本发明涉及农业生物技术领域。具体地，本发明涉及热稳定性提高的甘露聚糖酶pman5a突变体及其基因和应用。
背景技术：
甘露聚糖是植物半纤维素的主要成分，主要存在于软木植物及一些特殊结构如植物种子中，它也是重要的植物性饲料原料。甘露聚糖的结构比较复杂，其主链是以1,4-β-d-吡喃甘露糖苷键连接而成的线性多糖，并且主链或侧链具有多种取代基。由于甘露聚糖的多样性与结构的复杂性，它的完全水解需要多种酶的协同作用才能完成，包括内切-β-甘露聚糖酶、外切-β-甘露糖苷酶、β-葡萄糖苷酶、乙酰甘露聚糖酯酶和α-半乳糖苷酶等。其中，内切-β-甘露聚糖酶能够降解甘露聚糖主链的β-1,4-糖苷键，是甘露聚糖降解过程中发挥最重要作用的酶。
按照糖苷水解酶家族分类，目前所发现的内切-β-甘露聚糖酶可以分为gh5，26，113或134家族，其中，有关gh5家族β-甘露聚糖酶的报道已有许多，但由于其对极端环境耐受性差、催化活性低和对底物亲和力弱等缺点极大地限制了其在各领域的应用，并催生了新型酶基因的开发及酶分子改造方面的研究。影响β-甘露聚糖酶结构与特性因素有很多，包括氢键、盐桥、二硫键，蛋白质的热稳定性与多种结构特性因素相关。
技术实现要素：
针对现有的β-甘露聚糖酶的耐受能力差、催化活性低的问题，本发明通过将pman5a中第93、94、356和389位点的氨基酸h、f、l和a分别突变为y、y、h和p，或组合突变，获得热稳定性提高的甘露聚糖酶突变体。
本发明的目的是提供包含单点h93y、f94y、l356h、a389p及组合h93y/f94y、h93y/l356h、h93y/a389p、l356h/a389p、h93y/f94y/l356h、h93y/l356h/a389p、h93y/f94y/l356h/a389p突变的突变体。
本发明的再一目的是提供编码上述突变体的基因。
本发明的再一目的是提供包含上述突变体基因的重组载体。
本发明的再一目的是提供包含上述突变体基因的重组菌株。
根据本发明的技术方案，野生甘露聚糖酶pman5a的氨基酸序列如seqidno.1所示：
其中，该酶n端前19个氨基酸为信号肽序列“mksaililpflshlavsqt”。
根据本发明的技术方案，对野生型甘露聚糖酶pman5a成熟蛋白进行改造，其氨基酸序列如seqidno.2所示：
根据本发明的耐热性提高的甘露聚糖酶pman5a突变体，是通过将成熟的甘露聚糖酶pman5a进行h93y、f94y、l356h和/或a389p点突变而获得，其中，将甘露聚糖酶成熟蛋白的n端第93位组氨酸(h)突变为酪氨酸(y)，获得突变体h93y，其氨基酸序列如seqidno.3所示：
根据本发明具体实施方式，突变体f94y的甘露聚糖酶成熟蛋白的n端第94位苯丙氨酸(f)突变为酪氨酸(y)，其氨基酸序列如seqidno.4所示：
根据本发明具体实施方式，突变体l356h的甘露聚糖酶成熟蛋白的c端第356位亮氨酸(l)突变为组氨酸(h)，其氨基酸序列如seqidno.5所示：
根据本发明具体实施方式，突变体a389p的甘露聚糖酶成熟蛋白的c端第389位丙氨酸(a)突变为脯氨酸(p)，其氨基酸序列如seqidno.6所示：
根据本发明具体实施方式，突变体h93y/f94y的甘露聚糖酶成熟蛋白的n端第93位组氨酸(h)和第94位苯丙氨酸(f)均突变为酪氨酸(y)，其氨基酸序列如seqidno.7所示：
根据本发明具体实施方式，突变体h93y/l356h的甘露聚糖酶成熟蛋白的n端第93位组氨酸(h)突变为酪氨酸(y)，及c端第356位亮氨酸(l)突变为组氨酸(h)，其氨基酸序列如seqidno.8所示：
根据本发明具体实施方式，突变体h93y/a389p的甘露聚糖酶成熟蛋白的n端第93位组氨酸(h)突变为酪氨酸(y)，及c端第389位丙氨酸(a)突变为脯氨酸(p)，其氨基酸序列如seqidno.9所示：
根据本发明具体实施方式，突变体l356h/a389p的甘露聚糖酶成熟蛋白的c端第356位亮氨酸(l)突变为组氨酸(h)，及第389位丙氨酸(a)突变为脯氨酸(p)，其氨基酸序列如seqidno.10所示：
根据本发明具体实施方式，突变体h93y/f94y/l356h的甘露聚糖酶成熟蛋白的n端第93位组氨酸(h)和第94位苯丙氨酸(f)均突变为酪氨酸(y)，及c端第356位亮氨酸(l)突变为组氨酸(h)，其氨基酸序列如seqidno.11所示：
根据本发明具体实施方式，突变体h93y/l356h/a389p的甘露聚糖酶成熟蛋白的n端第93位组氨酸(h)突变为酪氨酸(y)，及c端第356位亮氨酸(l)突变为组氨酸(h)和第389位丙氨酸(a)突变为脯氨酸(p)，其氨基酸序列如seqidno.12所示：
根据本发明具体实施方式，突变体h93y/f94y/l356h/a389p的甘露聚糖酶成熟蛋白的n端第93位组氨酸(h)和第94位苯丙氨酸(f)均突变为酪氨酸(y)，及c端第356位亮氨酸(l)突变为组氨酸(h)和第389位丙氨酸(a)突变为脯氨酸(p)，其氨基酸序列如seqidno.13所示：
本发明还提供了一种制备热稳定提高的甘露聚糖酶的方法，包括以下步骤：
(1)用上述包含突变体编码基因的重组载体转化宿主细胞，获得重组菌株；
(2)培养重组菌株，诱导重组甘露聚糖酶pman5a突变体表达；
(3)回收并纯化所表达的甘露聚糖酶pman5a突变体。
本发明的有益效果：
本发明通过对成熟的甘露聚糖酶pman5a的第93h、94f、356l和389a位点进行单点及组合突变，构建了11个突变体，并将突变体重组载体转化毕赤酵母gs115，实现了突变体酵母工程菌的构建。对突变体和野生甘露聚糖酶pman5a进行大瓶诱导，蛋白浓缩与纯化，并进行了耐热性质与催化效率的测定与分析。
分析实验结果表明，单点突变体pman5a-h93y和pman5a-a389p在高温条件下的酶活要明显高于野生甘露聚糖酶pman5a，而突变体pman5a-f94y和突变体pman5a-l356h的最适温度没有发生变化，且在各个温度下表现出与野生酶pman5a相似的相对酶活力。两点组合突变体h93y/f94y、h93y/l356h、h93y/a389p和l353/a389p的最适温度比野生甘露聚糖酶pman5a提高了10℃、10℃、15℃和5℃，其最适温度的提高相对于单点突变体h93y和a389p表现出叠加效应。同样组合突变体h93/l353/a389p与h93/f94y/l353/a389p表现出相同的叠加效应，最适温度提高到85℃。
耐热性测定中，甘露聚糖酶pman5a突变体h93y、l356h和a389p对70℃耐受性提高，并且h93和a389两位点突变后提高更明显。组合突变体的热稳定性与野生甘露聚糖酶pman5a相比都是提高的。
单点突变体中h93y、l356h和a389p的t50值比野生甘露聚糖酶pman5a分别提高了7℃、2℃和4℃，而突变体f94y的t50值并没有变化，可见三个位点是促进gh5甘露聚糖酶pman5a热稳定性提高关键位点，并且其效应h93y>a389p>l356h。组合突变在5甘露聚糖酶pman5a的耐热性上表现出明显的叠加效应。多位点组合的突变体h93y/f94y/l356h、h93y/l356h/a389p、h93y/f94y/l356h/a389p表现出更高的热稳定性，其t50值分别比野生酶pman5a提高了10℃、13℃和14℃。
在70℃下，四个单点突变体h93y、f94y、l356h和a389p的耐热性均增强。在75℃下，组合突变体对高温的耐受能力为l353/a389p<h93y/f94y<h93y/f94y/l356h<h93y/a389p<h93y/l356h/a389p<h93y/f94y/l356h/a389p组合突变体的耐热性提高有叠加效应。
位点h93和a389对甘露聚糖酶pman5a热稳定性至关重要，并具有叠加效应。虽然f94y和l356h的突变并没有提高野生酶的tm值，但当与其他位点组合后又表现出tm值的叠加。
所有突变体的催化效率与比活均较野生甘露聚糖酶pman5a有所提高，并且提高的幅度呈现单点突变体<双点突变体<多点突变体的规律。组合突变体h93y/f94y/l356h/a389p的比活约提高了0.7倍，催化效率也提高了约0.5倍。
本发明确认了甘露聚糖酶pman5a中的关键耐热性位点h93、f94、h356和a389，并证明了这些位点对于该酶高耐热性的重要性，对于研究甘露聚糖酶pman5a的热稳定机制提供了重要的线索，同时对于提高gh5家族的其他甘露聚糖酶的热稳定性提供了可靠的参考依据。
附图说明
图1显示野生酶pman5a及突变体的最适温度，其中，a表示单点突变体；b表示组合突变体；
图2显示野生酶pman5a及突变体的温度稳定性，其中，a：70℃；b：75℃；c：80℃。
具体实施方式
试验材料和试剂
1、菌株及载体：表达宿主pichiapastorisgs115，表达质粒载体ppic9。
2、酶类及其它生化试剂：点突变试剂盒及其它生化试剂均购自生化试剂公司。
3、培养基：
(1)lb培养基：0.5％酵母提取物，1％蛋白胨，1％nacl，ph7.0；
(2)ypd培养基：1％酵母提取物，2％蛋白胨，2％葡萄糖；
(3)md固体培养基：2％葡萄糖，1.5％琼脂糖，1.34％ynb，0.00004％biotin；
(4)bmgy培养基：1％酵母提取物，2％蛋白胨，1％甘油(v/v)，1.34％ynb，0.00004％biotin；
(5)bmmy培养基：1％酵母提取物，2％蛋白胨，1.34％ynb，0.00004％biotin，0.5％甲醇(v/v)。
实施例1甘露聚糖酶突变体工程菌的构建
(1)表达载体的构建及在毕赤酵母中的表达
以penicilliumsp.wn1来源的gh5家族甘露聚糖酶pman5a-ppic9为模板，在基因的h93、f94、l356和a389位点处设计突变引物h93yf/r、f94yf/r、l356hf/r和a389pf/r(如表1所示)，利用点突变试剂盒首先进行pcr扩增，然后pcr产物经过dmt酶进行去甲基化并转化dmt感受态细胞，最后挑取单克隆验证，挑选阳性转化子进行dna测序，测序表明序列正确的转化子用于大量制备重组质粒。
表1以野生甘露聚糖酶pman5a为母本构建突变体所用引物
将与表达载体ppic9连接测序正确的重组载体用内切酶drai线性化并转化毕赤酵母gs115感受态细胞，30℃培养2-3天，挑取在md平板上生长的转化子进行进一步的表达实验，获得重组酵母菌株。
(2)高甘露聚糖酶活性转化子的筛选
用灭过菌的牙签从长有转化子的md板上挑取单菌落，按照编号先点到md平板上，将md平板置于30℃培养箱中培养1～2天，至菌落长出。按编号从md平板上挑取转化子接种于装有3mlbmgy培养基的离心管中，30℃，220rpm摇床培养48h；将摇床培养48h的菌液3,000×g离心15min，去上清，离心管中再加入1ml含有0.5％甲醇的bmmy培养基，在30℃、220rpm诱导培养；诱导培养48h后，3,000×g离心5min，取上清用于酶活性检测，从中筛选出高甘露聚糖酶活性转化子，具体操作请参考毕赤酵母表达操作手册。
实施例2甘露聚糖酶突变体及野生酶酶液的制备
(1)突变体基因在毕赤酵母中摇瓶水平的表达
挑取酶活最高的转化子，接种于30mlypd培养基中培养48h进行种子放大培养，后按1％接种体积接种于300mlbmgy培养基的1l三角瓶中，置于30℃，220rpm摇床培养48h；后将培养液3000g离心5min，弃上清，沉淀用200ml含有0.5％甲醇的bmmy培养基重悬，并再次置于30℃，220rpm条件下诱导培养。每隔12h补加1ml甲醇，使菌液中的甲醇浓度保持在0.5％，同时取上清用于酶活性检测。
(2)突变体甘露聚糖酶的纯化
收集摇瓶表达的突变体甘露聚糖酶上清液，通过10kda膜包进行浓缩，同时用低盐缓冲液置换其中的培养基，然后用10kda超滤管进一步的浓缩，浓缩能稀释到一定倍数的酶液，利用离子交换层析进行纯化。具体地，取突变体及野生甘露聚糖酶酶pman5a浓缩液2.0ml经预先用10mm柠檬酸-磷酸氢二钠(ph7.2)平衡过的hitrapqsepharosexl阴离子柱，然后用0-1mol/l的nacl进行线性梯度洗脱，对分步收集的洗脱液检测酶活性和进行蛋白浓度的测定。
实施例3甘露聚糖酶突变体及野生酶pman5a的活性分析及性质测定
采用dns法对本发明的甘露聚糖酶进行活性分析，具体方法如下：
在给定的ph、温度条件下，1ml的反应体系包括100μl适当的稀释酶液，900μl底物，反应10min，加入1.5mldns终止反应，沸水煮5min。冷却后540nm测定od值。1个酶活单位(u)定义为在给定的条件下，每分钟分解角豆胶生成1μmol还原糖所需的酶量。
(1)突变体及野生酶pman5a的最适温度及温度稳定性测定
在ph5.0柠檬酸-磷酸氢二钠缓冲体系及不同温度(40-90℃)下进行酶促反应，测定突变体及野生酶pman5a的最适温度。
结果表明，如图1、2所示，在ph5.0条件下，单点突变体h93y、f94y、l356h和a389p的最适温度分别为80℃、70℃、70℃和75℃，其中突变体h93y和a389p比野生酶pman5a分别提高了10℃和5℃。f94y和l356h的最适温度没有发生变化，在各个温度下两者表现出与野生酶pman5a相似的相对酶活力。因此，单点突变体pman5a-h93y和pman5a-a389p在高温条件下的酶活要明显高于野生酶pman5a。
两点组合突变体h93y/f94y、h93y/l356h、h93y/a389p和l353/a389p的最适温度分别为80℃、80℃、85℃和75℃，比野生酶pman5a提高了10℃、10℃、15℃和5℃。与单点突变体h93y相比，只有h93y/a389p的最适温度仅仅提高了5℃。两点组合体的最适温度的提高相对于单点突变体h93y和a389p表现出叠加效应。
组合突变体h93/l353/a389p与h93/f94y/l353/a389p表现出与两点突变体相同的叠加效应，最适温度提高到85℃。
(2)突变体及野生酶tm值的测定
取0.25mg的蛋白样品溶于1ml10mm的柠檬酸-磷酸氢二钠(ph7.2)缓冲液中，在25-100℃下扫描，扫描速度为1℃/min，具体结果见表2：
表2野生酶pman5a与突变体的tm值
结果显示，野生甘露聚糖酶pman5a的tm值为61.8℃，单点突变体h93y和a389p的tm值分别为69.2℃和66.8℃，分别较野生酶提高了7.5℃和5.0℃，此两点组合突变后的tm值提高到71.9℃，比野生酶提高了10.1℃，表明位点h93和a389对甘露聚糖酶pman5a热稳定性至关重要，并具有叠加效应。
虽然f94y和l356h的突变并没有提高野生酶的tm值，但当与其他位点组合后又表现出tm值的叠加，如h93y/l356h/a389p的tm值为75.3℃，组合f94y后又提高了0.3℃。
(3)突变体及野生甘露聚糖酶pman5a的t50值与半衰期测定
将突变体及野生酶pman5a用ph5.0的na2hpo4-柠檬酸缓冲液稀释至70μg/ml，在无底物存在的情况下，置于不同温度下(60-80℃)热处理30min后迅速置于冰上，并在ph5.0和各自最适温度的条件下测定剩余酶活，结果见表3。
将突变体及野生酶pman5a用ph5.0的na2hpo4-柠檬酸缓冲液稀释至70μg/ml，在无底物存在的情况下，分别置于70℃、75℃及80℃下热处理不同时间后立即置于冰上，并在ph5.0和各自最适温度的条件下测定剩余酶活。计算某温度下剩余酶活为最高酶活一半时所对应的时间，为此酶在这一温度下的半衰期，结果见表3。
表3野生酶pman5a与突变体的t50和t1/2值
注：“/”表示处理时间太长无法测定；“－”表示处理2min内无酶活而无法测定
结果显示，野生酶pman5a的t50值为66℃，单点突变体中h93y、l356h和a389p的t50值分别为73℃、68℃和70℃，比野生酶pman5a分别提高了7℃、2℃和4℃，而突变体f94y的t50值并没有变化。由此可见，h93y、l356h和a389p三个位点是促进gh5甘露聚糖酶热稳定性提高关键位点，并且其效应表现为h93y>a389p>l356h。
组合突变体h93y/l356h和h93y/a389p的t50值分别比h93y提高了2℃和6℃，组合突变体l356h/a389p比l356h提高了2℃，可见组合突变在酶的耐热性上表现出明显的叠加效应。
多位点组合的突变体h93y/f94y/l356h、h93y/l356h/a389p、h93y/f94y/l356h/a389p表现出更高的热稳定性，其t50值达76℃、79℃和80℃，分别比野生酶pman5a提高了10℃、13℃和14℃。
在70℃下，四个单点突变体h93y、f94y、l356h和a389p的t1/2值分别为64、4、14、45min，表现出突变体耐热性的增强。在75℃下，组合突变体对高温的耐受能力为l353/a389p<h93y/f94y<h93y/f94y/l356h<h93y/a389p<h93y/l356h/a389p<h93y/f94y/l356h/a389p，组合突变体的耐热性的提高有叠加效应，最耐热的突变体h93y/f94y/l356h/a389p在75℃下能够耐受3h酶活剩余一半，其在80℃下的半衰期为27min。
(4)突变体及野生酶pman5a的动力学参数测定
确定测定km及vmax的反应时间为5min。用不同浓度的角豆胶(5,2.5,2,1,0.75,0.5,0.375mg/ml)为底物，在同一条件(85℃/80℃/70℃、ph5.0)下测定酶活性，计算出相应的反应速度，利用grafit7软件计算km值及vmax，具体结果见表4：
表4野生酶pman5a与突变体的动力学参数
结果显示，所有突变体的催化效率与比活均比野生酶pman5a有所提高，并且提高的幅度呈现单点突变体<双点突变体<多点突变体的规律。比活由野生酶pman5a1276u/mg提高到组合突变体h93y/f94y/l356h/a389p的2226u/mg，约提高了0.7倍，同时催化效率也提高了约0.5倍。
序列表
<110>中国农业科学院饲料研究所
<120>耐热性提高的甘露聚糖酶pman5a突变体及其基因和应用
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trpglygluthraspsertrpglysersertrpilethralahisgly
325330335
glnalacyslysasnalaglylysprocysleuleugluglutyrgly
340345350
serthrserleucysserserglualaprotrpglnthrthralaile
355360365
serservalalaalaaspleuphetrpglntrpglyaspthrleuser
370375380
thrglyglnserprohisaspglutyrserilephetyrglyserser
385390395400
asptyrthrcysleuvalthrasphisvalseralaileaspserala
405410415
<210>10
<211>416
<212>prt
<213>青霉菌wn1(penicilliumsp.wn1)
<400>10
alaasntrpglyglncysglyglygluasntrpasnglyaspthrthr
151015
cysasnproglytrptyrcyssertyrleuasnprotrptyrsergln
202530
cysvalproglyserglyserserserserserthrthrleuserthr
354045
valvalserserglnthrserserileargthrthrseralathrser
505560
thrleualaalaseralaserthrthralaglyserleuproserala
65707580
serglythrserphevalileaspglylyslysglyhisphealagly
859095
thrasnsertyrtrpleupropheleuthrasnasnalaaspvalasp
100105110
leuvalmetglyhisleuglnglnserglyleulysileleuargval
115120125
trpglypheasnaspvalasnalavalproserserglythrvaltrp
130135140
pheglnleuleualaasnglyglnglnthrileasnthrglyserasp
145150155160
glyleuglnargleuasptyrvalvallysseralaglualahisgly
165170175
ilelysleuileileasnphevalasnasntrpasnasptyrglygly
180185190
metasnalatyrvalglnasntyrglyglyasnglnthrsertrptyr
195200205
thrasnasnalaalaglnaspalatyrlysthrtyrilelysthrval
210215220
ileserargtyrileglyserseralailephealatrpgluleuala
225230235240
asngluproargcyslysglycysglythraspvaliletyrasntrp
245250255
alaglnserthrserglntyrilelysserleugluproglyargmet
260265270
valcysileglyaspgluglymetglyleuservalaspseraspgly
275280285
sertyrpropheglytyrsergluglyasnasppheglulysthrleu
290295300
alaileprothrileasppheglythrilehisleutyrprosergln
305310315320
trpglygluthraspsertrpglysersertrpilethralahisgly
325330335
glnalacyslysasnalaglylysprocysleuleugluglutyrgly
340345350
serthrserhiscysserserglualaprotrpglnthrthralaile
355360365
serservalalaalaaspleuphetrpglntrpglyaspthrleuser
370375380
thrglyglnserprohisaspglutyrserilephetyrglyserser
385390395400
asptyrthrcysleuvalthrasphisvalseralaileaspserala
405410415
<210>11
<211>416
<212>prt
<213>青霉菌wn1(penicilliumsp.wn1)
<400>11
alaasntrpglyglncysglyglygluasntrpasnglyaspthrthr
151015
cysasnproglytrptyrcyssertyrleuasnprotrptyrsergln
202530
cysvalproglyserglyserserserserserthrthrleuserthr
354045
valvalserserglnthrserserileargthrthrseralathrser
505560
thrleualaalaseralaserthrthralaglyserleuproserala
65707580
serglythrserphevalileaspglylyslysglytyrtyralagly
859095
thrasnsertyrtrpleupropheleuthrasnasnalaaspvalasp
100105110
leuvalmetglyhisleuglnglnserglyleulysileleuargval
115120125
trpglypheasnaspvalasnalavalproserserglythrvaltrp
130135140
pheglnleuleualaasnglyglnglnthrileasnthrglyserasp
145150155160
glyleuglnargleuasptyrvalvallysseralaglualahisgly
165170175
ilelysleuileileasnphevalasnasntrpasnasptyrglygly
180185190
metasnalatyrvalglnasntyrglyglyasnglnthrsertrptyr
195200205
thrasnasnalaalaglnaspalatyrlysthrtyrilelysthrval
210215220
ileserargtyrileglyserseralailephealatrpgluleuala
225230235240
asngluproargcyslysglycysglythraspvaliletyrasntrp
245250255
alaglnserthrserglntyrilelysserleugluproglyargmet
260265270
valcysileglyaspgluglymetglyleuservalaspseraspgly
275280285
sertyrpropheglytyrsergluglyasnasppheglulysthrleu
290295300
alaileprothrileasppheglythrilehisleutyrprosergln
305310315320
trpglygluthraspsertrpglysersertrpilethralahisgly
325330335
glnalacyslysasnalaglylysprocysleuleugluglutyrgly
340345350
serthrserhiscysserserglualaprotrpglnthrthralaile
355360365
serservalalaalaaspleuphetrpglntrpglyaspthrleuser
370375380
thrglyglnseralahisaspglutyrserilephetyrglyserser
385390395400
asptyrthrcysleuvalthrasphisvalseralaileaspserala
405410415
<210>12
<211>416
<212>prt
<213>青霉菌wn1(penicilliumsp.wn1)
<400>12
alaasntrpglyglncysglyglygluasntrpasnglyaspthrthr
151015
cysasnproglytrptyrcyssertyrleuasnprotrptyrsergln
202530
cysvalproglyserglyserserserserserthrthrleuserthr
354045
valvalserserglnthrserserileargthrthrseralathrser
505560
thrleualaalaseralaserthrthralaglyserleuproserala
65707580
serglythrserphevalileaspglylyslysglytyrphealagly
859095
thrasnsertyrtrpleupropheleuthrasnasnalaaspvalasp
100105110
leuvalmetglyhisleuglnglnserglyleulysileleuargval
115120125
trpglypheasnaspvalasnalavalproserserglythrvaltrp
130135140
pheglnleuleualaasnglyglnglnthrileasnthrglyserasp
145150155160
glyleuglnargleuasptyrvalvallysseralaglualahisgly
165170175
ilelysleuileileasnphevalasnasntrpasnasptyrglygly
180185190
metasnalatyrvalglnasntyrglyglyasnglnthrsertrptyr
195200205
thrasnasnalaalaglnaspalatyrlysthrtyrilelysthrval
210215220
ileserargtyrileglyserseralailephealatrpgluleuala
225230235240
asngluproargcyslysglycysglythraspvaliletyrasntrp
245250255
alaglnserthrserglntyrilelysserleugluproglyargmet
260265270
valcysileglyaspgluglymetglyleuservalaspseraspgly
275280285
sertyrpropheglytyrsergluglyasnasppheglulysthrleu
290295300
alaileprothrileasppheglythrilehisleutyrprosergln
305310315320
trpglygluthraspsertrpglysersertrpilethralahisgly
325330335
glnalacyslysasnalaglylysprocysleuleugluglutyrgly
340345350
serthrserhiscysserserglualaprotrpglnthrthralaile
355360365
serservalalaalaaspleuphetrpglntrpglyaspthrleuser
370375380
thrglyglnserprohisaspglutyrserilephetyrglyserser
385390395400
asptyrthrcysleuvalthrasphisvalseralaileaspserala
405410415
<210>13
<211>416
<212>prt
<213>青霉菌wn1(penicilliumsp.wn1)
<400>13
alaasntrpglyglncysglyglygluasntrpasnglyaspthrthr
151015
cysasnproglytrptyrcyssertyrleuasnprotrptyrsergln
202530
cysvalproglyserglyserserserserserthrthrleuserthr
354045
valvalserserglnthrserserileargthrthrseralathrser
505560
thrleualaalaseralaserthrthralaglyserleuproserala
65707580
serglythrserphevalileaspglylyslysglytyrtyralagly
859095
thrasnsertyrtrpleupropheleuthrasnasnalaaspvalasp
100105110
leuvalmetglyhisleuglnglnserglyleulysileleuargval
115120125
trpglypheasnaspvalasnalavalproserserglythrvaltrp
130135140
pheglnleuleualaasnglyglnglnthrileasnthrglyserasp
145150155160
glyleuglnargleuasptyrvalvallysseralaglualahisgly
165170175
ilelysleuileileasnphevalasnasntrpasnasptyrglygly
180185190
metasnalatyrvalglnasntyrglyglyasnglnthrsertrptyr
195200205
thrasnasnalaalaglnaspalatyrlysthrtyrilelysthrval
210215220
ileserargtyrileglyserseralailephealatrpgluleuala
225230235240
asngluproargcyslysglycysglythraspvaliletyrasntrp
245250255
alaglnserthrserglntyrilelysserleugluproglyargmet
260265270
valcysileglyaspgluglymetglyleuservalaspseraspgly
275280285
sertyrpropheglytyrsergluglyasnasppheglulysthrleu
290295300
alaileprothrileasppheglythrilehisleutyrprosergln
305310315320
trpglygluthraspsertrpglysersertrpilethralahisgly
325330335
glnalacyslysasnalaglylysprocysleuleugluglutyrgly
340345350
serthrserhiscysserserglualaprotrpglnthrthralaile
355360365
serservalalaalaaspleuphetrpglntrpglyaspthrleuser
370375380
thrglyglnserprohisaspglutyrserilephetyrglyserser
385390395400
asptyrthrcysleuvalthrasphisvalseralaileaspserala
405410415