葡萄糖异构酶突变体的应用的制作方法

专利名称：葡萄糖异构酶突变体的应用的制作方法
技术领域：
本发明涉及分子生物技术领域，具体地说，涉及利用基因突变技术制备高活力、或高活力并耐热的葡萄糖异构酶突变体的方法、所获得的突变体及其应用。
背景技术：
葡萄糖异构酶(Xylose isomerase，E.C.5.3.1.5，简称XI)、或称木糖异构酶(Glucose isomerase)是戊糖发酵途径的关键酶。该酶是最重要的酶制剂之一(Kaneko，et al.，Biosci Biotechnol Biochem，64940-947，2000)。在生物能源工业中，人们正在试图使用该酶并连同其它纤维素与半纤维素降解途径中的关键酶商业化地生产生物乙醇。例如，从木聚糖至5-磷酸木酮糖的酶解途径如下木聚糖在内-β-1，4-木聚糖酶的作用下生成低聚木聚，后者再在β-木糖苷酶的作用下生成木糖，该木糖又可被葡萄糖异构酶(即木糖异构酶)催化生成木酮糖，最后该木酮糖在β-木酮糖激酶的作用下，生成5-磷酸木酮糖。
迫于环境污染和石油紧缺的压力，世界上许多国家，如巴西、美国、加拿大，早已以甘蔗或玉米为原料工业化生产燃料乙醇。中国也已利用玉米或其他粮食生产生物乙醇。由于自然界中生物质资源十分丰富，世界上多个国家的科学家正在探索以饱含纤维素与半纤维素的残余农作物秸秆等为原料，工业化地生产生物乙醇。葡萄糖异构酶是生物降解半纤维素的关键酶之一。高活力、或高活力并耐热的葡萄糖异构酶有利于降低生物乙醇的生产成本，进而实现工业化生产生物乙醇。
世界各地的科学家对从耐热菌中筛选并通过蛋白质工程的方法培育耐热、高催化活性的葡萄糖异构酶进行了许多研究。如J.G.Zeikus及其合作者从Thermoanaerobacterium saccharolyticum、Thermotoga neapolitana等数种耐高温菌中分离并研究了耐高温葡萄糖异构酶(见Lee et al.，Journal of General Microbiology，1391227-1234，1993；Vieille et al.，Methods Enzymology，330215-24，2001；Lee et al.，Journal of General Microbiology，1391241-1243，1993；Scriprapundh et al.，Protein Engineering，13259-265，2000；Scriprapundh et al.，Protein Engineering，16683-690，2003；Zeikuset al.，US Patent No 5,656,497)。这些或其它来源的耐高温葡萄糖异构酶之活力较低，尚未见用于工业化生产生物乙醇。因此，目前仍存在高活力、或高活力并耐热的葡萄糖异构酶的需求。

发明内容
本发明人对利用遗传工程和蛋白质工程技术对来源于T.saccharolyticum的葡萄糖异构酶进行改良所获得的一系列突变体的酶活性进行了研究，从而提供了该突变体在较高温度下使用并能够高活性地催化D-木糖转变为木酮糖的应用。
本发明人通过对T.saccharolyticum葡萄糖异构酶基因进行定点突变，随后在MacConkey培养基上筛选，从而取得一系列高催化活性、或具高催化活性并耐热的葡萄糖异构酶突变体。具体而言，利用分子生物学技术，构建含有亲本葡萄糖异构酶基因的载体质粒，然后设定定点突变的位点以及突变后的氨基酸种类，再合成适当的引物，以所述的含亲本葡萄糖异构酶基因的载体质粒或其衍生质粒为模板，PCR扩增DNA片段、装配所扩增的DNA片段以及PCR扩增全长突变基因。通过将该全长突变基因克隆到适当的载体上并转化适当的宿主细胞，经培养筛选出具有葡萄糖异构酶活性的阳性克隆。从阳性克隆中提取质粒DNA，进行DNA序列测定分析，以确定引入的突变。最后，以D-木糖为底物测定具有所设定位点突变的葡萄糖异构酶的酶活力，将该酶活力与亲本的酶活力相比较，选取具有比亲本的活力高的催化活性的葡萄糖异构酶突变体，将之用于将D-木糖转变为木酮糖的工艺、降解纤维素的工艺以及生产生物乙醇的工艺。
在上述筛选突变体的过程中，所述载体可为任何适当的载体。例如，适用的载体包括但不限于原核表达载体pGEMT-Easy，pRSET和pET21；包括但不限于真核表达载体pYD1和pYES2/GS；包括但不限于克隆载体pUC18/19和pBluscript-SK。
所获得的葡萄糖异构酶突变体基因可以在原核细胞或真核细胞胞内表达，也可采用本领域已知的任何其它适当方法实现在原核细胞或真核细胞胞外表达。
所述载体的微生物宿主细胞为原核细胞或真核细胞。所述原核微生物包括但不限于大肠杆菌、结芽孢杆菌、枯草芽胞杆菌、巨大芽胞杆菌(如巨大芽胞杆菌BP931)、T.saccharolyticum和链霉菌(如Streptomyces diastaticus M1033)。所述真核微生物包括但不限于酿酒酵母和毕赤巴斯德酵母(如毕赤巴斯德酵母GS115/9891)。
在根据本发明的应用中，以序列表中的序列2为参考序列，所述葡萄糖异构酶突变体具有至少一个、至少两个、至少三个、至少四个、至少五个、至少六个、至少七个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的至少一个突变，并且在以D-木糖为底物生成木酮糖的反应中所述突变体具有比亲本高出至少50％的酶催化活性。所述的葡萄糖异构酶突变体还可用于半纤维素的降解过程，并适宜用于生产生物乙醇。
以序列表中的序列2为参考序列，优选的是，所述第87位的苯丙氨酸(Phe)突变为甲硫氨酸(Met)、或亮氨酸(Leu)；第139位的色氨酸突变为丝氨酸(Ser)、或赖氨酸(Lys)、或半胱氨酸(Cys)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或天冬酰胺(Asn)、或天门冬氨酸(Asp)；第182位的精氨酸突变为丙氨酸(Ala)、或脯氨酸(Pro)、或丝氨酸(Ser)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或缬氨酸(Val)；第187位的缬氨酸突变为甘氨酸(Gly)、或丙氨酸(Ala)、或脯氨酸(Pro)；第217位的缬氨酸(Val)突变为精氨酸(Arg)、或甘氨酸(Gly)；第260位的天门冬氨酸(Asp)突变为谷氨酸(Glu)、或丙氨酸(Ala)；第276位的苯丙氨酸(Phe)突变为甘氨酸(Gly)、或苏氨酸(Thr)；和/或第299位的苏氨酸突变为异亮氨酸(Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或甲硫氨酸(Met)、或谷氨酸(Glu)。
更为优选的是，在本发明的应用中，所述的突变体具有序列表中序列5至序列40之一(即SEQ ID NO.5至SEQ ID NO.40中之一)所示的氨基酸序列。
最优选的是，在本发明的应用中，所述葡萄糖异构酶突变体包含下文表2中所列的那些突变的氨基酸序列。因为表2所列的突变体具有高的催化活性并耐热。例如，一个具有七个点突变的突变体MGI4-34的比活性较亲本高305.9％，且在80℃反应19.2小时后仍保持50％或以上的活力。另一个具有七个点突变的突变体MGI4-35的比活性较亲本高296.3％，且在80℃反应大于24小时后仍保持50％或以上的活力。
在本发明的应用中，所述葡萄糖异构酶突变体可以未经纯化粗酶形式使用，也可以是经部分纯化的或完全纯化的形式。如果需要，还可利用本领域已知的固化技术将所述葡萄糖异构酶突变体制成固相酶或固相细胞。可以将表达葡萄糖异构酶突变体的表达载体转入细菌(如产酸克雷伯菌和运动发酵单胞菌)、酒精酵母菌或其它宿主(如毕赤巴斯德酵母)中以加速木糖的代谢。具体地讲，所述葡萄糖异构酶突变体的表达菌，包括天然或工程的生物乙醇发酵用的细菌，如产酸克雷伯菌(Klebsiella oxytoca)和运动发酵单胞菌(Zymomonas mobilis)；天然或工程的生物乙醇发酵用的酿酒酵母真菌；以及天然或工程的生物乙醇发酵用的毕赤巴斯德酵母真菌等。
定义本申请文本中所用的术语“亲本”系指来自Thermoanaerobacterium saccharolyticum ATCC 49915的葡萄糖异构酶，其核苷酸序列如序列1所示，氨基酸序列如序列2所示。本发明中亲本基因的核苷酸序列与公布的T.saccharolyticum葡萄糖异构酶的基因序列(Lee et al.，Journal of General Microbiology，1391227-1234，1993；GenBank L09699)相比有两个核苷酸的差异，即与基因库(GenBank L09699)的核苷酸序列相比，本发明中亲本基因第241-242位为GC，相应的第81位氨基酸序列为丙氨酸(Ala)，GenBankL09699对应处、第241-242位核苷酸序列为CG，相应的第81位氨基酸序列为精氨酸(Arg)。
本申请文本中所用的术语“参考序列”，当其为核苷酸序列时，系指序列表中的序列1，当其为氨基酸序列时，是指序列表中的序列2。在将参考序列和突变的葡萄糖异构酶序列进行排序比较时，可以手工进行，也可以用计算机进行(目前有许多可供利用的计算机软件，例如CLUSTALW，AMAS，DIALIGN程序等)。
本申请文本中所用的术语“位点”或“第x位”是指当本发明的葡萄糖异构酶突变体的序列与参考序列的排序对比达最大同源性时，被比较的葡萄糖异构酶突变体的各核苷酸或氨基酸所对应的参考序列中的核苷酸或氨基酸位置。
本申请文本所用的术语“葡萄糖异构酶突变体”是指一种以序列表中序列2所示氨基酸序列为参考序列，在第87位、第139位、第182位、第187位、第217位、第260位、第276位、第299位具有至少一个氨基酸的差异，并且以D-木糖为底物生成木酮糖时其具有比亲本高出至少50％的葡萄糖异构酶催化活性的酶。因此，在本专利申请中，所述葡萄糖异构酶突变体包括具有序列4所示氨基酸序列的突变体、序列4的保守取代形式、增加或缺失一个或几个氨基酸的形式、氨基端截断的形式、羧基端截断的形式、以及序列4的部分或全部串联重复形式。
在本申请文本所用的氨基酸三字母或单字母表达方式，采用IUPAC规定的氨基酸代码(Eur.J.Biochem.，1389-37，1984)。


图1亲本葡萄糖异构酶与葡萄糖异构酶突变体在80℃的热稳定。MGI-4、MGI4-34和MGI4-35为葡萄糖异构酶突变体，具体请参见实施例8和15。
具体实施例方式
下列实施例仅用于说明本发明而不应视为限定本发明的范围。
实施例中未注明具体条件者，按常规条件或制造商建议的条件进行。
实施例1亲本基因的扩增及pGEMT-TS的构建根据基因库(GenBank L09699)基因序列设计引物T1和T2(见表1)。利用引物对T1和T2从T.saccharolyticum ATCC 49915(购自ATCC，USA)中扩增葡萄糖异构酶亲本基因。
扩增条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1，400nM引物T2，1.5U Taq DNA聚合酶(Promega，USA)，用接种环挑取少许T.saccharolyticum菌体，再用无菌水调反应体积至50μl。
PCR扩增反应程序为95℃3分钟，40圈循环95℃50秒、50℃30秒和72℃1分钟，最后72℃10分钟。将扩增的产物(长约1.5kb)连接至载体pGEMT-Easy，得质粒pGEMT-TS。经DNA测序确定亲本葡萄糖异构酶的核苷酸序列为序列1，相应的氨基酸序列为序列2。与基因库(GenBank L09699)的核苷酸序列相比，本发明中亲本基因第241-242位为GC，相应的第81位氨基酸序列为丙氨酸(Ala)。GenBankL09699对应处、第241-242位核苷酸序列为CG，相应的第81位氨基酸序列为精氨酸(Arg)。
实施例2葡萄糖异构酶位点139之定点突变定点突变技术参考Ho et al.(Gene 7751-59，1989和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对139FF和139FR(见表1)，将亲本氨基酸序列中第139位点的Trp(W)突变为Phe(F)，获得突变体MGI-W139F。引物对T1和T2见实施例1。
利用引物对T1和139FR，扩增T1FR片段，引物对139FF和T2，扩增FFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物139FR或400nM引物139FF和400nM引物T2，1.5UPfu DNA聚合酶(Promega，USA)，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1FR片段和FFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μMdCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段与20ng FFT2片段，用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI-W139F。将MGI-W139F与载体pGEMT-Easy连接，得质粒pGEMT-MGI-W139F。将质粒pGEMT-MGI-W139F转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-W139F DNA，经DNA测序确定引入的点突变无误。MGI-W139F氨基酸序列见序列表序列5，比活力见表2。
实施例3葡萄糖异构酶位点182之定点突变定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对182AF和182AR(见表1)，将亲本氨基酸序列中第182位点的Arg(R)突变为Ala(A)，获得突变体MGI-R182A。引物T1和T2见实施例1。
利用引物对T1和182AR，扩增T1AR片段，引物对182AF和T2，扩增AFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物182AR或400nM引物182AF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到T1AR片段和AFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1AR片段与20ng AFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到全长突变基因MGI-R182A。将MGI-R182A与载体pGEMT-Easy连接，得质粒pGEMT-MGI-R182A。将质粒pGEMT-MGI-R182A转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-R182ADNA，经DNA测序确定引入的点突变无误。MGI-R182A的氨基酸序列见序列表序列6。
实施例4葡萄糖异构酶位点187之定点突变定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对187SF和187SR(见表1)，将亲本氨基酸序列中第187位点的Phe(F)突变为Ser(S)，获得突变体MGI-F187S。引物T1和T2见实施例1。
利用引物对T1和187SR，扩增T1SR片段，引物对187SF和T2，扩增SFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物187SR或400nM引物T2和400nM引物187SF，1.5UPfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到T1SR片段和SFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1SR片段与20ngSFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick ExtractionGel Kit回收，得到全长突变基因MGI-F187S。将MGI-F187S与载体pGEMT-Easy连接，得质粒pGEMT-MGI-F187S。将质粒pGEMT-MGI-F187S转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-F187SDNA，经DNA测序确定引入的点突变无误。MGI-F187S的氨基酸序列见序列表序列7。
实施例5葡萄糖异构酶位点299之定点突变定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对299QF和299QR(见表1)，将亲本氨基酸序列中第299位点的Thr(T)突变为Gln(Q)，获得突变体MGI-T299Q。引物T1和T2见实施例1。
利用引物对T1和299QR，扩增T1QR片段，引物对299QF和T2，扩增QFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物299QR或400nM引物299QF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到T1QR片段和QFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1QR片段与20ng QFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到全长突变基因MGI-T299Q。将MGI-T299Q与载体pGEMT-Easy连接，得质粒pGEMT-MGI-T299Q。将质粒pGEMT-MGI-T299Q转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-T299QDNA，经DNA测序确定引入的点突变无误。MGI-T299Q的氨基酸序列见序列表序列8，比活力见表2。
实施例6葡萄糖异构酶双突变组合MGI-2之构建定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按实施例2扩增和回收T1FR片段，按实施例3扩增和回收AFT2片段。用引物对139FF(如实施例2)和182AR(如实施例3)扩增和回收FFAR片段。FFAR片段扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 139FF和400nM 182AR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到FFAR片段。然后扩增全长基因。扩增反应条件为20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ngT1FR片段，20ng FFAR片段和20ng AFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick DNA回收试剂盒，得到全长突变基因MGI-2。将MGI-2与载体pGEMT-Easy连接，得质粒pGEMT-MGI-2。将质粒pGEMT-MGI-2转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-2 DNA，经DNA测序确定引入的点突变无误。MGI-2序列含有W139F和R182A的二个突变。MGI-2的氨基酸序列见序列表序列9，比活力见表2。
实施例7葡萄糖异构酶三突变组合MGI-3之构建定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按实施例2扩增和回收T1FR片段，按实施例5扩增和回收QFT2片段，按实施例6扩增和回收FFAR片段。用引物对182AF(如实施例3)和299QR(如实施例5)扩增和回收AFQR片段。AFQR片段扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM 182AF和400nM 299QR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到AFQR片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段，20ng FFAR片段，20ng AFQR片段和20ng QFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI-3。将MGI-3与载体pGEMT-Easy连接，得质粒pGEMT-MGI-3。将质粒pGEMT-MGI-3转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-3DNA，经DNA测序确定引入的点突变无误。MGI-3序列含有W139F、R182A和T299Q的三突变。MGI-3的氨基酸序列见序列表序列10，比活力见表2。
实施例8葡萄糖异构酶四突变组合MGI-4之构建定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按实施例2扩增和回收T1FR片段，按实施例5扩增和回收QFT2片段，按实施例6扩增和回收FFAR片段。用引物对182AF(如实施例3)和187SR(如实施例4)扩增AFSR片段。AFSR片段扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μMdCTP，50μM dGTP，400nM 182AF和400nM 187SR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到AFSR片段。用引物对187SF(如实施例4)和299QR(如实施例5)扩增SFQR片段。SFQR片段扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM 187SF和400nM 299QR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到SFQR片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段，20ng FFAR片段，20ng AFSR片段，20ng SFQR片段和20ng QFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI-4。将MGI-4与载体pGEMT-Easy连接，得质粒pGEMT-MGI-4。将质粒pGEMT-MGI-4转入感受态细菌细胞E.coliHB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-4DNA，经DNA测序确定引入的点突变无误。MGI-4序列含有W139F、R182A、F187S和T299Q的四突变。MGI-4的氨基酸序列见序列表序列11，比活力见表2。
实施例9葡萄糖异构酶四突变组合MGI-4之139位点的饱和突变定点突变技术参考Ho et al.(Gene 7751-59，1989和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例8)为模板，设计引物对139SF和139SR(见表1)，将亲本氨基酸序列中第139位点的Phe(F)突变为Ser(S)，获得突变体MGI4-F139S。引物对T1和T2见实施例1。
利用引物对T1和139SR，扩增T1SR片段，引物对139SF和T2，扩增SFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物139SR或400nM引物139SF和400nM引物T2，1.5UPfu DNA聚合酶(Promega，USA)，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1SR片段和SFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5UPfu DNA聚合酶，20ng T1SR片段与20ng SFT2片段，用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-F139S。将MGI4-F139S与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-F139S。将质粒pGEMT-MGI4-F139S转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-F139S DNA，经DNA测序确定引入的点突变无误。MGI4-F139S序列含有F139S、R182A、F187S和T299Q的四突变。MGI4-F139S氨基酸序列见序列表序列12，比活力见表2。
按照类似步构建突变体MGI4-F139K、MGI4-F139C、MGI4-F139I、MGI4-F139T、MGI4-F139N和MGI4-F139D，所用引物见表1。所得诸突变体的氨基酸序列见序列表序列13-18，比活力见表2。
实施例10葡萄糖异构酶四突变组合MGI-4之182位点的饱和突变定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例8)为模板，设计引物对182PF和182PR(见表1)，将亲本氨基酸序列中第182位点的Ala(A)突变为Pro(P)，获得突变体MGI4-A182P。引物T1和T2见实施例1。
利用引物对T1和182PR，扩增T1PR片段，引物对182PF和T2，扩增PFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物182PR或400nM引物182PF和400nM引物T2，1.5UPfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到T1PR片段和PFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1PR片段与20ng PFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到全长突变基因MGI4-A182P。将MGI4-A182P与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-A182P。将质粒pGEMT-MGI4-A182P转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-A182P DNA，经DNA测序确定引入的点突变无误。MGI4-A182P序列含有W139F、A182P、F187S和T299Q的四突变。MGI4-A182P的氨基酸序列见序列表序列19，比活力见表2。
按照类似步构建突变体MGI4-A182S、MGI4-A182I、MGI4-A182T和MGI4-A182V，所用引物见表1。所得诸突变体的氨基酸序列见序列表序列20-23，比活力见表2。
实施例11葡萄糖异构酶四突变组合MGI-4之187位点的饱和突变定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例8)为模板，设计引物对187GF和187GR(见表1)，将亲本氨基酸序列中第187位点的Ser(S)突变为Gly(G)，获得突变体MGI4-S187G。引物T1和T2见实施例1。
利用引物对T1和187GR，扩增T1GR片段，引物对187GF和T2，扩增GFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％ Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物187GR或400nM引物T2和400nM引物187GF，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到T1GR片段和GFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1GR片段与20ng GFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到全长突变基因MGI4-S187G。将MGI4-S187G与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-S187G。将质粒pGEMT-MGI4-S187G转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-S187G DNA，经DNA测序确定引入的点突变无误。MGI4-S187G序列含有W139F、R182A、S187G和T299Q的四突变。MGI4-S187G的氨基酸序列见序列表序列24，比活力见表2。
按照类似步构建突变体MGI4-S187A和MGI4-S187P，所用引物见表1。所得诸突变体的氨基酸序列见序列表序列25-26，比活力见表2。
实施例12葡萄糖异构酶四突变组合MGI-4之299位点的饱和突变定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例8)为模板，设计引物对299IF和299IR(见表1)，将亲本氨基酸序列中第299位点的Gln(Q)突变为Ile(I)，获得突变体MGI4-Q299I。引物T1和T2见实施例1。
利用引物对T1和299IR，扩增T1IR片段，引物对299IF和T2，扩增IFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物299IR或400nM引物299IF和400nM引物T2，1.5U PfuDNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到T1IR片段和IFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1IR片段与20ngIFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick ExtractionGel Kit回收，得到全长突变基因MGI4-Q299I。将MGI4-Q299I与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-Q299I。将质粒pGEMT-MGI4-Q299I转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-Q299IDNA，经DNA测序确定引入的点突变无误。MGI4-Q299I序列含有W139F、R182A、F187S和Q299I的四突变。MGI4-Q299I的氨基酸序列见序列表序列27，比活力见表2。
按照类似步构建突变体MGI4-Q299Y、MGI4-Q299C、MGI4-Q299M和MGI4-Q299E，所用引物见表1。所得诸突变体的氨基酸序列见序列表序列28-31，比活力见表2。
实施例13葡萄糖异构酶五突变组合MGI4-F87L、MGI4-F87M、MGI4-V217R、MGI4-D260E和MGI4-F276G之构建定点突变技术参考Ho et al.(Gene 7751-59，1989和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.Humana Press，1993)之描述。
以质粒pGEMT-MGI-4(见实施例8)为模板，设计引物对87LF和87LR(见表1)，将MGI-4氨基酸序列中第87位点的Phe(F)突变为Leu(L)，获得突变体MGI4-F87L。引物对T1和T2见表1。
用引物对T1和87LR，扩增T1LR片段，用引物对87LF和T2，扩增LFT2片段。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物87LR或400nM引物87LF和400nM引物T2，1.5U PfuDNA聚合酶(Promega，USA)，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1LR片段和LFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μMdCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段与20ng LFT2片段，用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-F87L。将MGI4-F87L与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-F87L。将质粒pGEMT-MGI4-F87L转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-F87L DNA，经DNA测序确定引入的点突变无误。所得突变体序列含有F87L、W139F、R182A、F187S和T299Q五个突变。MGI4-F87L氨基酸序列见序列表序列32，比活力见表2。
按照类似步构建突变体MGI4-F87M，所用引物见表1。MGI4-F87M突变体序列含有F87M、W139F、R182A、F187S和T299Q五个突变。MGI4-F87M的氨基酸序列见序列表序列33，比活力见表2。按照类似步构建突变体MGI4-V217R引物见表1。所得突变体序列含有W139F、R182A、F187S、V217R和T299Q五个突变。MGI4--V217R的氨基酸序列见序列表序列34，比活力见表2。按照类似步构建突变体MGI4-D260E所用引物见表1。所得突变体序列含有W139F、R182A、F187S、D260E和T299Q五个突变。MGI4-D260E的氨基酸序列见序列表序列35，比活力见表2。按照类似步构建突变体MGI4-F276G所用引物见表1。所得突变体序列含有W139F、R182A、F187S、F276G和T299Q五个突变。MGI4-F276G的氨基酸序列见序列表序列36，比活力见表2。
实施例14葡萄糖异构酶六突变组合MGI4-24和MGI4-25之构建定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按实施例13扩增和回收T1LR片段。用引物对87LF和260AR(见表1)扩增和回收LFAR片段。LFAR片段扩增反应条件为20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM 87LF和400nM 260AR，1.5U Pfu DNA聚合酶，20ngpGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到LFAR片段。用引物对260AF和T2(见表1)扩增和回收AFT2片段。AFT2片段扩增反应条件为20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM 260AF和400nM T2，1.5U Pfu DNA聚合酶，20ngpGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到AFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段，20ng LFAR片段和20ng AFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick DNA回收试剂盒，得到全长突变基因MGI4-24。将MGI4-24与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-24。将质粒pGEMT-MGI4-24转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-24DNA，经DNA测序确定引入的点突变无误。MGI4-24的氨基酸序列见序列表序列37。所得突变体序列含有F87L、W139F、R182A、F187S、T299Q和D260A六个突变，比活力见表2。
按照类似步构建突变体MGI4-25。突变体MGI4-25所用引物对为T1和87LR、87LF和276TR、276TF和T2(均见表1)，所得突变体序列含有F87L、W139F、R182A、F187S、T299Q和F276T六个突变。所得突变体的氨基酸序列见序列表序列38，比活力见表2。
实施例15葡萄糖异构酶七突变组合MGI4-34和MGI4-35之构建定点突变技术参考Ho et al.(Gene 7751-59，1989)和White et al.(PCR Protocolcurrent methods and applications.Totowa，N.J.HumanaPress，1993)之描述。
按实施例13扩增和回收T1LR片段。用引物对87LF和217GR(见表1)扩增和回收LFGR片段。LFGR片段扩增反应条件为20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μMdGTP，400nM 87LF和400nM 217GR，1.5U Pfu DNA聚合酶，20ngpGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到LFGR片段。用引物对217GF和276TR(见表1)扩增和回收GFTR片段。GFTR片段扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM (NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 217GF和400nM 276TR，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到GFTR片段。用引物对276TF和T2(见表1)扩增和回收TFT2片段。TFT2片段扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 276TF和400nM T2，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到TFT2片段。然后扩增全长基因。扩增反应条件为20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段，20ng LFGR片段，20ng GFTR片段和20ng TFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为95℃3分钟，35圈循环95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-34。将MGI4-34与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-34。将质粒pGEMT-MGI4-34转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-34DNA，经DNA测序确定引入的点突变无误。MGI4-34序列含有F87L、W139F、R182A、F187S、T299Q、V217G和F276T七个突变。MGI4-34的氨基酸序列见序列表序列39，比活力见表2。
按照类似步骤构建七突变体MGI4-35。突变体MGI4-35所用引物对为T1和87LR、87LF和217GR、217GF和260AR、260AF和T2(均见表1)，所得突变体序列含有F87L、W139F、R182A、F187S、T299Q、V217G和D260A七突变。所得突变体的氨基酸序列见序列表序列40，比活力见表2。
扩增亲本葡萄糖异构酶(亲本)与实施例1-12所葡萄糖异构酶突变体所用的引物如下表1所列。
表1



实施例16亲本葡萄糖异构酶的提取与纯化葡萄糖异构酶的提取与纯化主要参考Lee et al.，Journal ofGeneral Microbiology，1391227-1234(1993)。
将含亲本葡萄糖异构酶基因的质粒pGEMT-TS转化感受态细菌细胞E.coli HB101，在MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上、37℃培养36小时。接种单个克隆在5ml LB液体培养基(含50mg/L氨苄青霉素)中培养16小时。离心收集菌体，并悬浮于1ml 20mM磷酸钠缓冲液(pH 6.5)中，加CoCl2和MgCl2至终浓度分别为250μM和5mM。然后用超声波裂解细菌细胞。离心(10℃，17，800g，15分钟)并收集上清液为粗提蛋白。粗提蛋白经80℃热处理10分钟后，离心(10℃，17，800g，15分钟)去沉淀，即为部分纯化的葡萄糖异构酶，可用于酶活性的测定。
实施例17葡萄糖异构酶突变体的提取与纯化葡萄糖异构酶突变体MGI4-35的提取与纯化与实施例16同，惟所用质粒为pGEMT-MGI4-35。其它葡萄糖异构酶突变体也照实施例16所述提取与纯化。
实施例18亲本葡萄糖异构酶活性的测定配底物溶液A含1.0M的D-木糖、20mM磷酸钠缓冲溶液(pH6.5)、终浓度为250μM之CoCl2和终浓度为5mM之MgCl2，调pH至6.5。取底物溶液A 90μl，然后加入10μl按实施例16制备的葡萄糖异构酶。反应于80℃进行10分钟。将反应物置冰上以终止反应。反应产物D-木酮糖通过半胱氨酸-咔唑法测定。测定方法参见Dische et al.，J.Biol.Chem，192583-587，1951)以及Nakamura，Agr.Biol.Chem.32701-706，1968)。用CoomassiePlusProtein Assay Reagent Kit(PIERCE，USA)并结合SDS聚丙烯酰胺胶电泳测定酶蛋白浓度。一单位酶比活性定义为在上述条件下每分钟转化一微摩尔D-木糖为木酮糖所需之酶量。下列表2显示亲本葡萄糖异构酶之比活性。
实施例19葡萄糖异构酶突变体活性的测定葡萄糖异构酶突变体MGI4-35活性测定与实施例18同。其它葡萄糖异构酶突变体的活性也照实施例18所述测定。下表2显示诸葡萄糖异构酶突变体与亲本葡萄糖异构酶(亲本)比活性之差异。
表2亲本葡萄糖异构酶与葡萄糖异构酶突变体比活性之差异


实施例20亲本葡萄糖异构酶热稳定性测定置按实施例16获得的部分纯化的亲本葡萄糖异构酶于7管1.5ml离心管中。每管离心管分别加入200μl酶液，并加入200μl矿物油。将离心管置于80℃水浴中，0小时、4小时、12小时、24小时后分别取出一管酶液，离心(10℃，17，800g，20分钟)后，取上清液，按实施例18所述、测定葡萄糖异构酶残余蛋白和残余比活性。图1显示亲本葡萄糖异构酶在80℃的热稳定。
实施例21葡萄糖异构酶突变体热稳定性测定葡萄糖异构酶突变体MGI-4、MGI4-34、或MGI4-35热稳定性测定与实施例20同。图1显示葡萄糖异构酶突变体MGI-4、MGI4-34、或MGI4-35在80℃的热稳定。如图所示，亲本葡萄糖异构酶的活性在80℃的半衰期为13.4小时；MGI-4的活性在80℃的半衰期为21.4小时；MGI4-34的活性在80℃的半衰期为19.2小时；MGI4-35的活性在80℃的半衰期大于24小时。
本发明不受上述具体文字描述的限制，本发明可在权利要求书所概括的范围内做各种改变。这些改变均在本发明的范围之内。
序列表<110>百瑞全球有限公司(BioRight Worldwide Company Ltd.)<120>葡萄糖异构酶突变体的应用(Use of Glucose Isomerase Mutants)<130>FI-050438-59<160>40<170>PatentIn version 3.2<210>1<211>1320<212>DNA<213>Thermoanaerobacterium saccharolyticum<400>1atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat60ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag 120catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa 240gcaagggtag aagcagcatt tgagtttttt gataagataa atgcaccttt cttctgcttc 300catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat 360acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgtggggt 420accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct 480gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt 540ggccgcgaaa actacgtatt ttggggtgga agagaagggt acgagacgct tctcaataca 600gatatggagt tagagcttga taactttgca agatttttgc acatggctgt tgactatgca 660aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa 720catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttgac 780aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcattcca cgacttccaa 840catgagctaa gatacgccag aataaacggt gtattaggat caattgacgc aaatacaggc 900gacatgcttt tgggatggga tacggaccag ttccctacag atatacgcat gacaacgctt 960gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca 1020aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg 1080gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac 1140aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc 1200ggtaaagctg acttcaagag ccttgaaaag tatgcattag agcacagcca gattgtaaac 1260aaatcaggca gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa 1320<210>2<211>439
<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>2Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435
<210>3<211>1320<212>DNA<213>Thermoanaerobacterium saccharolyticum<220>
<221>misc_feature<222>(259)..(261)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for phenylalanine.
<220>
<221>misc_feature<222>(415)..(417)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for typtophan.
<220>
<221>misc_feature<222>(544)..(546)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid Arginine.
<220>
<221>misc_feature<222>(559)..(561)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for phenylalanine.
<220>
<221>misc_feature<222>(649)..(651)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid valine.
<220>
<221>misc_feature<222>(778)..(780)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for aspartic acid.
<220>
<221>misc_feature
<222>(826)..(828)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for phenylalanine.
<220>
<221>misc_feature<222>(895)..(897)<223>″n″represents a，t，g or c；and″nnn″represents any other aminoacid code except for threonine.
<400>3atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat60ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag 120catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa 240gcaagggtag aagcagcann ngagtttttt gataagataa atgcaccttt cttctgcttc 300catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat 360acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgnnnggt 420accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct 480gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt 540ggcnnngaaa actacgtann ntggggtgga agagaagggt acgagacgct tctcaataca 600gatatggagt tagagcttga taactttgca agatttttgc acatggctnn ngactatgca 660aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa 720catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttnnn 780aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcannnca cgacttccaa 840catgagctaa gatacgccag aataaacggt gtattaggat caattgacgc aaatnnnggc 900gacatgcttt tgggatggga tacggaccag ttccctacag atatacgcat gacaacgctt 960gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca 1020aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg 1080gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac 1140aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc 1200ggtaaagctg acttcaagag ccttgaaaag tatgcattag agcacagcca gattgtaaac 1260aaatcaggca gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa 1320<210>4<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<220>
<221>MISC_FEATURE<222>(87)..(87)<223>Xaa represents any amino acid residue other than phenylalanine.
<220>
<221>MISC_FEATURE<222>(139)..(139)<223>Xaa represents any amino acid residue other than tryptophan.
<220>
<221>MISC_FEATURE<222>(182)..(182)<223>Xaa represents any amino acid residue other than Arginine.
<220>
<221>MISC_FEATURE<222>(187)..(187)<223>Xaa represents any amino acid residue other than phenylalanine.
<220>
<221>MISC_FEATURE<222>(217)..(217)<223>Xaa represents any amino acid residue other than valine.
<220>
<221>MISC_FEATURE<222>(260)..(260)<223>Xaa represents any amino acid residue other than aspartic acid.
<220>
<221>MISC_FEATURE<222>(276)..(276)<223>Xaa represents any other amino acid residue other than phenylalanine.
<220>
<221>MISC_FEATURE<222>(299)..(299)<223>Xaa represents any other amino acid residue other than threonine.
<400>4Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Xaa Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Xaa Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Xaa Glu Asn Tyr Val Xaa Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Xaa Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Xaa Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Xaa His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Xaa Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>5<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>5Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Ghy Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>6<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>6
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>7<211>439<212>PRT
<213>Thermoanaerobacterium saccharolyticum<400>7Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435
<210>8<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>8Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430
Asn Gln Tyr Leu Phe Ala Glu435<210>9<211>439<212>PRT<213>Thermoanaerobacter Iactoethylicum<400>9Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>10<211>439<212>PRT<213>Thermoanaerobacter Iactoethylicum<400>10Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>11<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>11Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu
130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>12<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>12Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ser Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>13<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>13Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Lys Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>14<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>14Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Cys Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>15<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>15Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ile Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>16<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>16Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Thr Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>17<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>17Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Asn Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>18<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum
<400>18Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Asp Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>19
<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>19Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Pro Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu
435<210>20<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>20Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ser Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>21<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>21Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175Thr Lys Glu Leu Gly Ile Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>22<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>22Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Thr Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>23<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>23Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Val Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>24<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>24Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Gly Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Va Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>25<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>25Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ala Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>26<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>26Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Pro Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>27<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>27Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Ile Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>28<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>28Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Tyr Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>29<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>29Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Cys Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>30<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>30
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Met Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>31<211>439
<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>31Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Glu Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435
<210>32<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>32Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430
Asn Gln Tyr Leu Phe Ala Glu435<210>33<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>33Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Met Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>34<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>34Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Arg Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>35<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>35Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Glu Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>36<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>36Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Gly His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>37<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>37Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Ala Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>38<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>38Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Thr His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>39<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>39Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Gly Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Thr His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu435<210>40<211>439<212>PRT<213>Thermoanaerobacterium saccharolyticum<400>40Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro1 5 10 15Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val20 25 30Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr50 55 60Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys65 70 75 80Ala Arg Val Glu Ala Ala Leu Glu Phe Phe Asp Lys Ile Asn Ala Pro85 90 95Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu100 105 110Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp115 120 125Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu130 135 140Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala145 150 155 160Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile165 170 175Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu180 185 190Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn195 200 205Phe Ala Arg Phe Leu His Met Ala Gly Asp Tyr Ala Lys Glu Ile Gly210 215 220Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys225 230 235 240His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys245 250 255Tyr Asp Leu Ala Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala260 265 270Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu290 295 300Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu305 310 315 320Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu325 330 335Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu340 345 350Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys355 360 365Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu370 375 380Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser385 390 395 400Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser405 410 415Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu420 425 430Asn Gln Tyr Leu Phe Ala Glu43权利要求
1.一种葡萄糖异构酶突变体的应用，其特征在于以序列表中的序列2为参考序列所述的突变体具有至少一个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的至少一个突变，并且在以D-木糖为底物生成木酮糖的反应中所述突变体具有比亲本高出至少50％的酶催化活性。
2.权利要求1所述的应用，其中第87位的苯丙氨酸(Phe)突变为甲硫氨酸(Met)、或亮氨酸(Leu)。
3.权利要求1所述的应用，其中第139位的色氨酸突变为苯丙氨酸(Phe)、或丝氨酸(Ser)、或赖氨酸(Lys)、或半胱氨酸(Cys)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或天冬酰胺(Asn)、或天门冬氨酸(Asp)。
4.权利要求3所述的应用，其中所述的突变体具有序列表中SEQID NO.5所示的氨基酸序列。
5.权利要求1所述的应用，其中第182位的精氨酸突变为丙氨酸(Ala)、或脯氨酸(Pro)、或丝氨酸(Ser)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或缬氨酸(Val)。
6.权利要求5所述的应用，其中所述的突变体具有序列表中SEQID NO.6所示的氨基酸序列。
7.权利要求1所述的应用，其中第187位的苯丙氨酸突变为甘氨酸(Gly)、或丙氨酸(Ala)、或丝氨酸(Ser)、或脯氨酸(Pro)。
8.权利要求7所述的应用，其中所述的突变体具有序列表中SEQID NO.7所示的氨基酸序列。
9.权利要求1所述的应用，其中第217位的缬氨酸(Val)突变为精氨酸(Arg)、或甘氨酸(Gly)。
10.权利要求1所述的应用，其中第260位的天门冬氨酸(Asp)突变为谷氨酸(Glu)、或丙氨酸(Ala)。
11.权利要求1所述的应用，其中第276位的苯丙氨酸(Phe)突变为甘氨酸(Gly)、或苏氨酸(Thr)。
12.权利要求1所述的应用，其中第299位的苏氨酸突变为谷氨酰胺(Gln)、或异亮氨酸(Ile)、或酪氨酸(Tyr)、或半胱氨酸(Cys)、或甲硫氨酸(Met)、或谷氨酸(Glu)。
13.权利要求12所述的应用，其中所述的突变体具有序列表中SEQ ID NO.8所示的氨基酸序列。
14.权利要求1所述的应用，其中所述的突变体具有至少两个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突变。
15.权利要求14所述的应用，其中所述的突变体具有序列表中SEQ ID NO.9所示的氨基酸序列。
16.权利要求14所述的应用，其中所述的突变体具有至少三个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突变。
17.权利要求16所述的应用，其中所述的突变体具有序列表中SEQ ID NO.10所示的氨基酸序列。
18.权利要求16所述的应用，其中所述的突变体具有至少四个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突变。
19.权利要求18所述的应用，其中所述的突变体具有序列表中SEQ ID NO.11至SEQ ID NO.31之一所示的氨基酸序列。
20.权利要求18所述的应用，其中所述的突变体具有至少五个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突变。
21.权利要求20所述的应用，其中所述的突变体具有序列表中SEQ ID NO.32至SEQ ID NO.36之一所示的氨基酸序列。
22.权利要求20所述的应用，其中所述的突变体具有至少六个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突变。
23.权利要求22所述的应用，其中所述的突变体具有序列表中SEQ ID NO.37或SEQ ID NO.38所示的氨基酸序列。
24.权利要求23所述的应用，其中所述的突变体具有至少七个选自第87位、第139位、第182位、第187位、第217位、第260位、第276位和第299位的突变。
25.权利要求24所述的应用，其中所述的突变体具有序列表中SEQ ID NO.39或SEQ ID NO.40所示的氨基酸序列。
26.权利要求1所述的应用，其中所述的突变体用于从半纤维素转化成乙醇。
全文摘要
本发明披露应用基因突变技术改良的一系列高催化活性Thermoanaerobacterium saccharolyticum葡萄糖异构酶突变体的应用。这些葡萄糖异构酶突变体可以木糖为底物高效率地生成木酮糖。
文档编号C12N9/92GK1966702SQ200510123630
公开日2007年5月23日 申请日期2005年11月18日 优先权日2005年11月18日
发明者王骏, 傅荣昭, 张天佐 申请人:百瑞全球有限公司