.050.050.080.050.31R453Q0.100.121.610.050.250.060.620.400.63R453S0.050.101.520.050.060.280.730.260.18R453T0.170.050.050.050.050.050.110.050.09R453V0.150.050.050.050.050.050.120.050.16R453W0.190.050.050.050.050.080.050.050.25R453Y0.180.050.050.050.050.060.050.050.12N454A1.320.980.920.480.940.910.910.850.94N454C0.801.000.900.470.980.700.940.950.97N454D0.770.880.960.050.930.900.870.880.83N454F0.270.950.920.301.451.941.341.501.08N454G0.580.810.870.090.861.610.740.830.80N454K0.610.920.840.520.921.170.820.910.86N454L0.520.810.860.320.881.650.790.850.69N454M0.490.830.780.330.881.120.800.850.72N454N1.001.001.001.001.001.001.001.001.00N454R0.620.770.880.350.791.080.790.840.65N454S0.620.880.860.390.881.660.810.850.82N454T0.620.820.860.300.851.190.780.830.72N454V0.490.960.860.430.991.140.931.030.84N455A1.000.941.000.540.931.010.900.930.96N455C0.811.141.060.271.130.931.151.221.21N455D0.431.481.110.941.752.431.792.081.82N455E0.650.810.980.210.891.580.780.810.91N455F0.700.840.960.400.891.250.830.830.83N455G0.730.810.910.260.822.690.770.820.75N455H0.590.930.950.560.981.350.950.990.89变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN455I0.490.641.010.050.841.760.770.860.66N455L0.600.780.870.160.781.320.760.820.59N455M0.480.780.930.250.861.480.790.910.78N455N1.001.001.001.001.001.001.001.001.00N455R0.790.790.960.210.770.730.750.780.57N455S0.540.950.940.391.031.091.001.090.82N455T0.410.700.920.220.881.180.820.890.71N455V0.370.760.910.111.001.320.941.050.58N455W0.381.250.910.451.441.441.491.651.45N455Y0.520.820.940.380.901.050.900.980.83H460A0.210.450.840.050.930.361.081.200.76H460C0.240.560.820.050.980.491.161.210.82H460D0.600.890.900.050.970.561.040.961.01H460E0.330.720.940.051.020.671.161.120.93H460F0.510.940.810.050.990.871.151.031.03H460G0.390.840.920.051.000.261.221.201.10H460H1.001.001.001.001.001.001.001.001.00H460I0.430.830.780.050.940.681.021.030.92H460K0.520.910.880.050.960.901.061.081.03H460L0.240.640.820.050.980.421.201.260.71H460M0.340.850.770.051.010.531.121.200.96H460N0.611.000.940.050.960.790.971.050.95H460Q0.410.910.870.051.070.811.161.261.00H460R0.270.650.800.051.000.441.171.300.97H460S0.250.650.930.051.010.481.191.340.81H460V0.110.050.050.050.050.170.050.120.05H460W0.450.830.860.050.970.611.081.140.96H460Y0.530.920.890.050.980.951.041.090.93Q467A0.120.472.350.100.831.100.810.730.54Q467C0.180.541.020.140.820.910.770.620.58Q467D0.180.502.630.110.760.770.770.560.30Q467E0.130.421.040.100.710.660.720.570.67Q467H0.140.452.010.090.900.590.690.560.33Q467I0.100.390.830.060.850.780.760.490.42Q467K0.070.301.100.050.620.800.640.380.10Q467L0.180.410.850.110.610.580.540.440.40Q467N0.130.491.020.230.770.950.780.610.43Q467P0.080.290.050.111.070.860.590.340.63Q467Q1.001.001.001.001.001.001.001.001.00Q467S0.150.571.040.171.040.900.940.800.65Q467T0.090.340.050.050.680.520.530.280.81Q467V0.130.401.260.090.810.900.780.510.46Q467W0.160.441.370.070.720.630.730.500.60Q467Y0.150.322.100.050.530.550.550.400.38N473A0.740.900.990.811.001.340.880.810.92N473C0.940.920.910.800.921.040.830.780.83变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN473E0.670.980.990.871.021.390.910.840.92N473F0.140.221.050.060.601.250.560.600.25N473G0.610.960.970.940.971.100.940.830.89N473H0.830.900.950.830.891.140.870.770.75N473K0.330.590.940.520.881.190.800.770.75N473L0.560.930.980.841.001.350.940.830.84N473M0.580.860.960.840.931.120.840.800.69N473N1.001.001.001.001.001.001.001.001.00N473P0.290.421.080.300.761.170.630.610.44N473Q0.670.771.010.790.811.120.780.710.63N473R0.300.681.010.541.121.490.900.980.76N473S1.021.041.011.200.941.280.910.840.99N473T0.640.961.020.920.981.040.980.900.79N473V0.420.771.000.600.941.260.830.840.67N473W0.050.190.970.081.062.360.981.000.20S474A0.890.991.000.891.011.030.961.081.00S474C0.761.100.940.821.111.011.171.091.14S474D1.960.761.251.531.001.081.030.930.91S474E0.860.960.980.760.950.860.981.041.01S474F0.911.040.960.940.981.031.101.021.04S474G1.390.981.051.221.011.041.050.940.96S474I0.981.090.971.130.961.081.060.991.05S474K1.380.941.041.191.021.071.101.050.99S474L0.561.021.030.741.050.981.051.050.97S474M0.911.051.021.010.951.011.070.991.13S474N1.401.021.051.401.001.091.051.021.10S474P0.871.070.971.000.960.981.071.130.96S474Q0.950.961.000.940.921.021.000.900.90S474R1.231.021.001.350.971.151.090.910.99S474S1.001.001.001.001.001.001.001.001.00S474T1.031.050.971.190.971.091.081.031.04S474V1.370.981.081.300.991.081.060.991.05S474W0.040.040.040.040.040.040.040.040.04S474Y1.061.000.931.000.930.910.990.991.00N475I0.274.501.010.371.013.431.291.110.87N475K0.080.051.060.050.302.111.020.430.19N475L0.332.361.030.450.891.991.150.900.79N475M0.422.030.980.611.002.861.121.030.98N475N1.001.001.001.001.001.001.001.001.00N475P0.312.601.040.440.973.031.300.960.79N475Q0.591.460.950.721.001.521.060.910.91N475R0.621.310.940.700.981.541.100.940.84N475S0.2316.690.990.531.383.291.961.701.24N475T0.21256.401.040.321.032.631.471.170.80N475V0.342.391.040.460.981.831.110.930.78N475W0.402.031.010.540.991.801.191.040.88变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCN475Y0.412.021.020.501.071.561.261.050.76E489A0.040.040.040.040.040.040.040.040.04E489D1.160.940.970.990.990.940.941.010.96E489E1.001.001.001.001.001.001.001.001.00E489F0.040.040.040.040.040.040.040.040.04E489G0.040.040.040.040.040.040.040.040.04E489H0.040.040.040.040.040.040.040.040.04E489I0.130.050.050.050.050.050.050.110.33E489K0.040.040.040.040.040.040.040.040.04E489L0.040.040.040.040.040.040.040.040.04E489M0.040.040.040.040.040.040.040.040.04E489N0.140.231.020.130.821.050.621.070.56E489P0.040.040.040.040.040.040.040.040.04E489Q0.040.040.040.040.040.040.040.040.04E489R0.040.040.040.040.040.040.040.040.04E489S0.050.050.050.050.050.050.050.050.05E489T0.040.040.040.040.040.040.040.040.04E489V0.190.050.050.050.050.100.050.140.26E489W0.040.040.040.040.040.040.040.040.04E489Y0.040.040.040.040.040.040.040.040.04Q490A0.811.130.990.900.960.920.890.950.91Q490C0.616.601.010.791.120.951.021.121.11Q490E0.652.930.970.680.910.980.910.931.13Q490F0.5110.910.920.610.990.910.900.861.00Q490G0.543.161.000.580.970.910.950.981.19Q490H0.479.130.900.621.040.860.960.981.08Q490K0.643.190.940.790.950.790.830.910.83Q490L0.542.260.960.841.070.891.031.001.17Q490P0.500.430.920.670.991.060.920.890.95Q490Q1.001.001.001.001.001.001.001.001.00Q490R0.571.920.990.751.010.860.910.950.96Q490S0.591.700.950.740.990.890.900.920.95Q490T0.741.360.960.961.000.850.931.000.88Q490V0.445.380.990.671.100.851.011.181.05Q490W0.400.480.990.741.361.051.301.401.32Q490Y0.571.450.970.761.040.971.021.141.01L492A0.561.100.910.571.171.231.181.140.95L492D0.461.050.970.611.291.191.251.170.89L492F0.270.740.910.301.031.040.801.080.69L492G0.210.430.960.180.760.680.630.780.38L492H0.461.010.900.450.940.931.041.030.87L492I0.590.970.980.580.971.031.031.060.84L492K0.130.340.880.090.550.780.550.750.27L492L1.001.001.001.001.001.001.001.001.00L492M0.450.910.980.520.691.050.970.910.52L492N0.371.040.900.560.910.971.141.050.55变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCL492P0.170.430.960.050.420.690.630.800.56L492Q0.480.970.930.460.761.001.031.000.79L492R0.170.500.950.170.620.820.801.010.30L492S0.120.050.050.050.080.050.050.120.17L492T0.390.990.950.480.861.041.071.000.54L492V0.130.050.050.050.050.160.050.130.05L492W0.370.850.860.351.011.050.970.970.54L492Y0.530.921.050.551.050.961.011.050.74Q496A0.170.611.020.150.900.900.920.760.67Q496C0.160.400.810.090.610.800.600.490.53Q496D0.160.430.960.050.670.710.670.430.51Q496F0.090.050.050.050.420.500.270.100.36Q496G0.170.531.070.161.001.040.800.600.53Q496K0.180.291.520.050.380.520.400.300.64Q496L0.130.050.050.050.350.270.290.100.56Q496N0.170.491.030.140.650.740.660.550.59Q496P0.130.361.370.160.560.600.830.440.16Q496Q1.001.001.001.001.001.001.001.001.00Q496S0.250.780.990.281.001.031.100.930.74Q496T0.150.442.520.100.740.520.690.450.64Q496V0.210.341.750.090.480.500.450.330.48Q496W0.070.561.480.091.341.371.461.020.47V497A0.781.070.981.050.961.010.900.840.90V497C0.740.961.090.771.020.980.970.950.95V497D0.050.050.050.050.070.460.050.050.17V497E0.050.050.050.050.050.050.050.050.05V497F0.050.050.050.050.050.100.050.050.07V497G0.070.050.050.100.170.280.190.050.46V497H0.040.040.040.040.040.040.040.040.04V497I0.590.890.941.070.971.030.890.800.88V497K0.080.050.050.050.050.110.050.050.13V497M0.510.990.961.161.010.900.970.830.96V497N0.170.461.030.350.780.810.780.420.48V497Q0.050.050.050.050.050.330.050.050.13V497R0.060.050.050.050.090.470.190.050.17V497S0.050.050.050.050.050.050.050.050.05V497T0.451.050.941.211.081.051.080.901.04V497V1.001.001.001.001.001.001.001.001.00V497W0.040.040.040.040.040.040.040.040.04V497Y0.040.040.040.040.040.040.040.040.04K498A0.870.991.130.761.051.080.981.031.07K498C0.630.911.150.561.060.981.090.970.96K498E0.451.001.070.601.150.941.061.071.15K498F0.290.811.040.381.161.031.101.040.83K498G0.370.811.010.401.000.871.010.880.81K498H0.660.921.120.680.991.080.990.970.94变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK498I0.430.911.070.531.091.011.010.950.72K498K1.001.001.001.001.001.001.001.001.00K498L0.691.010.980.790.890.890.890.880.59K498M0.441.010.950.621.120.940.971.031.18K498N0.590.930.980.680.961.040.950.950.78K498P0.050.050.050.050.050.050.050.050.05K498Q0.670.931.090.810.971.070.940.940.87K498R0.600.851.060.660.990.890.990.960.82K498S0.540.881.030.630.970.880.760.810.85K498T0.480.901.020.601.040.970.920.930.98K498V0.551.010.990.630.980.920.951.020.97K498W0.060.230.050.050.210.090.100.050.20K498Y0.440.901.000.521.020.900.980.920.77D521A1.041.020.971.051.031.310.890.781.03D521C0.150.550.930.251.482.371.631.160.76D521D1.001.001.001.001.001.001.001.001.00D521E0.850.881.040.880.921.000.880.700.94D521F0.820.871.070.900.871.180.810.690.91D521G0.440.700.990.560.931.090.920.670.85D521H0.890.940.970.900.931.050.850.750.91D521I0.470.710.970.490.861.430.860.690.79D521K0.470.780.980.570.921.280.940.750.72D521L0.720.891.000.820.921.260.890.720.95D521M0.620.870.900.750.941.430.900.750.99D521P0.240.401.060.260.831.220.770.610.80D521R0.430.730.980.500.951.340.910.730.81D521S0.920.980.941.050.971.220.870.791.05D521T0.660.881.020.790.911.070.890.831.00D521V0.560.891.020.760.951.030.920.660.90D521W0.630.951.000.761.061.661.030.761.16D521Y0.220.580.870.050.871.601.210.881.13V522A0.861.081.000.731.051.261.021.090.94V522C0.901.211.000.891.081.431.091.171.09V522F0.511.420.990.510.922.441.030.970.77V522G0.110.051.000.130.915.721.531.320.54V522H0.731.270.900.810.991.291.000.930.84V522I0.561.320.920.570.971.800.980.920.70V522K0.362.441.010.431.032.091.131.120.81V522L0.461.830.990.571.031.741.261.130.92V522M0.601.450.950.701.011.571.041.060.83V522N0.190.051.020.210.683.161.080.860.48V522P0.180.050.840.140.712.341.180.870.59V522Q0.372.260.940.421.042.411.311.200.76V522R0.303.230.940.391.023.231.281.150.92V522S0.461.690.960.551.052.111.191.051.04V522T0.511.500.980.590.971.511.061.010.98变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCV522V1.001.001.001.001.001.001.001.001.00V522W0.2411.890.980.491.482.652.092.051.30V522Y0.561.370.960.561.002.101.211.031.15K534A0.690.890.900.490.950.950.920.850.90K534C0.630.990.920.621.071.231.101.061.11K534D0.190.501.010.181.131.431.181.090.87K534E0.480.971.040.641.121.101.201.091.16K534F0.681.020.990.651.051.131.040.971.07K534G0.220.461.040.050.861.001.000.870.77K534H0.550.761.010.430.900.990.850.900.81K534I0.660.841.040.570.930.960.870.890.83K534K1.001.001.001.001.001.001.001.001.00K534M0.530.790.940.410.900.880.860.880.76K534N0.650.911.080.561.011.090.970.951.06K534P0.040.040.040.040.040.040.040.040.04K534Q1.170.971.050.830.940.910.890.980.97K534R0.840.980.980.811.021.050.981.100.98K534S0.530.781.030.480.880.930.850.880.84K534T0.620.781.020.520.880.950.820.890.80K534V0.751.031.060.701.021.181.081.071.07K534W0.160.050.050.050.050.170.110.050.26R542A1.340.981.020.910.951.051.091.091.36R542C0.761.171.040.470.931.140.981.091.11R542D1.080.951.080.440.891.151.011.031.20R542E0.511.790.900.370.901.100.921.161.19R542F0.551.571.050.380.921.300.951.151.08R542G0.831.090.990.470.931.070.971.051.21R542H0.601.620.920.490.891.010.941.090.86R542I0.541.781.040.050.911.351.021.150.98R542K0.811.071.050.600.811.110.960.991.09R542L0.541.760.860.410.931.191.031.181.35R542M0.551.690.890.420.961.180.971.171.24R542N0.901.280.880.700.901.351.101.101.57R542P0.541.171.130.180.891.320.690.910.64R542Q0.621.371.010.390.871.480.871.021.02R542R1.001.001.001.001.001.001.001.001.00R542S0.681.331.060.551.021.090.981.150.85R542T0.751.160.990.530.971.171.031.121.30R542V0.741.320.820.570.901.190.971.051.05R542W0.601.641.030.450.991.171.091.201.08R542Y0.621.331.040.440.941.040.961.050.90G547A0.381.471.081.551.731.271.912.151.67G547C0.560.951.020.881.060.960.931.120.88G547E1.250.961.041.310.850.970.710.570.74G547F0.540.761.100.830.950.740.800.790.75G547G1.001.001.001.001.001.001.001.001.00变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCG547I0.630.800.990.860.900.760.780.820.79G547K0.810.871.051.020.900.870.800.870.77G547L1.261.111.081.520.931.010.790.860.91G547N0.370.591.040.610.950.760.730.820.80G547P1.021.091.081.440.970.890.910.900.92G547Q0.450.671.110.740.950.800.760.800.73G547R0.770.961.021.060.960.910.860.940.84G547T0.630.821.100.960.960.810.830.860.83G547V0.750.931.091.190.970.910.820.830.91G547W0.040.040.040.040.040.040.040.040.04G547Y0.610.801.040.870.960.780.820.860.92S548A0.050.050.050.050.050.050.050.050.05S548C0.871.140.941.071.050.921.030.961.04S548E1.051.121.021.201.050.891.020.911.01S548F1.021.191.031.411.050.931.100.921.03S548H1.141.010.991.200.970.860.900.840.79S548I0.911.121.061.170.980.821.000.880.97S548K0.500.980.970.600.880.780.840.840.69S548L1.101.141.051.471.031.021.050.910.95S548M0.741.170.971.011.000.940.960.880.88S548N0.501.260.980.830.960.921.120.960.79S548Q0.461.121.100.731.010.961.050.970.73S548R0.531.040.930.730.840.890.860.860.61S548S1.001.001.001.001.001.001.001.001.00S548T0.561.341.020.971.150.941.231.110.89S548V0.651.171.011.010.910.920.980.900.74S548W0.771.361.021.131.160.941.250.961.04S548Y0.551.090.950.810.980.890.940.960.72E553A0.420.500.870.130.700.710.750.850.67E553C0.330.350.950.120.540.590.560.680.27E553D0.380.301.150.140.590.560.560.510.47E553E1.001.001.001.001.001.001.001.001.00E553H0.410.330.950.050.530.490.420.470.44E553I0.110.221.080.080.750.830.991.180.94E553K0.250.221.040.130.400.420.460.610.39E553M0.240.260.940.100.470.480.520.610.55E553N0.280.260.880.250.460.660.480.631.09E553Q0.190.141.220.090.350.470.280.440.90E553R0.250.200.870.070.330.360.400.510.22E553V0.190.160.930.080.360.380.290.450.79E553W0.360.281.040.120.470.490.440.410.56E553Y0.100.231.250.050.880.510.751.040.65G554A0.871.001.020.690.981.151.041.000.98G554C0.620.951.080.591.031.451.091.171.10G554D0.991.031.070.751.031.101.091.010.96G554F0.430.951.040.441.071.401.171.201.14变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCG554G1.001.001.001.001.001.001.001.001.00G554H0.590.941.030.601.011.051.081.020.94G554K0.800.841.040.740.820.950.860.830.75G554L0.741.041.040.560.971.051.110.970.89G554M0.621.031.020.741.010.971.030.960.77G554P0.040.040.040.040.040.040.040.040.04G554Q0.861.051.121.121.040.871.101.061.00G554R0.840.851.040.860.830.970.820.770.89G554S0.310.711.010.400.920.960.940.760.61G554T0.620.951.000.710.931.000.980.900.78G554V0.550.880.950.470.941.010.941.000.66G554W0.471.071.020.541.171.431.271.241.00L555A0.690.970.630.510.971.010.950.920.82L555C0.881.030.770.801.001.051.010.980.90L555D0.690.860.770.591.031.110.981.020.77L555E0.360.710.600.441.041.041.061.060.75L555F0.490.840.600.540.981.070.961.220.78L555G0.500.950.640.531.121.161.111.160.87L555H0.450.860.640.421.031.151.081.080.97L555I0.691.000.660.730.951.050.930.960.82L555K0.470.890.680.451.101.241.201.180.89L555L1.001.001.001.001.001.001.001.001.00L555M0.600.970.660.661.000.931.001.050.87L555N0.630.940.680.631.011.080.890.960.76L555P0.430.920.590.531.021.151.121.070.84L555Q0.610.900.640.530.971.121.011.020.77L555R0.160.050.050.050.050.210.050.050.05L555S0.580.910.660.510.930.850.990.930.75L555T0.620.970.670.650.990.990.971.040.77L555V0.901.090.760.820.991.061.001.050.91L555W0.390.730.590.250.891.130.941.020.87L555Y0.580.910.630.430.971.140.981.050.78K560A0.630.801.180.441.060.960.840.811.00K560C0.590.731.160.320.930.930.780.830.93K560D0.050.050.050.050.050.050.050.050.05K560E0.430.691.160.051.010.820.800.740.97K560G0.400.741.080.291.050.940.810.740.95K560H1.010.901.100.420.981.010.870.831.00K560I0.190.331.190.110.760.600.550.420.49K560K1.001.001.001.001.001.001.001.001.00K560L0.630.831.100.491.000.880.820.820.96K560M0.670.841.110.520.970.870.830.860.94K560N0.250.511.040.150.900.720.660.590.50K560P0.120.551.090.101.491.121.310.940.89K560Q0.650.801.200.090.970.790.820.820.79K560R0.720.981.030.901.011.050.920.940.78变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK560S0.370.571.240.270.980.920.760.690.96K560T0.390.661.120.260.940.740.730.680.74K560V0.190.391.110.100.830.680.590.360.58K560W0.640.900.990.071.001.020.910.760.94K560Y0.500.781.050.450.980.870.850.900.66H561A0.680.050.050.370.941.040.911.011.00H561C0.630.801.040.750.971.180.961.060.91H561D0.540.501.200.530.931.320.901.010.85H561E0.360.050.050.471.001.210.880.920.88H561F0.430.050.050.540.891.150.840.851.00H561G0.570.941.100.871.001.110.991.031.08H561H1.001.001.001.001.001.001.001.001.00H561I0.510.561.000.531.051.101.070.971.09H561M0.611.311.100.821.101.191.151.001.66H561N0.361.111.011.391.171.161.180.941.02H561P0.300.050.050.480.220.200.120.050.25H561Q0.550.490.930.611.141.111.200.920.87H561R0.890.420.050.270.840.750.880.740.91H561S0.400.410.970.491.191.181.151.011.20H561T0.650.341.030.560.900.950.900.791.13H561V0.520.270.990.351.001.021.020.871.42H561W0.640.481.020.611.181.281.141.201.16D563A0.891.011.041.201.061.171.071.051.10D563C0.280.660.940.160.871.210.890.670.68D563D1.001.001.001.001.001.001.001.001.00D563E0.820.980.971.030.980.910.981.080.89D563F0.390.810.950.470.921.120.960.930.89D563I0.390.791.020.420.991.321.020.890.84D563L0.560.980.980.650.981.051.031.070.90D563M0.791.020.930.990.940.940.931.060.96D563Q0.531.010.990.961.081.061.101.090.90D563R0.240.631.000.380.991.411.020.830.74D563S0.450.971.060.781.031.051.211.131.01D563T0.450.940.960.731.041.261.071.090.85D563V0.430.791.030.540.951.001.041.010.88D563W0.420.820.940.300.951.121.021.090.80D563Y0.470.871.060.511.031.031.131.120.96D564A0.621.071.080.701.141.071.221.161.11D564C0.771.011.020.671.071.071.121.160.78D564D1.001.001.001.001.001.001.001.001.00D564E0.490.741.040.500.950.950.920.850.76D564F0.651.011.040.741.101.021.181.101.02D564G0.520.891.050.561.060.991.070.990.79D564I0.050.050.050.050.050.050.050.050.05D564K0.490.850.970.481.011.011.111.060.73D564L0.561.100.940.691.081.141.111.070.89变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCD564M0.601.050.900.661.040.961.090.970.83D564N0.640.981.030.741.020.911.071.000.86D564P0.160.470.910.130.980.780.710.790.36D564Q0.560.900.930.551.000.970.981.110.77D564R0.630.940.970.650.971.151.071.130.90D564S0.670.990.980.760.990.970.760.981.75D564T0.591.160.980.781.111.021.221.311.33D564V0.401.170.960.721.371.111.501.660.88D564Y0.580.991.020.631.060.911.091.110.92R570A0.581.040.920.711.171.111.131.080.96R570C0.310.780.930.291.190.961.181.160.87R570D0.200.611.000.171.291.301.101.330.62R570E0.480.861.050.571.040.981.031.060.77R570F0.120.220.950.090.650.560.460.560.45R570G0.180.451.060.171.071.010.810.940.65R570H0.190.470.950.170.821.090.730.920.58R570I0.220.690.960.281.151.131.170.970.68R570M0.270.671.000.340.990.991.051.090.51R570N0.110.291.050.070.910.760.650.930.31R570P0.150.050.050.050.100.200.050.140.05R570Q0.520.911.010.671.050.971.031.100.57R570R1.001.001.001.001.001.001.001.001.00R570S0.220.630.960.231.051.050.931.080.42R570T0.360.851.010.441.070.901.091.180.68R570V0.230.621.020.271.211.041.071.200.59R570W0.040.040.040.040.040.040.040.040.04R570Y0.180.400.880.140.820.900.680.800.42Y571A0.050.050.050.050.050.050.050.050.05Y571D0.050.050.050.050.050.050.050.050.05Y571E0.050.050.050.050.050.050.050.050.05Y571H0.140.051.090.281.021.071.430.880.98Y571K0.050.050.050.050.050.050.050.050.05Y571L0.050.050.050.050.050.050.050.050.05Y571M0.150.051.060.291.121.011.570.780.91Y571N0.050.050.050.170.210.661.421.250.75Y571P0.050.050.050.050.050.050.050.050.05Y571Q0.040.040.040.040.040.040.040.040.04Y571R0.050.050.050.120.050.761.320.050.87Y571S0.050.050.050.050.050.050.050.050.05Y571T0.040.040.040.040.040.040.040.040.04Y571V0.050.050.050.050.050.050.050.050.05Y571W0.651.741.020.590.940.980.970.970.98Y571Y1.001.001.001.001.001.001.001.001.00K581A0.691.620.960.801.060.851.041.011.02K581C0.390.051.030.511.050.971.031.101.17K581D0.532.941.000.641.070.941.031.071.13变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK581E0.494.420.790.611.080.711.060.990.88K581F0.475.150.940.641.070.781.001.101.20K581G0.513.701.020.701.010.881.091.171.14K581H0.542.970.970.771.060.910.880.931.01K581I0.552.770.980.681.050.970.911.010.90K581K1.001.001.001.001.001.001.001.001.00K581L0.661.871.030.851.080.930.960.980.99K581M0.632.030.990.731.030.921.020.901.00K581N0.552.781.020.691.040.890.930.890.81K581P0.400.050.910.440.970.860.910.791.03K581R0.572.560.920.731.040.961.000.990.90K581S0.671.771.000.801.020.890.921.031.07K581T0.602.021.050.641.000.790.910.991.07K581V0.691.520.980.800.980.880.921.171.08K581W0.622.570.800.901.121.071.001.221.13K581Y0.811.320.960.901.060.950.961.110.97N583A0.821.050.920.951.011.021.061.071.01N583C0.571.180.880.971.191.371.381.431.29N583D1.081.041.031.191.041.031.091.021.05N583E0.600.860.910.660.991.080.941.030.92N583F0.620.850.920.650.951.160.911.010.90N583G0.661.020.920.911.041.271.141.170.96N583H0.560.920.830.751.051.330.961.130.89N583I0.480.830.890.670.991.210.931.180.87N583K0.600.880.890.670.931.110.931.070.84N583L0.530.810.920.750.971.090.941.080.63N583M0.510.820.840.710.911.120.911.080.68N583N1.001.001.001.001.001.001.001.001.00N583P0.210.430.770.290.791.490.741.200.32N583R0.851.030.971.170.961.430.921.130.90N583S0.530.800.900.710.941.110.931.090.67N583T0.530.810.850.710.941.160.901.080.67N583V0.751.050.880.980.990.801.031.120.84N583W0.700.920.890.820.891.110.911.000.78N583Y0.660.880.830.570.921.240.911.110.81R586C0.130.050.050.050.050.180.050.050.26R586D1.250.981.051.181.000.941.101.081.06R586E0.731.000.900.720.941.140.970.940.94R586F0.641.040.980.670.991.191.001.090.96R586G0.380.840.930.480.921.100.901.030.87R586H0.080.140.050.050.361.050.160.380.34R586I0.040.040.040.040.040.040.040.040.04R586K0.610.940.970.600.930.990.900.980.87R586L0.451.020.960.560.981.210.961.050.90R586N0.661.100.980.821.001.031.061.071.01R586P0.181.030.900.371.401.881.541.871.09变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR586Q0.100.050.050.050.050.290.050.050.09R586R1.001.001.001.001.001.001.001.001.00R586S0.100.050.050.050.050.160.050.050.06R586V0.531.470.960.991.221.411.411.461.44R586W0.491.060.890.551.021.151.111.090.96R586Y0.510.980.940.520.991.091.041.071.01S591C0.290.261.120.160.440.660.450.610.32S591D0.250.421.030.220.821.010.871.030.48S591F0.230.181.050.090.330.530.380.470.25S591G0.200.191.080.140.440.800.490.630.44S591H0.230.271.400.360.610.790.560.720.27S591I0.270.201.320.200.390.550.370.470.14S591K0.170.141.270.170.360.550.330.410.26S591M0.230.151.120.180.320.330.320.450.30S591N0.260.280.970.350.530.810.510.520.31S591P0.130.321.020.360.900.110.640.670.38S591Q0.220.221.160.290.440.770.520.560.43S591R0.040.040.040.040.040.040.040.040.04S591S1.001.001.001.001.001.001.001.001.00S591V0.290.291.110.390.500.730.530.650.36V603A0.931.010.991.470.891.010.970.981.83V603C0.951.020.940.750.971.001.061.401.18V603D0.541.030.960.820.761.100.821.352.32V603E0.941.110.961.230.940.961.000.751.01V603F0.711.010.910.810.771.000.841.191.72V603G0.401.050.940.721.001.061.282.792.16V603H0.591.310.911.151.171.251.203.341.32V603L2.390.590.970.930.640.830.600.420.59V603M1.960.720.921.740.710.870.691.390.59V603N0.820.950.941.250.881.040.871.360.87V603P0.210.820.900.280.540.830.658.070.56V603Q1.070.900.881.500.841.060.832.370.73V603R0.620.850.860.860.771.050.702.420.59V603S1.031.030.911.410.870.860.861.420.67V603T0.481.020.950.570.700.910.691.580.97V603V1.001.001.001.001.001.001.001.001.00V603W0.162.110.050.130.160.260.251.570.72V603Y0.336.940.960.872.221.862.538.791.55F611A0.671.320.931.261.211.181.551.361.52F611C0.380.650.940.411.021.111.171.060.86F611D0.210.330.920.190.791.151.050.930.65F611G0.670.741.000.580.840.960.920.830.77F611I0.160.291.040.170.770.980.950.990.52F611K0.320.570.950.420.911.171.091.110.77F611L0.700.921.020.770.891.010.970.900.82F611M0.580.820.990.700.901.041.000.970.83变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCF611N0.650.801.000.660.861.030.930.860.81F611P0.540.700.980.530.860.860.910.950.66F611Q0.450.610.930.460.820.790.920.950.74F611R0.230.411.060.300.911.391.061.220.75F611S0.600.761.010.630.861.010.920.970.89F611T0.520.661.030.500.800.870.920.990.69F611V0.530.691.130.510.810.920.961.030.87F611W0.610.780.960.580.941.041.011.110.86F611Y0.700.801.000.700.960.930.961.090.94Q612C0.791.171.120.841.181.261.261.351.28Q612D1.410.941.161.271.000.771.031.080.97Q612F0.660.920.950.700.931.640.911.000.72Q612G1.311.061.041.161.011.111.011.010.84Q612H0.420.771.040.500.972.040.941.140.84Q612I0.750.761.020.670.811.430.800.830.68Q612K1.050.960.950.730.871.180.870.920.80Q612L0.530.780.950.560.823.330.800.840.76Q612M0.560.810.940.570.871.140.810.900.65Q612P0.420.640.970.450.830.940.830.920.66Q612Q1.001.001.001.001.001.001.001.001.00Q612R0.980.910.990.840.851.100.880.900.80Q612S0.471.030.920.391.111.471.101.240.89Q612T0.660.740.950.280.790.800.730.790.60Q612V1.090.960.990.970.920.940.900.940.91Q612W0.720.781.010.660.811.100.830.890.76Q612Y0.880.750.990.660.810.930.750.760.72A622A1.001.001.001.001.001.001.001.001.00A622D0.880.991.021.060.900.850.981.081.06A622E0.871.361.010.911.030.951.130.661.20A622F0.681.000.950.930.800.790.840.541.73A622G0.561.220.921.030.950.950.992.101.06A622H0.711.280.911.090.981.041.011.681.66A622I0.830.950.911.150.780.880.821.910.91A622K0.691.110.950.930.781.020.821.120.98A622L1.331.061.001.860.910.970.901.250.91A622M0.481.030.900.680.600.830.671.581.37A622N0.900.980.951.470.840.840.790.631.08A622P0.110.050.050.050.050.190.1016.240.74A622R0.991.160.951.410.920.950.930.621.15A622S0.851.140.991.280.900.980.981.511.03A622T0.521.160.970.570.730.840.821.561.05A622V0.471.191.000.590.780.900.821.691.28A622W1.510.930.961.110.840.860.830.380.87A622Y1.150.940.901.400.780.890.891.240.83Q626D0.040.040.040.040.040.040.040.040.04Q626E0.821.390.991.021.070.891.140.731.39变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCQ626F0.531.190.960.870.940.931.080.691.58Q626G0.471.010.880.510.700.800.771.571.53Q626H0.090.910.930.242.262.172.552.771.31Q626I4.620.050.050.050.050.120.050.050.07Q626K0.050.050.050.050.050.050.050.050.05Q626L1.180.950.961.580.810.880.751.140.85Q626M0.311.030.870.360.661.030.580.811.97Q626P0.040.040.040.040.040.040.040.040.04Q626Q1.001.001.001.001.001.001.001.001.00Q626R0.040.040.040.040.040.040.040.040.04Q626S0.040.040.040.040.040.040.040.040.04Q626T1.051.040.961.640.890.920.881.420.88Q626V1.060.950.920.960.800.890.791.400.80Q626W0.400.660.880.540.370.600.430.930.47Q626Y0.120.050.050.050.090.790.080.410.64V627D0.290.171.540.060.360.470.320.440.35V627K0.260.153.950.300.280.650.310.370.12V627P0.110.201.230.130.761.250.880.860.57V627Q0.280.211.120.210.370.670.400.300.78V627R0.260.211.250.240.390.590.430.360.52V627S0.310.251.110.310.380.530.420.520.72V627V1.001.001.001.001.001.001.001.001.00V627Y0.270.212.020.070.420.550.420.540.18T638A0.911.051.020.941.011.071.001.071.09T638D1.121.021.081.031.051.061.061.070.85T638E0.731.021.000.740.901.030.900.930.93T638F0.430.990.900.400.821.180.890.920.80T638G0.841.151.000.851.011.141.051.091.05T638I0.871.010.960.780.901.240.980.920.98T638K0.691.030.980.600.951.200.950.970.98T638L0.771.020.910.770.881.190.850.960.86T638M0.661.180.960.770.931.121.071.020.92T638P0.160.620.970.130.591.060.680.840.52T638Q0.711.031.000.740.931.241.071.010.95T638R0.891.061.030.790.991.161.101.020.93T638S0.871.080.980.940.981.201.041.090.95T638T1.001.001.001.001.001.001.001.001.00T638V0.761.030.960.730.951.141.051.020.91T638W0.063.960.980.384.865.157.448.322.49T638Y0.551.150.970.550.941.031.121.110.97S642A0.910.920.980.710.931.040.870.920.77S642C0.851.050.980.690.971.371.131.040.71S642D1.380.731.090.800.980.790.960.930.92S642E0.591.541.000.771.091.571.041.130.90S642F0.304.471.040.471.332.281.701.591.02S642G1.000.961.050.861.021.211.000.940.71变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCS642H0.491.700.950.631.071.661.131.100.88S642I0.541.451.070.521.011.931.110.990.95S642K0.990.860.990.710.941.220.890.820.79S642L0.431.991.050.571.042.931.191.221.02S642M0.481.621.020.530.911.761.221.000.70S642N0.891.051.020.821.001.210.950.930.73S642P0.551.430.940.671.001.531.081.010.75S642Q0.741.270.990.861.031.541.281.090.91S642R0.971.020.990.851.011.170.980.980.75S642S1.001.001.001.001.001.001.001.001.00S642T0.671.270.980.671.011.211.041.030.70S642V0.761.150.980.671.001.711.130.980.82S642W0.352.990.920.461.232.991.511.461.41S642Y0.276.120.970.451.394.902.031.740.98A643A1.001.001.001.001.001.001.001.001.00A643C0.770.820.970.600.991.091.000.910.90A643D0.040.040.040.040.040.040.040.040.04A643E0.960.981.060.960.960.991.020.910.94A643F0.360.511.020.170.821.171.030.940.73A643G0.520.601.110.430.901.040.950.870.85A643H0.550.751.010.680.961.121.021.020.78A643K0.250.481.030.281.091.411.261.290.89A643L0.831.030.990.980.981.071.111.070.99A643M1.111.121.041.120.961.171.051.000.93A643N0.660.761.100.620.920.991.020.970.90A643Q0.480.581.110.440.921.110.991.070.80A643R0.640.770.910.620.931.110.930.960.77A643S0.870.881.080.880.921.070.990.970.87A643T0.920.961.040.950.931.041.011.030.90A643V0.890.921.110.871.021.071.091.021.01A643W0.430.541.050.440.880.960.891.050.89A643Y0.540.701.040.450.971.061.081.210.89R645A0.570.871.030.791.041.270.981.120.81R645C0.180.320.960.270.880.860.730.800.33R645D0.800.891.121.030.951.090.931.030.82R645E0.370.491.150.600.930.820.760.850.57R645F0.520.761.050.650.981.020.941.100.77R645G0.891.170.981.231.041.221.081.191.02R645H0.520.681.010.720.951.010.820.970.85R645I0.440.651.000.601.021.030.891.030.73R645K0.851.071.001.061.061.061.111.160.98R645L0.450.711.040.701.051.000.971.280.74R645M0.460.821.070.891.171.091.071.320.97R645P0.950.781.000.930.881.080.770.820.77R645Q0.650.631.030.620.811.020.680.760.59R645R1.001.001.001.001.001.001.001.001.00变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR645S0.690.731.060.890.910.980.860.910.80R645T0.560.791.040.710.981.010.901.060.79R645V0.420.631.050.531.001.050.931.170.71R645W0.300.661.090.561.341.061.281.530.95R645Y0.570.891.040.801.141.161.131.190.98K649A1.241.041.010.710.791.030.880.891.07K649C0.691.080.960.801.021.041.070.971.02K649E0.400.780.900.420.891.000.860.800.96K649F0.771.060.970.770.971.050.950.901.00K649I0.671.010.960.880.970.970.920.900.82K649K1.001.001.001.001.001.001.001.001.00K649L0.861.040.981.300.991.070.980.900.92K649M0.941.120.931.330.920.950.910.940.88K649N0.090.370.860.141.331.571.540.820.58K649P0.410.200.770.070.230.320.180.140.30K649Q0.821.021.011.150.931.020.910.910.83K649S0.891.080.980.850.971.061.020.961.09K649T0.480.970.980.731.041.090.991.000.99K649W1.031.001.011.090.941.000.950.840.92K649Y0.801.100.961.000.991.030.990.990.84Q650A0.820.990.900.811.000.980.970.921.00Q650C0.970.970.990.731.051.181.010.980.99Q650D1.160.941.010.681.051.161.010.950.93Q650E0.520.800.920.590.951.070.921.131.07Q650F0.260.500.900.281.051.190.800.990.73Q650G0.800.960.940.711.081.200.971.031.03Q650H0.610.910.910.770.941.170.931.001.00Q650I0.500.840.920.510.971.120.931.060.83Q650K0.390.620.930.341.091.220.901.150.76Q650L0.570.890.940.670.981.120.961.020.96Q650M0.500.790.910.550.971.070.911.000.97Q650N0.540.880.940.531.091.271.001.261.00Q650Q1.001.001.001.001.001.001.001.001.00Q650R0.410.680.830.431.001.260.881.180.74Q650T0.760.940.970.651.010.981.021.040.98Q650V0.890.980.960.921.031.231.041.001.03Q650W0.260.530.820.260.860.900.790.960.75Q650Y0.550.910.870.611.041.171.031.111.04K656A0.070.050.050.050.050.360.050.050.35K656C0.070.050.050.050.060.250.050.050.44K656D0.070.050.050.050.080.140.050.050.37K656E0.060.050.050.090.070.120.050.050.33K656F0.050.050.050.050.050.050.050.050.05K656G0.050.050.050.050.050.350.050.050.28K656I0.080.050.050.050.080.310.050.050.37K656K1.001.001.001.001.001.001.001.001.00变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK656L0.060.050.050.050.070.130.050.050.44K656M0.050.050.050.050.050.050.050.050.05K656P0.070.050.050.050.050.160.050.050.43K656Q0.050.050.050.050.050.050.050.050.05K656R0.400.581.190.460.930.970.920.950.82K656S0.050.050.050.050.050.050.050.050.05K656T0.050.050.050.050.050.050.050.050.05K656V0.050.050.050.050.050.050.050.050.05K656W0.050.050.050.050.050.050.050.050.05K656Y0.050.050.050.050.050.050.050.050.05T660C0.591.061.041.081.171.161.251.200.90T660D0.851.080.991.241.050.981.001.010.95T660E0.860.921.081.240.950.840.910.820.98T660F0.610.861.061.080.970.961.010.960.86T660G0.830.921.080.301.000.800.930.930.86T660H0.620.920.951.200.970.930.950.910.80T660I0.901.011.111.600.990.990.990.950.80T660K0.680.901.021.060.950.780.860.920.76T660L0.420.800.970.920.980.910.980.920.58T660M0.610.841.101.130.950.830.900.890.69T660N0.470.881.060.951.070.941.041.030.88T660P0.160.331.250.300.660.550.570.700.32T660Q0.610.871.021.061.000.970.910.890.67T660R0.180.421.160.320.870.800.750.870.38T660S0.520.941.030.971.050.801.011.050.76T660T1.001.001.001.001.001.001.001.001.00T660V0.660.931.041.090.940.870.900.900.90T660W0.291.331.081.141.831.521.992.121.44T660Y0.620.971.091.191.011.111.041.010.83P661A0.820.941.020.991.030.971.001.171.00P661C0.621.221.031.051.281.091.371.511.52P661D0.751.041.031.031.040.861.071.111.16P661E0.450.981.000.931.180.881.141.060.86P661F0.231.071.010.751.711.481.881.741.43P661G0.660.871.070.930.910.980.951.090.94P661H0.360.860.980.661.181.061.171.240.90P661I0.281.071.050.871.521.141.641.741.56P661K0.470.830.980.741.041.031.021.141.05P661L0.191.180.940.841.941.292.282.241.63P661M0.150.790.880.511.611.171.721.901.02P661P1.001.001.001.001.001.001.001.001.00P661Q0.370.990.980.811.291.161.411.481.10P661R0.470.910.950.831.091.091.081.261.17P661S0.371.160.931.041.330.981.611.431.28P661T0.240.950.980.751.491.091.551.641.21P661V0.481.130.991.071.141.001.241.281.12变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCP661W0.141.060.930.592.401.512.612.811.70G662A0.301.330.960.961.811.341.931.811.46G662C0.161.770.991.273.342.494.244.022.74G662D1.270.970.991.440.980.910.930.881.05G662E1.330.861.021.490.910.980.780.760.81G662F0.591.361.011.711.351.141.481.331.42G662G1.001.001.001.001.001.001.001.001.00G662H0.891.010.971.391.011.070.940.900.85G662I0.210.671.120.111.321.031.361.380.69G662K0.770.971.051.360.971.050.880.870.74G662L1.020.941.011.510.950.980.890.800.85G662M0.900.971.041.360.980.980.880.850.83G662N0.670.981.071.321.030.860.990.920.79G662P0.250.560.960.500.870.920.780.780.44G662Q0.950.911.121.410.920.870.840.800.63G662R0.760.910.991.180.921.000.880.840.71G662S1.130.841.131.260.900.950.740.740.80G662T1.001.030.991.580.950.800.920.890.80G662V0.540.871.001.000.950.810.940.870.67G662W0.991.021.021.200.970.930.920.750.88G662Y0.731.021.001.240.991.200.930.970.80Q663A0.841.070.820.700.921.070.960.991.33Q663C0.771.010.970.640.961.060.991.031.44Q663D1.101.090.960.780.981.211.051.031.49Q663E0.301.240.920.541.461.611.561.731.80Q663F0.660.960.930.490.901.250.951.011.33Q663G1.131.040.850.980.950.961.051.001.31Q663H0.500.940.980.610.900.990.921.031.03Q663I0.601.090.850.620.931.301.061.090.97Q663K0.891.040.840.670.840.910.920.940.99Q663L0.680.940.790.680.871.110.940.971.09Q663M0.700.970.770.710.881.180.970.960.99Q663N0.780.970.780.740.881.100.920.921.23Q663Q1.001.001.001.001.001.001.001.001.00Q663R0.811.000.830.530.841.130.940.881.19Q663S0.851.000.980.840.911.070.950.961.17Q663T0.040.040.040.040.040.040.040.040.04Q663V0.890.891.000.720.861.090.920.871.12Q663W0.961.091.000.820.911.160.991.000.93Q663Y0.040.040.040.040.040.040.040.040.04T666A1.011.021.081.180.991.010.940.951.04T666C0.431.281.030.901.451.381.561.501.27T666D0.650.901.030.751.031.000.940.950.82T666E0.700.891.030.830.940.920.850.850.79T666F0.820.771.050.770.840.950.770.760.65T666G0.510.791.040.630.930.870.870.840.77变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCT666H0.560.821.010.720.911.020.840.880.72T666I0.640.791.000.800.840.960.810.900.77T666K0.670.890.970.740.901.100.870.970.85T666L0.650.931.040.860.810.960.860.830.83T666M0.620.971.000.890.880.990.950.860.84T666N0.491.170.990.911.091.151.211.191.01T666P0.470.850.980.770.910.980.830.880.64T666R0.610.880.990.750.831.050.901.010.74T666S0.680.891.020.940.950.980.860.920.81T666T1.001.001.001.001.001.001.001.001.00T666V0.690.921.040.890.880.800.860.910.86T666W0.630.800.990.710.901.000.830.810.74T666Y0.770.901.010.770.900.920.820.810.81R672A0.560.800.840.530.970.970.910.980.92R672C0.360.750.830.421.071.081.201.241.06R672D0.070.240.650.061.221.621.751.830.78R672E0.120.310.740.050.910.951.151.290.73R672F0.370.700.800.390.890.911.021.040.83R672G0.270.610.810.291.011.121.121.200.94R672H0.510.920.840.610.990.861.151.090.98R672I0.230.520.810.270.951.051.161.250.86R672K0.761.080.840.770.961.041.021.021.05R672L0.490.880.890.551.010.921.101.090.93R672M0.180.671.240.051.010.941.391.680.79R672N0.480.930.850.481.000.921.031.080.98R672P0.140.110.630.060.240.490.320.550.28R672Q0.150.190.760.050.470.770.670.860.34R672R1.001.001.001.001.001.001.001.001.00R672S0.110.130.750.050.380.680.480.830.36R672T0.320.870.850.441.170.971.381.481.12R672V0.560.920.920.571.001.011.051.070.96R672W0.160.430.850.170.991.251.281.530.75R672Y0.160.290.790.070.680.980.821.060.60R673A0.770.950.850.740.941.001.010.941.03R673C0.900.990.890.820.981.161.090.971.10R673E0.480.800.860.590.990.931.071.050.95R673F0.430.431.450.050.370.110.320.470.41R673G0.570.880.910.521.071.071.031.021.06R673H0.700.990.910.860.981.161.090.981.10R673I0.720.990.910.651.001.071.011.001.03R673K0.981.050.980.781.001.121.070.961.02R673L0.370.940.900.551.191.351.321.451.15R673M0.620.930.910.620.991.001.031.011.02R673N1.111.081.040.961.030.971.040.940.98R673P0.200.310.770.160.670.920.890.990.64R673Q0.760.970.880.720.991.031.001.010.95变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR673R1.001.001.001.001.001.001.001.001.00R673S0.791.020.960.831.020.941.101.041.07R673T0.891.080.940.851.031.000.961.001.10R673V0.991.000.960.760.991.101.010.931.09R673W1.210.981.011.110.931.070.970.961.07R674A0.390.050.050.190.390.510.300.270.34R674C0.420.050.050.500.370.400.290.270.75R674D0.040.040.040.040.040.040.040.040.04R674E0.240.050.050.320.200.270.110.050.38R674G0.220.050.050.320.550.750.390.270.49R674H0.260.050.050.530.190.240.130.050.15R674I0.250.050.050.400.160.310.080.050.20R674K0.830.421.250.310.770.930.830.830.66R674L0.380.441.080.600.831.070.880.730.79R674M0.320.280.050.700.921.120.890.931.07R674P0.260.050.050.230.210.320.100.050.16R674Q0.280.431.150.940.710.820.620.630.38R674R1.001.001.001.001.001.001.001.001.00R674S0.260.050.050.290.280.400.190.090.60R674T0.060.050.050.121.592.221.501.021.18R674V0.390.050.050.190.780.720.580.551.04R674W0.290.050.050.360.630.760.590.540.96R674Y0.070.050.050.332.362.462.092.150.42D675A0.090.050.050.110.170.270.440.520.58D675C0.090.050.050.130.241.060.450.640.75D675D1.001.001.001.001.001.001.001.001.00D675E0.212.241.110.200.620.950.990.830.55D675F0.110.050.050.210.440.780.790.850.94D675G0.070.050.050.110.050.520.340.700.72D675H0.281.781.040.300.800.940.960.850.80D675I0.040.040.040.040.040.040.040.040.04D675K0.070.050.050.080.080.050.470.610.46D675L0.100.050.050.210.380.410.671.380.67D675M0.090.050.050.170.350.050.640.740.74D675N0.100.050.050.220.400.480.660.820.87D675P0.080.050.050.080.050.050.350.500.41D675R0.040.040.040.040.040.040.040.040.04D675S0.231.891.090.240.710.800.830.840.70D675T0.070.050.050.070.120.680.690.580.70D675V0.070.050.050.080.050.050.290.670.62D675W0.070.050.050.100.050.050.250.650.48D675Y0.221.921.150.230.660.980.920.950.66D680A0.601.040.910.630.971.051.071.101.00D680C0.881.100.910.921.051.091.181.131.18D680D1.001.001.001.001.001.001.001.001.00D680E0.771.060.980.750.991.021.081.020.99变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCD680F0.300.900.880.441.031.421.111.180.97D680G0.610.920.930.630.930.980.990.960.87D680H0.410.950.910.510.991.311.111.190.90D680I0.280.950.870.371.071.391.221.260.85D680K0.840.970.860.751.001.070.980.920.84D680L0.250.710.920.270.881.530.951.020.78D680M0.501.080.860.611.011.231.071.051.06D680N0.430.900.910.460.951.200.990.990.94D680P0.090.150.050.050.250.910.310.160.33D680Q0.651.060.900.701.011.101.031.101.01D680R0.871.000.920.861.001.111.080.950.93D680S0.200.480.870.190.691.030.700.690.58D680V0.661.080.900.721.081.151.091.101.16D680W0.290.850.900.360.991.271.121.111.03D680Y0.481.100.910.631.061.251.211.211.02T681A0.871.060.990.911.041.450.991.081.10T681F0.040.040.040.040.040.040.040.040.04T681G0.481.051.020.701.061.041.231.221.30T681H0.490.890.930.560.931.111.011.050.91T681K0.520.990.920.610.991.041.101.111.06T681L0.670.931.020.770.951.021.151.011.01T681M0.531.000.970.611.001.031.101.191.19T681N0.590.930.990.650.941.060.971.010.95T681P0.440.920.950.561.010.961.191.091.06T681Q0.570.980.960.651.021.051.191.181.04T681R0.690.860.950.660.931.060.960.920.97T681S0.601.040.990.761.040.951.161.081.15T681T1.001.001.001.001.001.001.001.001.00T681V0.661.000.990.781.040.951.071.091.15T681W0.731.080.980.861.021.101.071.131.11T681Y0.610.800.960.570.730.931.150.960.90A682A1.001.001.001.001.001.001.001.001.00A682C0.450.331.020.160.510.730.490.550.39A682D0.450.420.870.220.620.860.610.730.56A682E0.260.211.110.130.410.640.410.530.29A682F0.280.270.860.170.470.620.540.650.59A682I0.110.142.290.140.490.900.600.870.42A682L0.260.261.680.180.540.660.610.730.45A682M0.300.211.400.100.390.670.420.481.09A682N0.330.341.060.470.530.620.590.700.67A682P0.280.221.310.220.420.610.400.570.26A682R0.040.040.040.040.040.040.040.040.04A682S0.290.241.960.120.450.610.450.540.51A682T0.330.300.940.160.480.650.470.570.28A682V0.340.410.800.220.640.930.600.720.52A682W0.370.281.340.150.490.610.450.570.35变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCA682Y0.380.311.540.160.490.630.480.580.38S683A0.910.961.030.880.981.100.920.970.95S683C0.870.911.030.830.951.211.120.940.75S683D0.450.651.060.390.871.041.010.900.86S683E0.580.911.000.670.930.931.031.011.04S683F0.140.500.960.221.031.351.411.340.84S683G0.861.010.980.880.981.211.040.960.98S683I0.310.770.950.380.961.191.151.080.98S683K1.010.871.060.840.920.890.890.880.96S683L0.620.951.020.720.971.110.981.110.90S683M0.530.921.000.600.970.991.281.071.01S683P0.570.901.000.620.891.081.021.050.96S683Q0.660.870.990.690.940.981.100.960.96S683R0.960.901.050.860.951.560.950.970.92S683S1.001.001.001.001.001.001.001.001.00S683V1.001.001.021.061.011.331.101.071.03S683W0.511.260.880.821.141.271.371.471.35Q684A0.531.140.950.540.881.100.931.041.05Q684C0.373.311.020.891.930.052.271.801.67Q684D1.030.871.040.860.911.060.900.710.85Q684E0.860.941.050.750.880.990.920.700.90Q684F1.520.841.111.320.900.910.890.680.87Q684G0.237.481.050.832.670.113.162.432.20Q684H1.250.971.001.170.960.980.920.750.99Q684I0.660.981.110.610.850.850.890.710.83Q684K1.150.831.051.050.810.930.850.690.86Q684L1.400.881.021.230.910.970.790.690.89Q684M1.530.801.101.310.880.860.820.700.85Q684N0.691.371.140.921.090.121.191.001.17Q684P0.351.281.150.340.820.930.860.770.90Q684Q1.001.001.001.001.001.001.001.001.00Q684R1.120.861.030.980.850.900.810.730.75Q684S1.860.791.001.390.870.970.810.690.79Q684T1.180.861.131.020.890.900.880.740.79Q684W0.040.040.040.040.040.040.040.040.04K685A0.671.230.981.021.161.021.180.991.16K685E0.691.120.950.921.030.861.010.811.09K685F0.601.130.980.891.021.150.990.850.62K685G0.411.150.940.751.221.241.140.950.96K685I0.351.130.950.721.251.271.151.121.07K685K1.001.001.001.001.001.001.001.001.00K685L0.651.300.981.161.151.071.191.011.13K685M0.531.320.990.971.131.231.211.051.18K685N0.371.210.940.760.931.101.251.090.95K685P0.040.040.040.040.040.040.040.040.04K685Q0.461.140.980.791.041.131.161.000.90变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCK685R0.571.110.970.910.781.110.990.890.84K685S0.761.300.991.161.111.061.071.031.18K685T0.391.200.980.771.171.211.221.111.17K685V0.421.220.960.850.961.011.201.030.97K685W0.511.330.990.891.281.171.331.011.14K685Y0.531.180.980.811.111.071.011.071.01S692C0.771.310.940.951.110.901.151.181.29S692D0.821.000.930.710.960.840.970.940.94S692E0.771.030.980.770.941.010.920.880.96S692F0.040.040.040.040.040.040.040.040.04S692G0.600.980.980.670.940.940.860.980.99S692H0.861.201.031.031.031.031.041.151.19S692I0.561.170.920.771.031.001.031.081.07S692K0.891.151.001.030.981.030.940.960.99S692L0.901.211.001.211.021.211.040.961.12S692M0.701.220.981.061.030.951.041.061.09S692N0.911.021.000.970.910.980.840.830.90S692P0.601.050.890.630.920.920.850.911.01S692Q0.791.060.990.840.971.140.880.980.90S692R0.950.890.980.730.830.920.800.800.92S692S1.001.001.001.001.001.001.001.001.00S692T0.891.141.031.010.991.050.971.000.89S692V0.861.121.100.801.051.211.001.061.12S692W0.661.180.990.621.021.111.071.251.02S692Y0.040.040.040.040.040.040.040.040.04R702C0.560.901.080.521.051.101.111.021.03R702D0.240.791.010.201.241.611.241.110.89R702F0.280.801.060.161.121.241.110.960.55R702G0.461.101.050.571.221.341.321.201.11R702H0.380.940.940.471.011.311.090.950.81R702I0.570.911.050.610.951.041.040.940.75R702K0.990.901.070.830.941.061.040.990.98R702L0.511.001.020.690.991.281.101.010.96R702M0.530.861.090.590.911.010.970.870.73R702N0.530.961.000.490.941.291.070.940.77R702P0.050.050.050.050.050.800.050.050.28R702Q0.570.911.060.560.951.131.011.040.84R702R1.001.001.001.001.001.001.001.001.00R702S0.430.931.010.441.011.041.051.111.01R702T0.390.940.970.431.011.301.061.010.83R702V0.650.971.050.641.051.111.131.180.82R702W0.560.951.120.580.981.181.111.200.94R705C0.640.970.830.671.001.050.970.961.01R705D0.250.370.990.130.720.940.560.740.52R705E0.240.440.850.230.710.870.610.830.76R705F0.470.930.960.531.041.180.991.110.95变体HPLCCNPGGlucHeatPASCPCSG2pH5G2pH6G2CCR705G0.330.760.900.340.961.000.890.980.79R705H0.370.840.960.710.991.090.951.080.85R705I0.591.020.920.841.151.221.131.231.07R705L0.490.960.881.071.071.080.961.100.81R705M0.420.900.770.701.061.190.920.990.87R705N0.280.591.000.400.850.900.720.870.82R705P0.270.671.040.410.851.070.770.940.67R705R1.001.001.001.001.001.001.001.001.00R705S0.350.870.910.611.061.070.931.050.86R705T0.410.940.850.701.031.080.981.101.24R705V0.561.030.950.801.070.981.001.051.15R705W0.430.930.830.511.051.150.971.080.87R705Y0.350.800.920.340.940.930.850.840.89上表针对活性和/或稳定性使用各种术语，例如，Heat、CNPG、PASC、PCS、GLUC、G2pH5、G2pH6、G2CC来描述突变。上升突变的PI为1或更大；中性突变的PI大于或等于0.5；无害突变的PI大于0.05；有害突变的PI为0.05。发生突变的位置分类如下：非完全限制性位置具有对于至少一种特性的至少一种中性突变，而完全限制性位置不具有对于活性和稳定性的中性突变。如在本发明的开发过程中所确定，红褐肉座菌BGL1的位置441和452是完全限制性位置。表3-1提供的数据可用于对任何β-葡萄糖苷酶进行工程改造，即使要进行工程改造的BGL在特定位置具有不同于红褐肉座菌BGL1的氨基酸。例如，表3-1中的数据可用于通过鉴定最佳选择置换(包括对红褐肉座菌BGL1野生型氨基酸的置换)来找到将改变所需的BGL特性的置换。如上所述对所有BGL1变体(3,153)进行分类。可组合的突变是指这样的突变，它们可被组合以得到具有对于一种或多种所需特性而言适当的性能指数的蛋白质。简而言之，鉴定了对于至少一种特性而言PI大于或等于0.5的2,609种适度可组合的变体。此外，鉴定了对于蛋白质表达(HPLC)而言PI为至少0.5并且对于所有其他特性而言PI大于0.8的365种高度可组合的变体，同时发现这些高度可组合的变体中的213种对于所有特性而言PI大于0.8。不可组合的变体是所有PI值≤0.05的那些变体。具有上述相对于红褐肉座菌BGL1的置换中的一种的任何BGL1变体，可通过将该氨基酸突变为该位置处的可组合置换中的一种来改善。在表3-1中列出的3153种BGL1变体中，鉴定了对于至少一种特性而言PI为1.0或更大的2,268种上升变体，同时鉴定了对于至少一种特性而言PI大于1.1的1,836种上升变体。在一些实施方案中，变体为在至少一种所关注的特性中PI大于或等于1.0的改善的变体。然而，在其他实施方案中，所关注的BGL1变体对于表达而言PI介于0.1和1.0之间。具体地讲，在适度至高水平的BGL1变体表达对于在发酵罐中生产蛋白质有害的情况下，对于表达而言PI介于0.1和1.0之间的变体是合乎需要的。同样地，在期望重组宿主细胞的培养基中酶浓度比率最佳的情况下，可能合乎需要的是使用具有降低的但可测量的表达水平的变体(0.1<PI<1.0)。在另外的实施方案中，所关注的BGL1变体对于热稳定性而言PI介于0.1和1.0之间。对于变体相比野生型或参考BGL具有降低的热稳定性但在所关注的条件下具有改善的活性的情况，那么对于热稳定性而言PI介于0.1和1.0之间的变体是合乎需要的。同样地，在期望重组宿主细胞的培养基中酶活性比率最佳的情况下，合乎需要的是使用具有降低的但明显的热稳定性水平的变体(0.1<PI<1.0)。同样在一些实施方案中，所关注的BGL1变体对于活性而言PI介于0.1和1.0之间。对于变体相比野生型或参考BGL具有降低的活性但在所关注的条件下具有改善的热稳定性的情况，那么对于活性而言PI介于0.1和1.0之间的变体是合乎需要的。同样地，在期望重组宿主细胞的培养基中酶活性比率最佳的情况下，合乎需要的是使用具有降低的但明显的活性水平的变体(0.1<PI<1.0)。表3-2总结了从上述研究中鉴定的特别值得关注的具有许多相比野生型BGL1改善的活性的变体。表3-2实施例4另外的BGL变体的表达、活性和性能4-1.检测分析当与实施例1中所描述的那些BGL变体相比较研究这个库中的BGL变体时，使用改进的HPLC检测分析来测定蛋白质含量。具体程序在下文描述。也改进了葡萄糖抑制检测分析，因此所使用的程序在下文描述。用于蛋白质含量测定的HPLC检测分析通过配备有在50℃下校准的ProswiftRP-2H50×4.6mm柱的Agilent1200(AgilentTechnologies)HPLC来测定来自合并培养物上清液的BGL变体的浓度。将五十(50)μL样品与50μL的10％乙腈混合，5min后，使用0.22μmMilliporeMultiscreenHTS96孔过滤系统真空过滤。将十(10)μL经过滤的样品上样到柱上。使用两种缓冲液建立流速为1mL/min的洗脱梯度：(1)缓冲液A：去离子水中0.3％PEG1000、0.1％TFA；和(2)缓冲液B：去离子水中64.63％乙腈、35％2-丙醇、0.3％PEG1000、0.07％TFA。使用以下程序进行洗脱：用5％缓冲液B持续0min至0.25min，接着用5％缓冲液B至35％缓冲液B的梯度持续0.25min至1min，然后用35％缓冲液B至55％缓冲液B的梯度持续1min至5min。使用纯化的野生型BGL1建立校准曲线。使用该标准校准曲线确定BGL变体的浓度。为计算性能指数(PI)，将BGL变体的浓度除以在相同板中野生型BGL1(例如，参考酶)的平均浓度。葡萄糖抑制检测分析通过在2.5mM葡萄糖存在下如上所述进行CNPG酶活性检测分析，来确定葡萄糖对β-葡萄糖苷酶的水解活性的影响。通过在葡萄糖存在下的平均比活性与在葡萄糖不存在时的平均比活性的比率，来确定变体和野生型蛋白质的相对残余活性。通过将BGL变体的相对残余活性除以野生型BGL1(例如，参考酶)的相对残余活性，来确定BGL变体的性能指数(PI)。4-2.另外的红褐肉座菌BGL1位点评估库的构建将含有野生型红褐肉座菌BGL1编码序列(SEQIDNO:1)的pTTTpyrG-bgl1质粒送至BASEClear(荷兰莱顿)，其随后构建以下表4-1的各位点处的位置库。参考BGL1成熟蛋白SEQIDNO:3的残基编号对各位点进行编号。对于表4-1中75个位点的每一个，作出约14至16个置换变体。然后如实施例2所述产生这些变体。4-3BGL1变体的表达、稳定性、活性和性能如本文所述，使用HPLC检测分析来测量变体的表达水平。计算PI值并且列于表4-2中标记为“HPLC”的一列下。还测量了CNPG水解活性。计算相应的PI值并且列于表4-2中标记为“CNPG”的一列下。还确定了葡萄糖对活性的影响或葡萄糖抑制，并且结果列于标记为“Gluc”的一列下。热稳定性测定值列于标记为“Heat”的一列下，PASC的水解中的比活性列于标记为“PASC”的一列下，PCS的水解中的比活性列于标记为“PCS”的一列下。还在pH5.0下测量了这些变体的纤维二糖酶活性，其结果列于标记为“G2pH5”的一列下。同样测量了氨预处理玉米芯(CC)的水解的比活性，结果列于标记为“CC”的一列下。如在表下方空白处所标记，基于范围对表4-2中所列出的PI进行分类。具体地讲，PI是变体与亲本或参考β-葡萄糖苷酶的性能比率。表4-2列出在第二SEL库中测试的1501种另外的置换。表4-2表4-2BGL1变体的性能变体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手动选择一些所关注的变体。表4-3包括来自第二SEL库的35种另外的此类变体。表4-3变体InhHeatHPLCPASCPCSG2CNPGCCL266Ywtwt--wt+wtwtwtI567Swt----wt+wtwt++A270Dwtwt--wt++wtwtwtS550Dwtwt--wtwtwtwt+T258Swtwt--+++++++++P536Dwtwt--+++wt++P536Vwtwt--+wt+++++F260D--+--wt+++++++F260G--++--wt+++++++Y530Fwtwt--+wt+++++S624N------+wt+wt+P607Qwt++--wtwt++++G606Mwt-----+++++++Q406Hwtwt---wt+++++wtN400Qwt++--wtwtwt-+G300Mwt----wtwt++wt+N038L++wt----------wtN038M++wt----------wtA601Ywtwt--++++++L293Vwt---+++++++wtT568K--++--++++++wtS308E+++--wt++wtwt++A630Y+++--wtwt+++++N461Dwtwt--++wtwtwtN146Dwt+++--wt+wt-wtA450Ewt---++++++++V043L+++---wtwtwt--wtQ220Awt--------------A655Qwt++--wtwt+++++S482Awt----++++++wt++A667Lwt---+++++-++A485T-----+++++-++K206Awt----++++++wt变体InhHeatHPLCPASCPCSG2CNPGCCY678Q-wt--+++++wt++如上所述分析从第一(实施例3)和第二(实施例4)SEL筛选得到的置换的结果的各种活性，并将这些结果相应地分组为具有两种、三种、四种、五种或六种(或更多种)活性的那些变体。具有这些多种活性的变体示于下表4-4至表4-7中：概括地说，表4-8列出具有两种或更多种改善的活性的所有变体，所述两种或更多种改善的活性选自(1)Heat(热稳定性)、(2)HPLC(蛋白质表达)、(3)PCS、(4)(PASC)、(5)G2(纤维二糖水解酶活性)、(6)通过G2+CC或CC水解测量的β-葡萄糖苷酶活性。表4-8I567QI567KI567SL266AN261CN566LS550QS384GF260TA565FI567RG606EI567ET258CN566PP607RS384WP607SA565KA565EG606HS283FF392QN566WN400QN038EA655NA565QA565SG606NS283PS624EA270KV602FN038MI671KA565VA565YG606ST258EP607CA270NA601GN038PP607IF556EF392YL293AT258IP604MF556HA601LV043HA450PF260IQ406HS308RT258KA377QF556KL293KV043WT242HP607EQ406TI444CT258QN461AP604NY575CY068ET568EG605RP604CM201DP536TN461FN461DY575RY068GA630YG300CN038FR542NP536WN461PN463EA450QY068MA655DA377CT568AD259SI532YT436AK206GI486CL110CE170FA377DN461GT243VY530TT436CA468QI486YL110GA338DS308CY639LL266FP607DT436FA468YA655SL110QS308EN146HY639MI567YQ406MT436IF556GQ245FL110WS507GN146ST243AA270RQ406ST436MF260SD329AA655HF260EA655CT243CS384CV602TT436QP604EP536FN264LT568KA655GQ245HA630WG300MT436YP604VF392CN566HF260LP176LQ245ME128RA630SQ220CN146DS624LF556VA633CT209IQ245TN146MA630TA655LY639TS624RP604YS312CS283DT646AN146VT180HT646HT221CS624WL293VN455DA270DT646CN146WT180MY678FN473SI486FA630GP536CN146YI671FL181FA450MA468IN583RI486WN461CA630QN261EI671LV043CI444ED177MR645GA667GN463TD215SN261KP607HY639PI444FP661EG662YA667SD457CG372AN400AT011ES507FI444NP536GP536QS384EQ220MG547AV602KT011YQ245PI444WP607QN369EL181MT221AF611AL293IN146ER179VI444YA655QN369WV043AT221GG662CN461SG606DF260AV500QI567VN369YV043GT221IG662FD457AY068VS474RA633IN566GP607KV043NA655RL293FV043QA377ID564TS482PA630KN146AQ060DA468FQ303NN461YN566FA667VY639KN146QA655YA468SK320SK206DA565CA485LQ245NN369TT242SQ216IG662DA468GA601DA485WK320YY639GS474DD564VL293MY575AA630HY678RA347YK206SY530SS692LQ220PY575KV466TV603GT436EA468TQ684NY639V实施例5BGL1组合库变体及其活性5.1检测分析：用于测定蛋白质含量的HPLC检测分析使用配备有在35℃下平衡的ShodexHICPH-814PHM75×8mm凝胶柱(Phenomenex)的Agilent1200(AgilentTechnologies)HPLC，测定合并的培养物上清液中每种BGL多肽(野生型或变体)的浓度。将四十五(45)μL的上清液与15μL的80ppm重组表达的褶皱链霉菌(S.plicatus)糖苷酶EndoH(例如NEBP0702L)在200mM醋酸钠缓冲液(pH5.0)中温育，并且在37℃下温育过夜并伴以900rpm的振动。将六十(60)μL的1.6M(NH4)2SO4加入到上清液，并且在5min后，使用0.22μmMilliporeMultiscreenHTS96孔过滤系统真空过滤混合物。将四十(40)μL经过滤的样品上样到柱上。采用两种洗脱缓冲液来建立洗脱梯度：(1)缓冲液A：16mMNaH2PO4(pH6.75)，800mM(NH4)2SO4；和(2)缓冲液B：16mMNaH2PO4(pH6.75)。使用以下程序以1.8mL/min的流速进行洗脱：0％缓冲液B持续0min至0.5min，接着0％缓冲液B至50％的梯度持续0.5min至1min，接着50％缓冲液B至100％持续1min至6min，之后100％缓冲液B持续6至8min。使用由纯化的野生型BGL1建立的校准曲线确定BGL变体的蛋白质浓度。为计算性能指数(PI)，将BGL变体的浓度除以在相同板中野生型BGL1(例如，参考酶)的平均浓度。使用CNPG酶活性检测分析来测定检测分析所需的样品稀释度测量BGL变体对于氯-硝基酚-β-D-葡糖苷(CNPG)的活性以确定BGL1产生水平。将五(5)μL的上清液加入到95μL的在50mM醋酸钠缓冲液(pH5)中的1mMCNPG，并且在微板读板机中记录OD405读数3min。基于CNPG活性和相对于野生型BGL1对照的活性，将上清液稀释至介于25和300ppmBGL1的水平。CNPG酶活性检测分析确定BGL变体对于氯-硝基酚-β-D-葡糖苷(CNPG)的活性。将表达BGL变体的培养物上清液在含有0.1mg/mL牛血清半蛋白(BSA)的50mM醋酸钠缓冲液(pH5.0)中稀释5倍、6.67倍、10倍和20倍。将稀释的上清液的五十(50)μL等份试样加入到在50mM醋酸钠缓冲液(pH5.0)中的50μL的2mMCNPG，达到最终浓度为1mMCNPG。通过监测OD405来确定CNP释放动力学，所述OD405在微量滴定板读板机(Spectramax,MolecularDevices)中记录3min。通过将平均CNPG水解活性除以BGL多肽浓度来计算野生型BGL1和BGL变体的平均比活性。通过将BGL变体的比活性除以在相同板上野生型BGL1(例如，参考酶)的平均比活性来计算性能指数(PI)。热稳定性检测分析使用CNPG检测分析来测定热温育后BGL1变体的残余活性。将表达BGL变体的培养物上清液在含有0.1mg/mLBSA的50mM醋酸钠缓冲液(pH5.0)中稀释5倍、6.67倍、10倍和20倍。在66℃下，使一式四份的八十(80)μL等分试样在带裙边96孔PCR板中在热循环仪中温育1hr。在冰上冷却5min后，如上所述测定每种野生型和BGL1变体的残余比活性。通过温育后的平均比活性与温育前的平均比活性的比率来确定变体和野生型BGL1的残余活性。通过将BGL1变体的残余活性除以野生型BGL1(例如，参考酶)的相对残余活性来确定BGL变体的性能指数(PI)。葡萄糖抑制检测分析通过在18.75mM葡萄糖存在下，如上所述重复CNPG酶活性检测分析来确定葡萄糖对β-葡萄糖苷酶的水解活性的影响。通过在葡萄糖存在下的平均比活性与在葡萄糖不存在时的平均比活性的比率，来确定变体和野生型蛋白质的相对残余活性。通过将BGL变体的相对残余活性除以野生型BGL1(例如，参考酶)的相对残余活性，来确定BGL变体的性能指数(PI)。磷酸溶胀纤维素(PASC)水解检测分析中的比活性根据公布的方法从Avicel制备磷酸溶胀纤维素(PASC)(参见，例如，Walseth,Tappi,35:228,1971；和Wood,Biochem.J.,121:353-362,1971)。用醋酸钠缓冲液和水稀释该材料以获得1％w/v混合物，其中醋酸钠的最终浓度为50mM，并且pH为5.0。将一百五十(150)μL的在50mM醋酸钠缓冲液(pH5.0)中的1％PASC悬浮液分配到96孔微量滴定板(CostarFlatBottomPS)中。将十(10)μL来自bgl1缺失菌株的含有0.75mg/mL蛋白质的培养物上清液加入到PASC。然后将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经8倍稀释(在50mM醋酸钠缓冲液(pH5.0)中)后取5、10、20或40μL加入到PASC/缺失突变体上清液混合物。加入补偿体积的醋酸钠缓冲液以补足在总体积方面的差异。将微量滴定板密封并在50℃恒温培养箱中温育，并以900rpm连续振动。2小时后，通过向每孔加入100μL100mM甘氨酸缓冲液(pH10)来终止水解反应。将板密封并在室温下以3000rpm离心5min。通过ABTS检测分析来分析上清液中的水解反应产物。建立野生型BGL1蛋白质的剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。稀酸预处理玉米秸秆(PCS)水解检测分析中的比活性用2％w/wH2SO4预处理玉米秸秆(参见，Schell等人，JAppl.Biochem.Biotechnol.,105:69-86,2003)，然后用去离子水多次洗涤以获得pH为4.5的浆料。然后加入醋酸钠缓冲液(pH5.0)(至最终浓度为50mM醋酸钠)，并且如果必要的话，使用1NNaOH进一步将该混合物滴定至pH5.0。反应混合物中的纤维素浓度为大约7％。向96孔微量滴定板(NuncFlatBottomPS)每孔加入六十五(65)μL的此纤维素悬浮液。将十(10)μL来自bgl1缺失菌株的含有10mg/mL蛋白质的培养物上清液加入到PCS。然后将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经2倍稀释(在50mM醋酸钠缓冲液(pH5.0)中)后取5、10、20或40μL加入到PCS/缺失突变体上清液混合物。加入补偿体积的醋酸钠缓冲液以补足在总体积方面的差异。密封后，将板置于50℃恒温培养箱中，并且以900rpm连续振动。16小时后，将板置于冰上5min，并且通过向每孔加入100μL的100mM甘氨酸缓冲液(pH10)来终止水解反应。将板密封并在室温下以3,000rpm离心5min。通过ABTS检测分析(如上)来分析上清液中存在的水解反应产物。从纯化的野生型BGL1建立剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。纤维二糖酶活性检测分析测试了野生型BGL1和BGL变体在pH5.0下的纤维二糖水解能力。将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液经4倍稀释(在50mM醋酸钠缓冲液(pH5.0)中)后取不同的数量(5、10、15或20μL)加入到80μL的在50mM醋酸钠缓冲液(pH5.0)中的16.4mM(5.63mg/mL)纤维二糖溶液。加入补偿体积的醋酸钠缓冲液以补足在总体积方面的差异。将微量滴定板密封并且在50℃恒温培养箱中温育，并以900rpm连续振动。30min后，使板在冰上冷却并且向每孔加入100μL的100mM甘氨酸缓冲液(pH10)。通过ABTS检测分析(如上)来分析水解反应产物。使用纯化的野生型BGL1建立剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。氨预处理玉米芯水解检测分析中的β-葡萄糖苷酶比活性研磨玉米芯以通过0.9mm筛，并如PCT专利申请公布WO2006110901中所描述进行预处理。将经预处理的玉米芯作为在50mM醋酸钠缓冲液(pH5.0)中的7％纤维素悬浮液使用。向96孔微量滴定板(NuncFlatBottomPS)每孔加入六十五(65)μL的悬浮液。将过表达里氏木霉内切木聚糖酶基因xyn3、轮枝镰孢(Fusariumverticillioides)β-木糖苷酶基因Fv3A、轮枝镰孢β-木糖苷酶基因Fv43D，和轮枝镰孢α-阿拉伯呋喃糖苷酶基因Fv51A的含有在50mM醋酸钠缓冲液(pH5.0)中的0.76mg/mL蛋白质的十(10)μL里氏木霉菌株加入到经预处理的玉米芯。将来自表达野生型BGL1或BGL变体的红褐肉座菌细胞的合并的培养物上清液取不同的数量(5、10、20或40μL)加入。加入补偿体积的醋酸钠缓冲液以补足在总体积方面的差异。将微量滴定板密封并在50℃恒温培养箱中温育，并以900rpm连续振动。24小时后，通过向每孔加入100μL100mM甘氨酸缓冲液(pH10)来终止水解反应。混合后，使板以3,000rpm离心5min。通过ABTS检测分析(如上)来分析水解反应产物。使用纯化的野生型BGL1建立剂量反应曲线。为计算性能指数(PI)，将由变体BGL产生的(平均)总糖除以由相同剂量下野生型BGL1(例如，参考酶)产生的(平均)总糖。5-2.红褐肉座菌BGL组合变体的产生组合BGL变体是构建的，或者购自商业供应商，例如，SloningBiotechnologyGmbH(德国Puchheim)、BaseClear(荷兰莱顿)。表5-1列出被选择包含在BGL组合库中的置换。根据参考野生型BGL1成熟氨基酸序列SEQIDNO:3分配氨基酸残基编号。表5-1.被选择用于构建组合变体的BGL1置换。在大肠杆菌中产生衍自pTTTpyrG-bgl1的组合变体，并将其接种在具有100μg/mL氨苄青霉素的2×TY琼脂板(16g/LBacto胰蛋白胨(Difco,USA)、10g/LBacto酵母提取物(Difco,USA)、5g/LNaCl、16g/LBacto琼脂(Difco,USA))上。在37℃下过夜温育后，从含有100μg/mL氨苄青霉素的2×TY琼脂板挑选携带bgl1变体的大肠杆菌菌落，并使其在37℃下在含有具有100μg/mL氨苄青霉素和50μg/mL卡那霉素的1mL2×TY培养基的微量滴定板中生长24hr。使用细菌培养物来提纯质粒DNA。编码bgl1组合变体的纯化pTTTpyrG-bgl1衍生质粒以150-300ng/μL的浓度用于红褐肉座菌转化。这些在cbh1启动子下表达bgl1变体的复制型质粒使经转化的红褐肉座菌细胞能在乙酰胺上生长。如在例如美国专利申请公布US2006/0094080A1中所述，用五(5)μL的质粒进行真菌转化。用各个pTTTpyrG-bgl1c构建体转化红褐肉座菌菌株的原生质体(Δeg1、Δeg2、Δcbh1、Δcbh2、Δbgl1)(即，每次转化包括单一BGL1变体)，并使其在28℃下在含有乙酰胺的选择性培养基上在24孔微量滴定板中生长7天。收获来自各个变体的红褐肉座菌转化株初始群落的孢子，并在乙酰胺琼脂板上重新选择。使用生理盐水溶液收获孢子，在96孔微量滴定板上重新排布，并用于对多个生产培养基接种以产生BGL变体样品。为此目的，对每孔含有250μL甘氨酸生产培养基的96孔过滤板(Corning，商品号3505)以一式四份接种红褐肉座菌转化株的孢子悬浮液，所述培养基含有4.7g/L(NH4)2SO4；33g/L1,4-哌嗪双(丙磺酸)(pH5.5)；6.0g/L甘氨酸；5.0g/LKH2PO4；1.0g/LCaCl2×2H2O；1.0g/LMgSO4×7H2O；2.5mL/L的400X里氏木霉微量元素，含有5g/LFeSO4×7H2O、1.4g/LZnSO4×7H2O、1.6g/LMnSO4×H2O、3.7g/LCoCl2×6H2O；20g/L葡萄糖；和6.5g/L槐糖。使板在28℃和80％湿度下温育6至8天。通过真空过滤收获培养物上清液。使用如实施例1A所述的己糖激酶检测分析来测量这些上清液滤液中的残余葡萄糖。如上所述测试置换的组合的各种活性。此测试的结果示于下表5-2中。表5-2：组合BGL变体的性能进一步分析组合置换的结果以确定具有相比野生型BGL1改善的至少两种、三种、四种、五种或六种(或更多)活性的那些变体。具有这些多种改善的活性的变体示于下表5-3至表5-6。表5-3.具有至少两种改善的活性的包含置换的组合的变体表5-4.具有至少三种改善的活性的包含置换的组合的变体表5-5.具有至少四种改善的活性的包含置换的组合的变体HPLC+PASC+PCS+CCInh+HPLC+PSC+CCL167W|D177M|Q626FN238W|R265P|K656RL167W|D177M|D225Q|D564V|Q684GN264M|R265P(+G662F)D177M|D225Q|D564T|Q684NN264L|A338I|S474R|G662DD177M|Q626F|Q684RInh+Heat+HPLC+PCSHPLC+PASC+PCS+G2L167W|D225Q|D564V|Q626F|Q684ND177M|D225Q|D564T|Q684AD177M|D225Q|D564V|Q626F|Q684NInh+PASC+G2+CCInh+HPLC+PASC+PCSL167W|D177M|Q626F|Q684GR265M|K560SL167W|D177M|D564V|Q626F|Q684AHeat+PASC+G2+CCHPLC+PCS+G2+CCP176L|K320S|V522Y|G662CK345E|N369E|G372A|P661EN369T|P661L|S683WHeat+HPLC+PCS+G2N369T|G372A|P661L|S683WP176L|Q226W|K320Y|R363E表5-6.具有至少五种、六种或七种改善的活性的包含置换的组合的变体实施例6表现降低的葡萄糖抑制的BGL1组合变体选择多种BGL变体并测试它们在一系列浓度的葡萄糖的存在下水解CNPG的能力。将产生野生型BGL1或BGL变体的红褐肉座菌菌株的培养物上清液稀释至20mOD/min的最低CNPG活性。然后将野生型BGL1或BGL变体上清液与不同数量的葡萄糖混合，使最终葡萄糖浓度介于0和25mM之间。通过加入在50mM醋酸钠缓冲液(pH5.0)中的1mMCNPG来开始检测分析。通过在SpectraMax读板机(Moleculardevices)中记录OD405nm3min来进行动力学测量。使用公式y＝a/(1+x/b)测量IC50值，其中y表示CNPG比活性(单位为mOD/min)，x表示抑制性底物浓度(单位为mM葡萄糖)，a表示最大反应速率(CNPG活性，单位为mOD/min)，b表示酶活性降低一半时的抑制剂浓度。为计算抑制的降低，将给定BGL变体得出的IC50值除以野生型BGL1得出的IC50值。表6-1：葡萄糖抑制活性检测分析中BGL变体的性能指数。*‘+’、‘++’、‘+++’和‘++++’表示与用野生型BGL1观察到的葡萄糖抑制相比，葡萄糖抑制分别降低1.2至2倍、2至3倍、3至6倍、6至10倍。在不脱离本发明的范围和精神的情况下，本发明的各种修改形式和变型对本领域技术人员将显而易见。虽然已结合具体优选的实施方案描述了本发明，但应当理解受权利要求书所保护的本发明不应不当地限制于此类具体实施方案。实际上，本领域技术人员理解的用于实施本发明的所述方式的各种修改形式旨在落入权利要求的范围内。当前第1页1 2 3