葡萄糖异构酶突变体的利记博彩app

专利名称：：葡萄糖异构酶突变体的利记博彩app
技术领域：
：-本发明涉及分子生物学与生物
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(Glucoseisomerase，E.C.5.3丄5，简称GI)、或称木糖异构酶(Xyloseisomerase)是戊糖发酵途径的关键酶。该酶是最重要的酶制剂之一(Kaneko，Wa/.，所^d别o化c^oZ说oc/2em，64:940-947，2000)。食品工业使用该酶制备果葡糖浆。酶法生产果葡糖浆，产品中果糖的含量取决于反应的温度。温度越高，异构反应越趋向于果糖的生成。目前商用葡萄糖异构酶主要取自密苏里游动放线菌(J"/"印/朋^7w^owr/em7.力、凑菱结芽孢杆菌^gflcz.〃船ccwgw/am")、或链霉菌(5^e;to附少c^)。这些葡萄糖异构酶在高温(如高于65。C)下稳定性差。因此，目前工业上在60。C左右制备果葡糖浆，产物中果糖的含量也就较低，一般不超过44%。含更高浓度果糖的果葡糖浆通常依赖层析分离方法生产，由此增加了生产成本。世界各地的科，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777em70cmaera&c^^mracctora(^cwm、7Tzwwotoga"eapo/fto""等数种耐高温菌中分离并研究了耐高温葡萄糖异构酷(见LeeWa/.，Jow7a/Gem^a/Af/cro6/o/ogy，139:1227-1234，1993;Vieilleefa/.，A/W/zocfei拜附o/ogy，330:215-24,2001;LeeefJowma/o/Gewera/Mz.cra6z'o/ogy，139:1241-1243，1993;Scriprapundha/.，/Vofe/"五"g7'wee〃.wg，13:259-265，2000;Scriprapundh"a/.，/VoW"16:683-690，2003;Zeikus"a/.，USPatentNO5,656,497)。这些或其它来源的耐高温葡萄糖异构酶之耐热性能均不如人意，且活力较低，尚未见用于工业化生产。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。
发明内容本发明利用遗传工程和蛋白质工程技术对来源于i:racc/wra/j^c"m的葡萄糖异构酶进行改良，获得了一系列高催化活性的葡萄糖异构酶突变体，适宜生产含高果糖的果葡糖浆。本发明的目的在于提供高催化活性的葡萄糖异构酶突变体。本发明的目的还在于提供使用本发明所述的葡萄糖异构酶突变体直接生产果糖含量等于或高于55%的果葡糖浆。本发明的目的还在于提供使用本发明所述的葡萄糖异构酶突变体生产果糖含量低于55%的果葡糖浆。为实现本发明的上述目的，本发明人进行了大量深入的实验，通过对7:.racc/rara/卢/cwm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，利用本领域己知的技术，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。最后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。在本发明制备葡萄糖异构酶突变体的方法中，可采用任何适当的载体。例如，适用的载体包括但不限十原核表达载体pGEMT-Easy，pRSET和pET21;包括但不限于真核表达载体pYDl禾QpYES2/GS;包括但不限于克隆载体pUC18A9禾口pBluscript-SK。在本发明制备葡萄糖异构酶突变体的方法中，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。在本发明制备葡萄糖异构酶突变体的方法中，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.凝结芽孢杆菌、枯草芽胞杆菌、巨大芽胞杆菌(如巨大芽胞杆菌BP931)、Z^cc/7o尸o(v'rtr"m和链霉菌(如Sreptowyces'c^w似"cwM1033)。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)。本发明获得了一种葡萄糖异构酶突变体,其特征在于以序列表中序列2所示氨基酸序列为参考序列,至少存在一个氨基酸的差异,并且以D-葡萄糖为底物其具有比亲本至少高山50-150%、优选至少高出150-250%、更优选至少高出250%的葡萄糖异构酶催化活性。优选的是，以序列表中的序列2为参考序列，具有至少一个选自第139位的色氨酸到其他19个天然氨基酸、第182位的精氨酸到其他19个天然氨基酸、第187位的苯丙氨酸到其他19个天然氨基酸和第299位的苏氨酸到其他19个天然氨基酸的突变,并且以D-葡萄糖为底物其具有比亲本高出至少50%的葡萄糖异构酶催化活性。更优选所述第139位的色氨酸突变为赖氨酸(Lys)、或丝氨酸(Ser)、或半胱氨酸(Cys)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe);所述第182位的精氨酸突变为脯氨酸(Pro)、或丝氨酸(Ser)、或丙氨酸(Ala)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或缬氨酸(Val);所述第187位的苯丙氨酸突变为甘氨酸(Gly)、或丝氨酸(Ser)、或丙氨酸(Ala)、或脯氨酸(Pra);和/或所述第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)、或蛋氨酸(Met)、或谷氨酸(Glu)、或谷氨酰胺(Gln)。最优选的是，木发明的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3这些突变体具有高的催化活性、并耐热和对较低pH具有耐受性。例如，在本发明获得的一系列突变体中，一个具有四个点突变的突变体MGI-4的比活性较亲本高651%,且在80°C反应24小时后仍保持50%或以上的活力，且在pH5.0处的活性相当于其在最佳pH(pll7.0)时的活性的80%。另一个具有三个点突变的突变体MGI-3的比活性较亲本高412%，且在80°C反应16小时后仍保持50%或以上的活力，且在pH5.0处的活性相当于其在最佳pH(pH7.0)时的活性的70%。本发明获得的高催化活性或高催化活性并耐热的葡萄糖异构酶突变体可用于直接生产果糖含量等于或高于55%的果葡糖浆，或用于生产果糖含量低于55%的果葡糖浆。所述的葡萄糖异构酶突变体可以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本发明葡萄糖异构酶突变体制成固相酶或固相细胞形式的固化酶。定义本申请文本中所用的术语"亲本，，系指來自7^emzoofnaera6a"e〃'MmraccAara(yricwmATCC49915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7:racc/wra(KZcwm葡萄糖异构酶的基因序列(Leew，Jow"a/^Ge"era/A/kroWo/ogy，139:1227-1234，1993;GenBankL09699)相比有两个核苷酸的差异，即与基因库(GenBankL09699)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(目甜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序列与参考序列不匹配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139位、第182位、第187位、第299位具有至少.个氨基酸的差异,并且以D-葡萄糖为底物生成果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■/SzocAe附.，138:9-37，1984)。附图的简要说明图l葡萄糖异构酶突变体MGI-4聚丙烯酰胺凝胶电泳图。其中从左至右的四条泳道依次为蛋白分子量标准、粗提蛋白、牛血清白蛋白、部分纯化的葡萄糖异构酶突变休MGI-4。(粗提蛋白、部分纯化的葡萄糖异构酶突变体MGI-4的制备见实例10)图2葡萄糖异构酶突变体MGI-4在8(fC转化D-葡萄糖至果糖之转化率。其中黑色柱代表葡萄糖剩余量，黑色柱下方的白色柱代表果糖的生成量。该图显示出在反应的头两个小时内果糖的生成量几乎与反应时间呈线性关系增长，在随后的两个小时内，果糖生成量的速度减缓。图3亲本葡萄糖异构酶与葡萄糖异构酶突变体在80°C的热稳定。其中TS表示亲本葡萄糖异构酶，MGI-2，MGI-3和MGI-4分别代表具有两个、三个和四个突变位点的葡萄糖异构酶突变体，具体请参见实施例6-8。图4pH对亲本葡萄糖异构酶及葡萄糖异构酶突变体的影响。其中TS表示亲本葡萄糖异构酶，MGI-2，MGI-3和MGI-4分别代表具有两个、三个和四个突变位点的葡萄糖异构酶突变体，具体请参见实施例6-8。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(GenBankL09699)基因序列设i十引物Tl和T2(见表1)。利用引物对Tl和T2从ZMcc/wra/,'cwwATCC49915(购自ATCC，USA)中扩增葡萄糖异构酶亲本基因。扩增条件为20mMTris-HCl(pH8.8)，10mMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-IOO，50dATP，50dTTP,50dCTP,50dGTP，400nM引物Tl，400nM引物T2，1.5UPfuDNA聚合酶(Promega，USA),用接种环挑取少许7:M"/zara/卢/cww菌体，再用无菌水调反应体积至50|J。PCR扩增反应程序为95°C3分钟，40圈循环95。C50秒、50°C30秒和72°C1分钟，最后72°C10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(GenBankL09699)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"a,(Gene77:51-59,1989禾口Whiteefa/.(PCRProtocol:currentmethodsandapplications.Totowa，N.J.:HumanaPress,1993)之描述。以质粒pGEMT-TS(见实施例l)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Tl和T2见实施例1。利用引物对Tl和139FR，扩增T1FR片段;利用引物对139FF和T2，扩增FFT2片段。扩增反应条件为20mMTris-HCl(pH8.8)，10mMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-100,50pMdATP，50pMdTTP，50dCTP，50dGTP，400nM弓l物Tl禾口400nM引物139FR或400nM引物139FF和400nM引物T2，1.5UPfuDNA聚合酶(Promega，USA)，20ngpGEMT-TS,再用无菌水调反应体积至50pl。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72。C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit(QIAGEN，German)回收，得至ljT1FR片段和FFT2片段。然后扩增全长基因。扩增反应条件为20mMTris-HCl(pH8.8)，10mMKCl，lOmM(NH4)2S04，2mMMgS04,0.1%TritonX-IOO，50(iMdATP，50dTTP,50一dCTP，50jaMdGTP，400nM引物Tl和400nMT2，1.5UPfuDNA聚合酶，20ngT1FR片段4;20ngFFT2片段，用无菌水调反应体积至50nl。PCR扩增反应程序为95°C3分钟，35圈循坏95°C50秒、52°C30秒和72。C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit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￡.co/ZHBIOI，在1。/。MacConkey平板(含P/。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-W139FDNA，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。按照类似步骤构建突变体MGI-W139K、MGI-W139S、MGI-W139C、MGI陽W1391、MGI-W139T和MGI-W139N，所用引物见表1。所得诸突变体氨基酸序列见序列表序列6-1]。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"a/.(Gene77:51-59，1989)和White"a/.(PCRProtocol:currentmethodsandapplications.Totowa,N.J.:HumanaPress,1993:)之描述。以质粒pGEMT-TS(见实施例l)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Tl和T2见实施例1。利用引物对Tl和182AR,扩增T1AR片段;利用引物对182AF和T2，扩增AFT2片段。扩增反应条件为20mMTris-HCl(pH8.8)，lOmMKCl，10niM(NH4)2S04，2mMMgS04，0.1%TritonX-IOO，50dATP,50|iMdTTP，50^vldCTP,50fiMdGTP,400nM引物Tl和400nM引物182AR或400nM引物182AF禾D400nM引物T2，1.5UPfuDNA聚合酶，20ngpGEMT-TS，再用无菌水调反应体积至50^1。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit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20mMTris-HCl(pH8.8)，10mMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-100,50dATP，50dTTP,50(iMdCTP，50dGTP，400nM引物Tl和400nMT2'1.5UPfuDNA聚合酶，20ngT1AR片段与20ngAFT2片段，再用无菌水调反应体积至50pl。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAqiiickExtractionGelKit回收，得到全长突变基因MGI-R182A。将MGI-R1S2A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cWHB101，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12。按照类似步骤构建突变体MGI-R182P、MGI-R182S、MGI-R182I、MG1-R182T和MGI-R182V，所用引物见表l。所得诸突变体的氨基酸序列见序列表序列13-17。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(Gene77:51-59，1989)和Whitea/.(PCRProtocol:currentmethodsandapplications.Totowa,NJ.:HumanaPress,1993)之描述。以质粒pGEMT-TS(见实施例l)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Tl和T2见实施例1。利用引物对T1和187SR，扩增T1SR片段;利用引物对187SF和T2，扩增SFT2片段。扩增反应条件为20mMTris-HCl(pH8.8)，10mMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-IOO，50pMdATP,50dTTP,50dCTP，50dGTP，400nM引物Tl和400nM引物187SR或400nM引物T2和400nM引物187SF，1.5UPfoDNA聚合酶，20ngpGEMT-TS，再用无菌水调反应休积至50^1。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72。C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit回收，得到T1SR片段和SFT2片段。然后扩增全长基冈。扩增反应条件为20mMTris-HCl(pH8.8)，lOmMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-100,50|aMdATP,50dTTP,50dCTP,50|iMdGTP，400nM引物Tl和400nMT2，1.5UPfiiDNA聚合酶，20ngT1SR片段与20ngSFT2片段，再用无菌水调反应体积至50pl。PCR扩增反应程序为95。C3分钟，35圈循环95°C50秒、52。C30秒和72°C3分钟，最后72。C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit回收，得到全长突变基因MGI-F187S。将MGI-F187S与载体pGEMT-Rasy连接，得质粒pGEMT-MGI-F187S。将质粒pGEMT-MGI-F187S转入感受态细菌细胞￡.co/!'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18。按照类似步骤构建突变体MGI-F187G、MGI-F187P和MGI-F187A，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19-21。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""/.(Gene77:51-59,1989)和White"a/.(PCRProtocol:currentmethodsandapplications.Totowa,N.J.:HumanaPress,1993)之描述。以质粒pGEMT-TS(见实施例l)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Tl和T2见实施例1。利用引物对T1和299QR，扩增T1QR片段;利用引物对299QF和T2，扩增QFT2片段。扩增反应条件为20mMTris-HCi(pH8.8)，10mMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-100,50|_iMdATP,50dTTP，50)_lMdCTP，50dGTP，400nM弓l物Tl和400nM引物299QR或400nM引物299QF禾U400nM引物T2，1.5UPfuDNA聚合酶，20ngpGEMT-TS，再用无菌水调反应体积至50pl。PCR扩增反应程序为95。C3分钟，35圈循环95°C50秒、52°C30秒和72。C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit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20mMTris-HCl(pH8.8)，10mMKC1,10mM(NH4)2S04，2mMMgS04，0.1%TritonX-100,50dATP，50dTTP，50dCTP，50|_iMdGTP，400nM引物Tl和400nMT2，1.5UPfuDNA聚合酶，20ngT1QR片段与20ngQFT2片段，再用无菌水调反应体积至50pl。PCR扩增反应程序为95°C3分钟，35圈循环95。C50秒、52°C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit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co//HB101，在1%MacConkey平板(含1%D-木糖和50mg/L氨节青霉素)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22。按照类似歩骤构建突变体MGI-T299I、MGI-T299Y、MGI-T299C、MGI-T299M和MGI-T299E，所用引物见表l。所得诸突变体的氨基酸序列见序列表序列23-27。实施例6:葡萄糖异构酶双突变组合MGI-2、MGI-2AQ和MGI-2FQ之构建定点突变技术参考HoWa/.(Gene77:51-59,1989)禾UWhite"a/.(PCRProtocol:currentmethodsandapplications.Totowa,N丄HumanaPress,1993)之描述。按实施例2扩增和冋收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20mMTris-HCl(pH8.8)，10mMKCl，10mM(NH4)2S04，2mMMgS04,0.1%TritonX響100，50|iMdATP，50dTTP,50nMdCTP,50dGTP，400nM139FF和400nM182AR，1.5UPfuDNA聚合酶，20ngpGEMT-TS，再用无菌水调反应体积至50pl。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit回收，得到FFAR片段。然后扩增全长基因。扩增反应条件为20mMTris-HCl(pH8.8)，lOmMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-IOO，50dATP,50|iMdTTP,50dCTP,50|iMdGTP，400nM弓l物Tl禾口400nMT2，1.5UPfuDNA聚合酶，20ngTlFR片段，20ngFFAR片段和20ngAFT2片段，再用无菌水调反应体积至50|J。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52。C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickDNA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￡.co/!'HB101，在1%MacConkey平板(含P/。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-2DNA，经DNA测序确定引入的点突变无误。MGI-2的氨基酸序列见序列表序列28。按照类似步骤构建双突变体MGI-2AQ和MGI-2FQ。突变体MGI-2AQ所用引物对为Tl和182AR、182AF和299QR、299QF和T2(见表1)，所得突变体序列含有R182A和T299Q的双突变。突变体MGI-2FQ所用引物对为Tl和139AR、139AF和299QR、299Q和FT2(见表1)，所得突变体序列含有T299Q和W139F,所得突变体的氨基酸序列见序列表序列29-30。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"a/.(Gene77:51-59,1989)禾BWhite"a/.(PCRProtocol:currentmethodsandapplications.Totowa，N丄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20mMTris-HCl(pH8.8)，lOmMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-100,50dATP,50fiMdTTP，50dCTP，50|aMdGTP,400nM182AF和400nM299QR，1.5UPfuDNA聚合酶，20ngpGEMT-TS，再用无菌水调反应体积至50pl。PCR扩增反应程序为95。C3分钟，35圈循环95。C50秒、52°C30秒和72°C3分钟，最后72。C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit回收，得到AFQR片段。然后扩增全L(基因。扩增反应条件为20mMTris-HCl(pH8.8)，lOmMKCl，10mM(NH4)2SO4，2mMMgS04，0.1%TritonX-100，50dATP,50dTTP,50dCTP,50(iMdGTP，400nM弓l物Tl禾口400nMT2，1.5UPfuDNA聚合酶，20ngTlFR片段，20ngFFAR片段，20ngAFQR片段和20ngQFT2片段，再用无菌水调反应体积至50^d。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit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-MCH-3转入感受态细菌细胞co//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31。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"a/.(Gene77:51-59,1989)和White"a/.(PCRProtocol:currentmethodsandapplications.T。towa,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；20mMTris-HCl(pll8.8),10mMKC1，10mM(NH4)2SCU，2mMMgS04，0.1%TritonX-100,50dATP,50dTTP,50dCTP，50dGTP，400nM182AF和400nM187SR,1.5UPfuDNA聚合酶，20ngpGEMT-TS，再用无菌水调反应体积至50(il。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72°C3分钟，最后72。C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit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20mMTris-HCl(pH8.8)，10mMKCl，10mM(NH4)2S04，2mMMgS04，0.1%TritonX-100,50dATP，50一dTTP，50岸dCTP，50岸dGTP，400nM187SF和400nM299QR，1.5UPftiDNA聚合酶，20ngpGEMT-TS，再用无菌水调反应体积至50pl。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52。C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit回收，得到SFQR片段。然后扩增全长基因。扩增反应条件为20mMTris-HCl(pH8.8)，10mMKCl，10mM(NH4)2SO4，2mMMgS04，0.1%TritonX-100,50dATP，50|aMdTTP，50(_iMdCTP,50|aMdGTP，400nM引物Tl和400nMT2，1.5UPfuDNA聚合酶，20ngTlFR片段，20ngFFAR片段，20ngAFSR片段，20ngSFQR片段和20ngQFT2片段，再用无菌水调反应体积至50jil。PCR扩增反应程序为95°C3分钟，35圈循环95°C50秒、52°C30秒和72°C3分钟，最后72°C5分钟。经1%琼脂糖胶电泳分离并用QIAquickExtractionGelKit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￡.HB101，在1%MacConkey平板(含1%D-木糖和50mg/L氨卞青霉素)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32。扩增亲本葡萄糖异构酶(亲本)与实施例1-8所葡萄糖异构酶突变体所用的引物如下表1所列表l<table>tableseeoriginaldocumentpage12</column></row><table>实施例9: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Leed"/.，Jow/tw/Ge"era/MkroWo/ogy，139:1227-1234(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Zs'.a^HB101，在MacConkeyT板(含1。/。D-木糖和50mg/L氨节青霉素)上、37°C培养36小时。接种单个克隆在5mlLB液体一培养基(含50mg/L氨苄青霉素)中培养16小时。离心收集菌休，并悬浮于1ml20mM磷酸钠缓冲液(pH6.5)中，力nCoCl2和MgCl2至终浓度分别为250禾B5mM。然后用超声波裂解细菌细胞。离心(10。C，17，800g，15分钟)并收集上清液为粗提蛋白。粗提蛋白经80。C热处理10分钟后，离心(10"C，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和制备果葡糖浆。实施例10:葡萄糖异构酶突变体的提取与纯化葡萄糖异构酶突变体MGI-4的提取与纯化与实施例9同，惟所用质粒为pGEMT-MGI-4。部分纯化的葡萄糖异构酶见图1。其它葡萄糖异构酶突变体也照实施例9所述提取与纯化。实施例ll:亲本葡萄糖异构酶活性的测定配底物溶液A含1.0M的D-葡萄糖、20mM磷酸钠缓冲溶液(pH6.5)、终浓度为250之CoCl2和终浓度为5mM之MgCl2，调pH至6.5。取底物溶液A9(^1，然后加入10pi按实施例9制备的葡萄糖异构酶。反应于80。C进行10分钟。将反应物置冰上以终止反应。反应产物D—果糖通过半胱氨酸-咔唑法测定。测定方法参见Dische"J.5iW.Cfem，192:583-587，1951)以及Nakamura，爿grOe附.32:701-706，1968)。用CoomassiePlusProteinAssayReagentKit(PIERCE,USA)并结合SDS聚丙烯酰胺凝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-葡萄糖为果糖所需之酶量。下列表2显示亲本葡萄糖异构酶之比活性。实施例12:葡萄糖异构酶突变体活性的测定葡萄糖异构酶突变体MGI-4活性测定与实施例11同。其它葡萄糖异构酶突变体的活性也照实施例ll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。<table>tableseeoriginaldocumentpage14</column></row><table>实施例13:D-葡萄糖转化为果糖转化率之测定主要参考Kaneko."a/.，64:940-947，2000。配底物溶液B含50%(W/V)D-葡萄糖、20mM磷酸钠缓冲溶液(pH6.5)、终浓度为250之CoCl2和终浓度为5mM之MgCl2，调pH至6.5。取底物溶液B60pl，然后加入40^按实施例10制备的葡萄糖异构酶突变体MGI-4。80°C反应4小时后，加入100^20%三氯醋酸终止反应，离心(10。C，17，800g，15分钟)并收集上清液。取10nl上清液稀释100倍后，用HPLC测定产生的果糖含量。HPLC测定用色谱柱为nBONDAPAKNH2SSCOL3.9X300(Waters,USA)，流动相为乙腈-水(85:15)，流速0.5毫升/分钟。探测器为ELSD500。进样量为10^1。图2显示葡萄糖异构酶突变体MGI-4将D-葡萄糖转化至果糖之转化率。实施例14:亲本葡萄糖异构酶热稳定性测定置按实施例9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200nl酶液，并加入200^il矿物油。将离心管置于80。C水浴中，0小时、2小时、4小时、8小时、16小时、32小时、72小时后分别取出-管酶液，离心(l(fC，17，800g，20分钟)后，取上清液，按实施例11所述、测定葡萄糖异构酶残余蛋白和残余比活性。图3显示亲木葡萄糖异构酶在80°C的热稳定。实施例15: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-2、MGI-3、或MGI-4热稳定性测定与实施例14同。图3显示葡萄糖异构酶突变体MGI-2、MGI-3、或MGI-4在80。C的热稳定。如图所示，亲本葡萄糖异构酶的活性在80°C的半衰期为4.0小时；MGI-2的活性在80°C的半衰期为4.4小时；MGI-3的活性在80°C的半衰期为21小时；MGI-4的活性在80°C的半衰期为25.5小时。实施例16:pH对亲本葡萄糖异构酶的影响配制底物溶液C含1.0M的D-葡萄糖、20mM醋酸钠缓冲溶液(pH4.0)、终浓度为250之CoCl2和终浓度为5mM之MgCl2，调pH至4.0。配制底物溶液D含1.0M的D-葡萄糖、20mM醋酸钠缓冲溶液(pH4.5)、终浓度为250|iM之CoCl2和终浓度为5mM之MgCl2，调pH至4.5。配制底物溶液E含l.OM的D-葡萄糖、20mM醋酸钠缓冲溶液(pH5.0)、终浓度为250(iM之CoCl2和终浓度为5mM之MgCl2，调pH至5.0。配制底物溶液F含1.0M的D-葡萄糖、20mM醋酸钠缓冲溶液(pH5.5)、终浓度为250之CoCl2和终浓度为5mM之MgCl"调pH至5.5。配制底物溶液G含1.0M的D-葡萄糖、20mM磷酸钠缓冲溶液(pH6.0)、终浓度为250之CoCl2和终浓度为5mM之MgCl2，调pH至6.0。配制底物溶液H含1.0M的D-葡萄糖、20mM磷酸钠缓冲溶液(pH6.5)、终浓度为250之CoCl2和终浓度为5mM之MgCl2，调pH至6.5。配制底物溶液I含1.0M的D-葡萄糖、20mM磷酸钠缓冲溶液(pH7.0)、终浓度为250之CoCl2和终浓度为5mM之MgCl2，调pH至7.0。配制底物溶液J含1.0M的D-葡萄糖、20mM磷酸钠缓冲溶液(pH7.5)、终浓度为250之CoCl2和终浓!为5mM之MgCl2，调pH至7.5。配制底物溶液K含1.0M的D-葡萄糖、20mM磷酸钠缓冲溶液(pH8.0)、终浓度为250pM之CoCl2和终浓度为5mM之MgCl2，调pH至8.0。配制底物溶液L含1.0M的D-葡萄糖、20mMTris-HCl缓冲溶液(pH8.5)、终浓度为250,uM之CoCl2和终浓度为5mM之MgCl2，调pH至8.5。配制底物溶液M含1.0M的D-葡萄糖、20mMTris-HCL缓冲溶液(pH9.0)、终浓度为250|iM之CoCl2和终浓度为5mM之MgCl2，调pH至9.0。取反应管11管，每管lOOpl反应液包括按实施例9制备的D-葡萄糖异构酶以及底物溶液C、或底物溶液D、或底物溶液E、或底物溶液F、或底物溶液G、或底物溶液H、或底物溶液I、或底物溶液J、或底物溶液K、或底物溶液L、或底物溶液M90pl。反应于8(fC进行IO分钟。将反应物置冰上以终止反应。反应产物D—果糖的测定与实施例11同。图4显示pH对亲本葡萄糖异构酶的影响。实施例17:pH对葡萄糖异构酶突变体的影响pH对葡萄糖异构酶突变体MGI-2、MGI-3、或MGI-4的影响的测定与实施例16同。图4显示pH对葡萄糖异构酶突变体MGI-2、MGI-3、或MGI-4的影响。以亲本葡萄糖异构酶在最佳pH(pH7.0)的比活性为100%，葡萄糖异构酶突变体MGI-2、MGI-3、和MGI-4在pH5.0-9.0的范围内均保有大部分活性。如在pH5.0处，葡萄糖异构酶突变体MGI-2的相对比活性为365%,为其在最佳pH(pH7.0)的相对比活性的72%;在pH5.0处，葡萄糖异构酶突变体MGI-3的相对比活性是370%，为其在最佳pH(pH7.0)的相对比活性的70%;在pH5.0处，葡萄糖异构酶突变体MG1-4的相对比活性为600%，为其在最佳pH(pH7.0)的相对比活性的80%。实施例18:亲本葡萄糖异构酶生化常数测定配制底物溶液N含磷酸钠-MgCl2-CoCl2缓冲溶液[20mM磷酸钠(pH6.5)、250pMCoCl2、5mMMgCl2]和2MD-葡萄糖，调pH至6.5。用同样的磷酸钠-MgCl2-CoCl2缓冲溶液将底物溶液N稀释至含D-葡萄糖1.8M、1.6M、1.4M、1,2M、1.0M、0.8M、0.6M、0.4M、0.2M、0.1M、0.05M和0.025M。取10pl部分纯化的亲本葡萄糖异构酶(见实施例9)与90pi不同浓度的，物溶液N在65°C或80°C反应10分钟。然后，按米-曼(Michaelis-Menten)方程并采用Lineweaver-Burk双倒数作图法，测定米氏常数&,、饱和底物浓度下的最大反应速率^。A与转换数&。,。其结果如卜表3所列。实施例19:葡萄糖异构酶突变体MGI-4生化常数之测定葡萄糖异构酶突变体MGI-4生化常数测定与实施例18同。表3显示葡萄糖异构酶突变体MGI-4与亲本葡萄糖异构酶生化常数之比较。表3亲本葡萄糖异构酶(亲本)与葡萄糖异构酶突变体MGI-4(MGI-4)之生化常数<table>tableseeoriginaldocumentpage16</column></row><table>实施例20固相化葡萄糖异构酶突变体MGI-4主要参考Ge，W"/.，J;p/.肌oc/e柳.5,Wec/2"o/.69:57-69，1998。具体步骤是取固相酶载体氯化聚苯乙烯乙基胺(成都化l研究所提供)颗粒100克，加10mM磷酸盐缓冲液(pH8.0)1升和部分提纯之葡萄糖异构酶突变体MGI-48克(按实施例IO所述)，室温(22°C)搅拌(60~120转/分)反应18小时。抽滤收集固相酶，以水洗涤三次，得固相酶107克。固相酶活力测定如实施例11所述，惟所用酶为如上制备的固相酶0.01克，测得该固相酶的活性为820单位/克。实施例21固定化含葡萄糖异构酶突变体MGI-4的细胞在含50mg/L氨苄青霉素的LB培养液中过夜生长携有pGEMT-MGT-4的￡.HB101至OD6。o为7。离心收集菌体细胞。取菌体细胞10克，加3%海藻酸钠溶液20克，充分搅拌混合，然后将菌体细胞从管径约0.5mm的针头滴入500ml2%氯化钙溶液中，室温反应1小时，用蒸馏水浸泡洗涤3次，每次约半小时，得固定化细胞约30克。固定化细胞活力测定如实施例11所述，惟所用酶为如上制备的固定化细胞0.01克，测得同定化细胞的活性为370单位/克。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(BioRightWorldwideCompanyLtd.)<120>葡萄糖异构酶突变体《30〉F卜081146-59:58《50〉CN200410042773-2<151〉2004-05-26〈160〉32<170〉Patentlnversion3.2<2，0>1<211〉1320<212〉DNA<213>ThermoanaerobacterisaccharoIyticum<400>1atgaataaat3ttttgagaacgtatctaaaataaaatatgaaggaccaaaatcasat33t60ccttattccttt33atttt3C3atCC3g3ggS3gt33tCgatggca3gacgatgg3gg3g120C3tctccgcttttct3t3gC"tt3ttggC3Cactttt3CtgCtg3tgg33C3g3tcaattt180ggC33ggCt3ct3tgcaaagaccatggaacC3Ct3C3C3g3tcctatggat3tagCg333240gcaagggtaga3gcagcatttgagttttttgataagataaatgcacctttcttctgcttc300C3tg3t3ggg3t3ttgCCCCtg33gg3g3tactcttagag3g3C333C33aaacttagat360acaatagttgctatgataaaggattacttaaagaccagca3gacaa3agttttgtggggt420accgca33tcttttctCC33tccgagatttgt3catggtgcatcaacatcctgcaatgct480gacgtttttgcat3ttctgcagcgc33gtc333333gCCCttgagatt3ctaaggagctt540ggccgcgaaaactacgtattttggggtgga3g3g33gggtacgagacgcttctC33t3C3600g3t3tgg3gttagagcttgat33CtttgC33g3tttttgCacatggctgttgactatgc3660aaggaaatcggctttgaaggtC3gttcttgattgagccga3gccaaaggagCCt3C333S720catcaatacg3ctttgacgtggcasatgtattggcattcttg3gaaaatacgaccttgac780aaatatttca3agtaaatatcgaagcasaccatgcgacattggcattccaCg3CttCC33840catgagctaagatacgccagaat333cggtgtattaggatcaattgacgcaaatacaggc,gacatgcttttgggatgggatacggaccagttCCCt3C3g3tat3CgC3tgacaacgctt960gctatgtatg3agtcataaagatgggtggatttgacaaaggtggccttsactttgatgca1020333gt33g3Cgtgcttcatt"tg3gCC3g33g3tcttttcttaggtcacatagcaggaatg1080gatgcttttgcaaaaggcttt333gttgCttacaagcttgtgaaagatggcgtatttgac114033gttC3tCg3ag3a3gataCgC33gCt3C3a3gaaggcattggcgctg3"t3t"tgt33gC1200ggta33gctg3cttcaagagccttg3asagt3tgC3tt3g3gcacagccagattgt333C1260aaatcaggcagacaagagctatt3gaatca3tCCt333tCagt3UtgtttgC3g33t331320〈210〉2<211>439<212〉PRT<213〉Thermoanaerobacteriumsaccharolyticum<400>2MetAsnl_ysTyrPheGluAsnVaISerLyslieLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVaI202530<formula>formulaseeoriginaldocumentpage18</formula>340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrLysLeuVaILysAspGlyVaIPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPheLysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSer420425430leLeuAsnGinTyrLeuPheAlaGlu435〈210>3<211〉1320<212>陽<213〉ThermoanaerobacterisaccharoIyticum<220〉<221><222〉<223〉misc—feature〗4〕,5丁..(417),,,,"n"代表a,t，g或c，并且"nnn"代表编码除色氨酸之外的任何氨基酸的密码子。<220〉<221><222〉<223〉misc—featurej5，..(546),,,,〃n"代表a,t，g或c，并且"nnn"代表编码除精氨酸之外的任何氨基酸的密码子。<220〉<221〉<222〉<223>misc—feature,;559L.(561)""n"代表a't,g或c，并且"nnn〃代表编码除苯丙氨酸之外的任何氨基酸的密码子。<220><221><222〉<223>misc_feature,辨5L.(897),,,,'V代表a,t，g或c，并且"nnn〃代表编码除苏氨酸之外的任何氨基酸的密码子。<400>33tgaataaat3ttttg3g33Cgt3tct33333gg3CC3333tC333t33t60ccttsttcctttaaattttacaatccagaggaagtaatcgatggcaagacgatggaggag120catctccgcttttCt3t3gCtt3ttggC3Cacttttactgctg3tggaac3gatcaattt180ggcaaggctact3tgC3aag3CC3tggS3Ccactacacagatcct3tggatatsgcgaaa240gC33gggt3gaagcagcatttgagttttttg3ta3g3taaatgcacctttcttctgcttc300catgatagggatattgcccctg33gg3gatactcttagag3g3C333C33aaacttagat360acaatagttgCt3tg3t333ggattacttaaagaccagcaagacaaaagttttgnnnggt420accgcaaatcttttctccaatccgagatttgtacstggtgC3tC33C3tCctgcaatgct480gacgtttttgcatattctgc3gCgC33gtC333333gCCCttgagatt3Ctaagg3gctt540ggcnnngaaaactacgtannntggggtggaagagaagggtacg3gacgcttctC33t3C3600g3t3tgg3gtt3gagcttg3taactttgcaagatttttgcacatggctgttgact3tgca660aaggaaatcggCtttg33ggtcsgttcttg3ttgagccgaagccaaaggagcctacaaaa720catcaatacgactttgacgtggcaaatgtattggcattcttgagaaaatacgaccttgac780333t3tttC3a3gtaa3t3tcg3agC33accatgcgacattggC3ttCC3Cg3CttCC33840catgagctaagat3Cgccag33ta33Cggtgtatt3ggatcaattgacgcaaatnnnggc900gacatgcttttggg3tgggatacggaccagttCCCt3C3g3t3tacgcatgacaacgctt960gctatgtatgaagtcataaag3tgggtgg3tttgacaaaggtggccttaaCtttg3tgC31020aaagtaagacgtgcttcatttgagccagaag3tcttttcttaggtcac3tagC3gg33tg1080gatgcttttgcaaaaggctttaaagttgcttacaagcttgtgaaagatggcgtatttgac1兩aagttcatcgaagaaagatacgcaagctacaaagaaggcattggcgctgatattgta3gc1200ggtaaagctgacttcaagagccttgaaaagtatgcatt3gagcacagccag3ttgt3S3C1260333tC3ggC3gacaagagct3ttag33tC3atcctaaatcagtatttgtttgC3g3ataa1320<210><211〉<212><213><220〉<221〉<222><223><220〉<221><222〉<223><220〉<221><222><223><220><221><222〉<223>4439PRTThermoanaerobacteriumsaccharoIyticumMISC一FEATUREXaa代籴除色氨酸之外的任何氨基酸残基。MISC—FEATURE(182}"..(182)Xaa代表除精氨酸之外的任何氨基酸残基。MISC—FEATURE(187亍..(187)Xaa代表除苯丙氨酸之外的任何氨基酸残基。MlSCFEATURE(299^(299)Xaa代表除苏氨酸之外的任何氨基酸残基。<400〉4MetAsnLys1TyrPheGluAsnVaISerLyslieLysTyrGluGlyPro51015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVaI202530leAspGlyLysThrMetGluGluHisLeuArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyl_ysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlal_ys65707580AlaArgVaIGluAlaAlaPheGluPhePheAspl_yslieAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrl_eu100105110<image>imageseeoriginaldocumentpage21</image>AsnGinTyrLeuPheAlaGlu435<210>5<211>439<212〉PRT<213>ThermoanaerobacteriumsaccharoIyticum<400>5MetAsnLysTyrPheGluAsnVaISerLyslieLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVaI202530leAspGlyLysThrMetGluGluHisl_euArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlal_ys65707580AlaArgVaIGluAlaAlaPheGluPhePheAspLysIleAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnl_ysAsnLeuAspThrlieVaIAlaMetlieLysAsp115120125TyrLeuLysThrSerLysThrLysVaILeuPheGlyThrAlaAsnLeu130135140PheSerAsnProArgPheVaIHisGlyAlaSerThrSerGysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVaILysLysAlal_euGlu165170175Thrl_ysGluLeuGlyArgGluAsnTyrValPheTrpGlyGlyArgGh賜185190GlyTyrGluThrl_eul_euAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeuIleGluProLysProLysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnIleGluAlaAsnHisAla260265270Thrl_euAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerlieAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrl_eu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspLysGlyGlyLeu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrl_ysl_euValLysAspGlyValPheAspl_ysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400Glyl_ysAlaAspPhel_ysSerl_euGlul_ysTyrAlal_euGluHisSer405410415GinlieValAsnl_ysSerGlyArgGinGluLeuLeuGluSer420425430AsnGinTyrLeuPheAlaGlu435〈210〉6<211〉439<212〉PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400>6MetAsnLysTyrPheGluAsnValSerl_ysIleLysTyrGluGlyPro51015l_ysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisLeuArgPheSerlieAlaTyr354045IVpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspLyslieAsnAtaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrlieValAlaMetliel_ysAsp115120125Tyrl_euLysThrSerLysThrLysValLeul_ysGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlal_euGlulie165170175ThrLysGlul_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrl_eul_euAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlal_ysGlulieGly210215220PheGluGlyGinPhel_eulieGluProLysPro匕ysGluProThr匕ys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerIleAspAlaAsnThrGlyAspMetLeul_eu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluValliel_ysMetGlyGlyPheAspl_ysGlyGlyLeu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350Phel_euGlyHislieAlaGlyMetAspAlaPheAlaLysGlyPheLys355360365ValAlaTyrLysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPhel_ysSerLeuGlul_ysTyrAlaLeuGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSerlieLeu420425430AsnGinTyrl_euPheAlaGlu435<210>7<211〉439<212>PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400〉7MetAsnLysTyrPheGluAsnVaISerl_yslieLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVaI202530leAspGlyLysThrMetGluGluHisl_euArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyl_ysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlal_ys65707580AlaArgValGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrMeVaIAlaMetMe(_ysAsp115120125TyrLeuLysThrSerLysThrLysVaILeuSerGlyThrAlaAsnLeu130135140PheSerAsnProArgPheVaIHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVaILysLysAlaLeuGlu165170175ThrLysGlul_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGli180185190GlyTyrGluThrl_eu!_euAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeulieGluProLysProLysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArg275280285AsnGlyValLeuGlySerlieAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspl_ysGlyGlyLeu325330335AsnPheAspAaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHisIleAlaGlyMetAspAlaPheAlaLysGlyPheLys355360365ValAlaTyrI_ysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPhel_ysSerl_euGluL_ysTyrAlaLeuGluHisSer405410415GinIleValAsnLysSerGlyArgGinGluLeuLeuGluSerIleLeu420425430AsnGinTyrLeuPheAlaGlu435<210〉8<211〉439〈212〉PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400>8MetAsnLysTyrPheGluAsnValSerLyslieLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisLeuArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAsplieAiaProGluGlyAspThrLeu100105110ArgGluThrAsnl_ysAsnl_euAspThrIleValAlaMetIleLysAsp115120125Tyrl_euLysThrSerl_ysThrl_ysVall_euCysGlyThrAlaAsnl_eu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlulie165170175ThrLysGlul_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPhe匕euHisMetAlaValAspTyrAlal_ysGlulieGly210215220PheGluGlyGinPheLeulieGluProLysProl_ysGluProThrl_ys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnIleGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerlieAspAlaAsnThrGlyAspMetLeul_eu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluValliel_ysMetGlyGlyPheAspLysGlyGlyl_eu325330335AsnPheAspAlal_ysValArgArgAlaSerPheGluProGluAspl_eu340345350PheLeuGlyHislieAlaGlyMetAspAlaPheAlaLysGlyPheLys355360365ValAlaTyrl_ysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPheLysSerLeuGluLysTyrAlal_euGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSerlieLeu420425430AsnGinTyrLeuPheAlaGlu435<210〉9<211〉439<212〉PRT<213〉ThermoanaerobacteriumsaccharoIyticum〈400〉9MetAsnLysTyrPheGluAsnValSerLyslieLysTyrGluGlyPro151015l_ysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisLeuArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspLysIleAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnl_euAspThrIleValAlaMetIleLysAsp115120125TyrLeuLysThrSerLysThrl_ysValLeulieGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGluHe165170175ThrLysGlul_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPhe匕euIleGluProLysPro匕ysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgl_ys245250255TyrAspLeuAspLysTyrPheLysValAsnIleGluAlaAsnHisAl:260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerlieAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspl_ysGlyGlyUu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrLysLeuValLysAspGlyValPheAspI_ysPheMeGlu370375380GluArgTyrAlaSerTyrl_ysGluGlylieGlyAlaAsplieValSer385390395400Glyl_ysAlaAspPheLysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinIleValAsnLysSerGlyArgGinGluLeuLeuGluSerIleLeu420425430AsnGinTyrLeuPheAlaGlu435<210>10<211〉439<212〉PRT<213>ThermoanaerobacteriumsaccharoIyticum<400>10MetAsnLysTyrPheGluAsnValSerLyslieLysTyrGluGlyPro151015l_ysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisLeuArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspi_yslieAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGfuThrAsnLysAsnLeuAspThrlieValAlaMetlieLysAsp115120125TyrLeuLysThrSerLysThrLysValLeuThrGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVall_ysl_ysAlaLeuGlulie165170175Thrl_ysGlul_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGluIleGly210215220PheGluGlyGinPheLeulieGluProl_ysProLysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270Thrl_euAlaPheHisAspPheGinHisGluLeuArgTyrAlaArgIle275280285AsnGlyValLeuGlySerlieAspAlaAsnThrGlyAspMetl_eul_eu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluValliel_ysMetGlyGlyPheAspl_ysGlyGlyl_eu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHislieAlaGlyMetAspAlaPheAlaLysGlyPheLys355360365ValAlaTyrLysl_euVall_ysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAspMeValSer385390395400GlyLysAlaAspPheLysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinIleValAsnl_ysSerGlyArgGinGlul_euLeuGluSerIlel_eu420425430AsnGinTyrLeuPheAlaGlu435<210〉11<211>439<212>PRT<213>ThermoanaerobacteriumsaccharoIyticum<400>11MetAsnLysTyrPheGluAsnValSerLysIleLysTyrGluGlyPre151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530lieAspGlyLysThrMetGluGluHisLeuArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlal_ys65707580AlaArgValGluAlaAlaPheGluPhePheAspLysIleAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrIleValAlaMetIleLysAsp115120125TyrLeul_ysThrSerl_ysThrLysValLeuAsnGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlu165170175Thrt_ysGlul_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGit180185190GlyTyrGluThrl_euLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGluIleGly210215220PheGluGlyGinPheLeulieGluProLysPro匕ysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPhel_euArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPfieHisAspPheGinHisGluLeuArgTyrAlaArgIle275280285AsnGlyValLeuGlySerIleAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspLysGlyGlyl_eu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlal_ysGlyPhel_ys360365ValAlaTyrLysI_euValLysAspGlyValPheAspi_ysPhelieGlu370375380GluArgTyrAlaSerTyrl_ysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPhel_ysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinlieValAsnl_ysSerGlyArgGinGluLeul_euGluSerliel_eu420425430AsnGinTyrl_euPheAlaGlu435<210>12<211〉439<212>PRT<213>ThermoanaerobacteriumsaccharoIyticum<400>12MetAsnLysTyrPheGluAsnValSerLysMeLysTyrGluGlyPro51015LysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530leAspGlyL_ysThrMetGluGluHisLeuArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspLysIleAsnAlaPro859095<image>imageseeoriginaldocumentpage33</image><formula>formulaseeoriginaldocumentpage34</formula>TyrAspLeuAspLysTyrPheLysValAsnMeGluAlaAsnHisAl;260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArg275280285AsnGlyValLeuGlySerIleAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspLysGlyGlyLeu325330335AsnPheAspAlal_ysValArgArgAlaSerPheGluProGluAspl_eu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrLysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlyIleGlyAlaAspIleValSer385390395400Glyl_ysAlaAspPhel_ysSerLeuGlul_ysTyrAlal_euGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSerMel_eu420425430AsnGinTyrLeuPheAlaGlu435<210>14〈211〉439<212>PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400>14MetAsnLysTyrPheGluAsnValSerLysliel_ysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisl_euArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580/UaArgValGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrlieValAlaMetliel_ysAsp115120125TyrLeuLysThrSerLysThrLysValLeuTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlulie165170175Thrl_ysGluLeuGlySerGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPhel_euHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeulieGluProLysProLysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArgMe275280285AsnGlyVall_euGlySerIleAspAlaAsnThrGlyAspMetl_euLeu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluValliel_ysMetGlyGlyPheAspLysGlyGlyLeu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheL_euGlyHislieAlaGlyMetAspAlaPheAlaLysGlyPheLys355360365ValAlaTyrLysLeuValLysAspGlyValPheAspl_ysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPhel_ysSerLeuGlul_ysTyrAlaLeuGluHisSer405410415GinValAsnLysSerGlyArgGinGluLeuLeuGluSerlieLeu420425430AsnGinTyrLeuPheAlaGlu435<210〉15<211〉439〈212〉PRT<213>Thermoanaerobacteriumsaccharo1yticum<400〉15MetAsnLysTyrPheGluAsnValSer匕ysMeLysTyrGluGlyPro151015l_ysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluiHisLeuArgPheSerMeAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspL_yslieAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrlieValAlaMetlieLysAsp115120125TyrLeuLysThrSerLysThrLysVall_euTrpGlyThrAlaAsnl_eu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlulie165170175ThrLysGlul_euGlylieGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrl_euLeuAsnThrAspMetGlul_euGlul_euAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGluIleGly210215220PheGluGlyGinPheLeulieGluProLysPro匕ysGluProThrLys240225230235HisGinTyrAspPheAspValAlaAsnValLeuAlaPhe37LeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAl;260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerIleAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspLysGlyGlyLeu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHisMeAlaGlyMetAspAlaPheAlaLysGlyPheLys355360365ValAlaTyrLysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400Glyl_ysAlaAspPheLysSerLeuGluLysTyrAlal_euGluHisSer405410415GinlieValAsnl_ysSerGlyArgGinGlul_eul_euGluSerlieLeu420425430AsnGinTyrLeuPheAlaGlu435<210〉16<211〉439<212〉PRT<213〉Thermoanaerobacteriumsaccharo1yticum<400>16MetAsnLysTyrPheGluAsnValSerLyslieLysTyrGiuGlyPro151015LysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisLeuArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspMeAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrIleValAlaMetIleLysAsp115120125TyrLeuLysThrSerl_ysThrLysValLeuTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlulie165170175ThrLysGluLeuGlyThrGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeulieGluProLysPro匕ysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerIleAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluValIlel_ysMetGlyGlyPheAspLysGlyGlyl_eu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspl_eu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrLysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400Glyl_ysAlaAspPhel_ys405Serl_euGlul_ysTyrAlal_euGluHisSer410415GinIIeVaIAsnLysSer420GlyArgGinGlul_euLeuGluSerlieLeu425430AsnGinTyrLeuPheAlaGlu435<210〉17〈211〉439<212>PRT<213〉ThermoanaerobacteriumsaccharoIyticum〈400〉17MetAsnLys丁yrPheGluAsnVaISerLyslieLysTyrGluGlyPro51015l_ysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVaI202530leAspGlyLysThrMetGluGluHisLeuArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlaLys65707580AlaArgVaIGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrlieVaIAlaMetlieLysAsp115120125Tyrl_euLysThrSerLysThrLysVaILeuTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheVaIHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVaILysl_ysAlaLeuGlulie165170175ThrLysGluLeuGlyValGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPhel_euHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeulieGluProLysProLysGluProThrLys225230235240<image>imageseeoriginaldocumentpage41</image>AlaArgVaIGluAlaAlaPheGluPhePheAspLys8590leAsnAlaPro95PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrlieVaIAlaMetlieLysAsp115120125TyrLeuLysThrSerLysThrLysVaILeuTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheVaIHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVaIl_ysLysAlaLeuGlulie165170175ThrLysGluLeuGlyArgGluAsnTyrValSerTrpGlyGlyArgGlu180185190GlyTyrGluThrl_euLeuAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPhel_euHisMetAlaValAspTyrAlaLysGluIleGly210215220PheGluGlyGinPheLeulieGluProLysProLysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPhel_euArgl_ys245250255TyrAspl_euAspLysTyrPhel_ysValAsnMeGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArgMe275280285AsnGlyValLeuGlySerIleAspAlaAsnThrGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspHeArgMetThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspl_ysGlyGlyl_eu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrt_ysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400<formula>formulaseeoriginaldocumentpage43</formula><formula>formulaseeoriginaldocumentpage44</formula>AlaArgValGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrl_eu100105110ArgGluThrAsnLysAsnLeuAspThrIleValAlaMetIleLysAsp115120125TyrLeul_ysThrSerl_ysThrI_ysValI_euTrpGlyThrAlaAsnl_eu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVall_ysLysAlaLeuGlulie165170175ThrLysGlul_euGlyArgGluAsnTyrValProTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPhel_euHisMetAlaValAspTyrAlaLysGluIleGly210215220PheGluGlyGinPhel_eulieGluProLysProLysGluProThrl_ys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyVall_euGlySerIleAspAlaAsnThrGlyAspMetl_eul_eu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMet"ThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspUsGlyGlyl_eu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspl_eu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrl_ysl_euValLysAspGlyValPheAspl_ysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPhel_ysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinNeVaIAsnl_ysSerGlyArgGinGluLeul_euGluSerlieLeu420425430AsnGinTyrl_euPheAlaGlu435<210>21<211〉439<212〉PRT<213〉ThermoanaerobacteriumsaccharoIyticum〈400〉21MetAsnLysTyrPheGluAsnVaISerLysIleLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVaI202530leAspGlyLysThrMetGluGluHisLeuArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyl_ysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlaLys65707580AlaArgVaIGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrlieVaIAlaMetlieLysAsp115120125Tyrl_euLysThrSerLysThrLysVaILeuTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheVaIHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVaILysLysAlaLeuGlulie165170175ThrLysGluLeuGlyArgGluAsnTyrValAlaTrpGlyGlyArgGlu180185190GlyTyrGluThrLeul_euAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaVaiAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeulieGluPro匕ysPro匕ysGluProThrLys225230235240HisGinTyrAspPheAspVaIAlaAsnVaILeuAlaPheLeuArgLys245250255TyrAspLeuAspl_ysTyrPheLysVaIAsnIleGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArgIle275280285AsnGlyVaILeuGlySerIleAspAlaAsnThrGlyAspMetl_euLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluVaIliel_ysMetGlyGlyPheAspLysGlyGlyl_eu325330335AsnPheAspAlaLysVaIArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrLysLeuVaILysAspGlyVaIPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPheLysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinlieValAsnLysSerGlyArgGinGlul_euLeuGluSerliel_eu420425430AsnGinTyrLeuPheAlaGlu435<210〉22<211〉439<212>PRT<213>ThermoanaerobacteriumsaccharoIyticum<400〉22MetAsnLysTyrPheGluAsnValSerLysIleLysTyrGluGlyPro151015l_ysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisl_euArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlal_ys65707580AlaArgVaIGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrl_eu100105110ArgGluThrAsnLysAsnLeuAspThrlieVaIAlaMetlieLysAsp115120125TyrLeuLysThrSerLysThrLysVaIl_euTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheVaIHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVaIl_ysl_ysAlal_euGlulie165170175Thrl_ysGlul_euGlyArgGluAsnTyrVaIPheTrpGlyGlyArgGlu180185190GlyTyrGluThrI_euLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaVaIAspTyrAlaLysGluIleGly210215220PheGluGlyGinPhe匕eulieGluPro匕ysProLysGluProThrLys225230235240HisGinTyrAspPheAspVaIAlaAsnVaILeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyfPheLysVaIAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyVaILeuGlySerIleAspAlaAsnGinGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluVaIlieLysMetGlyGlyPheAspLysGlyGlyLeu325330335AsnPheAspAlaLysVaIArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHislieAlaGlyMetAspAlaPheAlaLysGlyPheLys355360365ValAlaTyrLysLeuVaIl_ysAspGlyVaIPheAspLysPheIleGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPheLysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinIleValAsnl_ysSerGlyArgGinGlul_euLeuGluSerIleLeu420425430AsnGinTyrLeuPheAlaGlu435<210>23<211〉439〈212〉PRT<213>ThermoanaerobacteriumsaccharoIyticum<400>23MetAsnLysTyrPheGluAsnValSerl_ysIleLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyLysThrMetGluGluHisLeuArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspLysIleAsnAlaPro859095PhePheGysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnl_ysAsnLeuAspThrlieValAlaMetlieLysAsp115120125Tyrl_euLysThrSerl_ysThrl_ysVall_euTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlu165170175ThrLysGluLeuGlyArgGluAsnTyrValPheTrpGlyGlyArgGli180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeulieGluProLysPro匕ysGluProThrLys49225230235240HisGinTyrAspPheAspValAlaAsnVall_euAlaPheArgLys245250255TyrAspl_euAspLysTyrPheLysValAsnIleGluAlaAsnHisAl〔260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArgIle275280285AsnGlyValLeuGlySerlieAspAlaAsnMeGlyAspMetl_eul_eu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrl_eu305310315320AlaMetTyrGluValliel_ysMetGlyGlyPheAspl_ysGlyGlyLeu325330335AsnPheAspAlal_ysValArgArgAlaSerPheGluProGluAspl_eu340345350PheLeuGlyHis355leAlaGlyMet八spAlaPheAlal_ysGlyPheLys360365ValAlaTyrl_ysLeuValLysAspGlyValPheAspl_ysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400Glyl_ysAlaAspPhel_ysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSerliel_eu420425430AsnGinTyrLeuPheAlaGlu435<210〉24<2">439〈212>PRT<213>ThermoanaerobacteriumsaccharoIyticum<400>24MetAsni_ysTyrPheGluAsnValSerLysliel_ysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530lieAspGlyl_ysThrMetGluGluHisLeuArgPheSerHeAlaTyr354045T卬HisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlal_ys<image>imageseeoriginaldocumentpage51</image>GluArgTyrAlaSerTyrl_ysGluGlylieGlyAlaAsplieValSer385390395400GlyLysAlaAspPheLysSerl_euGluLysTyrAlaLeuGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSerlieLeu420425430AsnGinTyrLeuPheAlaGlu435<210>25〈211〉439<212〉PRT〈213〉ThermoanaerobacteriumsaccharoIyticum<400〉25MetAsnLysTyrPheGluAsnValSerLyslieLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyL_ysThrMetGluGluHisLeuArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAiaAlaPheGluPhePheAspt_yslieAsnAtaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrl_eu100105110ArgGluThrAsnLysAsnLeuAspThrlieValAlaMetlieLysAsp115120125TyrLeuLysThrSerLysThrLysValLeuTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysl_ysAlal_euGlulie165170175ThrLysGlut_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220<formula>formulaseeoriginaldocumentpage53</formula>MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspl_yslieAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnl_euAspThr115120leValAlaMetlieLysAsp125TyrLeul_ysThrSerLysThrLysValLeuTrpGlyThrAlaAsnt_eu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlulie165170175ThrLysGlul_euGlyArgGluAsnTyrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAla(_ysGlulieGly210215220PheGluGlyGinPheLeulieGluProLysProLysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGlu(_euArgTyrAlaArgMe275280285AsnGlyValLeuGlySerIleAspAlaAsnMetGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluValliel_ysMetGlyGlyPheAspLysGlyGlyl_eu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrt_ysLeuVall_ysAspGlyValPheAspLysPheHeGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400GlyI_ysAlaAspPheLysSerl_euGluLysTyrAlaLeuGluHisSer405410415GinlieValAsnLysSerGlyArgGinGlu(_eu(_euGluSerlieLeu420425430AsnGinTyrLeuPheAlaGlu435<210>27<211>439<212>PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400>27MetAsnLysTyrPheGluAsnValSerLyslieLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyl_ysThrMetGluGluHisl_euArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlal_ys65707580AlaArgValGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrIleValAlaMetIleLysAsp115120125TyrLeul_ysThrSerLysThrl_ysVall_euTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerGysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGiulie165170175ThrLysGluLeuGlyArgGluAsn丁yrValPheTrpGlyGlyArgGlu180185190GlyTyrGluThrl_euLeuAsnThrAspMetGluLeuGlul_euAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPhe匕euIleGluProLysProLysGluProThrLys225230235240HisGinTyrAspPheAspVaiAlaAsnVall_euAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnIleGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerIleAspAlaAsnGiuGlyAspMetLeu(_eu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluVallieLysMetGlyGlyPheAspl_ysGlyGlyLeu325330335AsnPheAspAla!_ysValArgArgAlaSerPheGluProGluAspLeu340345350Phel_euGlyHisHeAlaGlyMetAspAlaPheAlaLysGlyPhel_ys355360365ValAlaTyrLysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrl_ysGluGlylieGlyAlaAsplieValSer385390395400Glyl_ysAlaAspPheLysSerl_euGluLysTyrAlal_euGluHisSer405410415GinlieValAsnLysSerGlyArgGinGlul_eul_euGluSerliel_eu420425430AsnGinTyrLeuPheAlaGlu435<210>28<211〉439<212>PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400〉28MetAsnl_ysTyrPheGluAsnValSerLysIlel_ysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyl_ysThrMetGluGluHisl_euArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr50556056<formula>formulaseeoriginaldocumentpage57</formula>GluArgTyrAlaSerTyrLysGluGlyIleGlyAlaAspIleVaISer385390395400Glyl_ysAlaAspPheLysSerLeuGluLysTyrAlal_euGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSer420425430Isl_6UAsnGinTyrLeuPheAlaGlu435<210>29<211>439<212〉PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400〉29MetAsnl_ysTyrPheGluAsnValSerl_yslieLysTyrGluGlyPro151015l_ysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVal202530leAspGlyl_ysThrMetGluGluHisl_euArgPheSerlieAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlal_ys65707580AlaArgValGluAlaAlaPheGluPhePheAspl_ysIleAsnAlaPro859095PhePheCysPheHisAspArgAsplieAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrIleValAlaMetIleLysAsp115120125TyrLeu!_ysThrSerLysThrLysValLeuTrpGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspVafPheAlaTyrSerAlaAlaGinValLysl_ysAlaLeuGlu165170175ThrLysGluLeuGlyAlaGluAsnTyrValPheTrpGlyGlyArgGk180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly210215220PheGluGlyGinPheLeulieGluProl_ysProl_ysGluProThrl_ys225230235240HisGinTyrAspPheAspVaIAlaAsnVaILeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysVaIAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyVaIl_euGlySerIleAspAlaAsnGinGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluVaIMeLysMetGlyGlyPheAspl_ysGlyGlyLeu325330335AsnPheAspAlaLysVaIArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPheLys360365ValAlaTyrl_ysLeuVaIl_ysAspGlyVaIPheAspl_ysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieVaISer385390395400GlyLysAlaAspPhel_ysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinIleValAsnLysSerGlyArgGinGluLeuLeuGluSerIleLeu420425430AsnGinTyrLeuPheAlaGlu435〈210〉30<211>439<212〉PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400〉30MetAsnl_ysTyrPheGluAsnValSerLyslieI_ysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyl_ysThrMetGluGluHisl_euArgPheSerlieAlaTyr354045T卬HisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560<formula>formulaseeoriginaldocumentpage60</formula>370375380GluArgTyrAlaSerTyrl_ysGluGlylieGlyAlaAsplieVaISer385390395400Glyl_ysAlaAspPheLysSerLeuGluLysTyrAlal_euGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSerlieLeu420425430AsnGinTyrl_euPheAlaGlu435<210>31<211>439<212〉PRT<213〉ThermoanaerobacteriumsaccharoIyticum<400〉31MetAsnLysTyrPheGluAsnVaISer匕yslieLysTyrGluGlyPro151015l_ysSerAsnAsnProTyrSerPhel_ysPheTyrAsnProGluGluVaI202530leAspGlyl_ysThrMetGluGluHisl_euArgPheSerHeAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAsplieAlaLys65707580AlaArgVaIGluAlaAlaPheGluPhePheAspLyslieAsnAlaPro859095PhePheCysPheHisAspArgAspIleAlaProGluGlyAspThrLeu100105110ArgGluThrAsnLysAsnLeuAspThrIleVaIAlaMetIleLysAsp115120125TyrLeuLysThrSerl_ysThrl_ysVaIl_euPheGlyThrAlaAsnLeu130135140PheSerAsnProArgPheVaIHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinVaILysLysAlaLeuGlulie165170175ThrLysGluUuGlyAlaGluAsnTyrVaiPheTrpGlyGlyArgGlu180185190GlyTyrGluThrl_eul_euAsnThrAspMetGlul_euGlul_euAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGlulieGly61210215220PheGluGlyGinPheLeulieGluProl_ysProl_ysGluProThrl_ys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspLeuAspLysTyrPheLysValAsnlieGtuAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGlul_euArgTyrAlaArglie275280285AsnGlyValLeuGlySerlieAspAlaAsnGinGlyAspMetLeuLeu290295300GlyTrpAspThrAspGinPheProThrAspIleArgMetThrThrLeu305310315320AlaMetTyrGluVaIIlei_ysMetGlyGlyPheAspl_ysGlyGlyl_eu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHisIleAlaGlyMetAspAlaPheAlal_ysGlyPhel_ys355360365ValAlaTyrLysLeuValLysAspGlyValPheAspLysPhelieGlu370375380GluArgTyrAlaSerTyrLysGluGlylieGlyAlaAsplieValSer385390395400Glyl_ysAlaAspPhei_ysSerLeuGluLysTyrAlaLeuGluHisSer405410415GinlieValAsnLysSerGlyArgGinGluLeuLeuGluSerlieLeu425430420AsnGinTyrl_euPheAlaGlu435<210〉32<211〉439<212〉PRT<213>ThermoanaerobacteriumsaccharoIyticum<400>32MetAsnLysTyrPheGluAsnValSerLyslieLysTyrGluGlyPro151015LysSerAsnAsnProTyrSerPheLysPheTyrAsnProGluGluVal202530leAspGlyl_ysThrMetGluGluHisLeuArgPheSerIleAlaTyr354045TrpHisThrPheThrAlaAspGlyThrAspGinPheGlyLysAlaThr505560MetGinArgProTrpAsnHisTyrThrAspProMetAspIleAlaLys65707580AlaArgValGluAlaAlaPheGluPhePheAspt_ysIleAsnAlaPro859095PhePheGysPheHisAspArgAsplieAlaProGluGlyAspThrl_eu100105110ArgGluThrAsnLysAsnLeuAspThr115120leValAlaMetIleLysAsp125TyrLeul_ysThrSerLysThrLysValLeuPheGlyThrAlaAsnLeu130135140PheSerAsnProArgPheValHisGlyAlaSerThrSerCysAsnAla145150155160AspValPheAlaTyrSerAlaAlaGinValLysLysAlaLeuGlulie165170175ThrLysGluLeuGlyAlaGluAsnTyrValSerTrpGlyGlyArgGlu180185190GlyTyrGluThrLeuLeuAsnThrAspMetGluLeuGluLeuAspAsn195200205PheAlaArgPheLeuHisMetAlaValAspTyrAlaLysGluIleGly210215220PheGluGlyGinPheLeulieGluProLysProLysGluProThrLys225230235240HisGinTyrAspPheAspValAlaAsnValLeuAlaPheLeuArgLys245250255TyrAspl_euAspl_ysTyrPhel_ysValAsnlieGluAlaAsnHisAla260265270ThrLeuAlaPheHisAspPheGinHisGluLeuArgTyrAlaArglie275280285AsnGlyValLeuGlySerIleAspAlaAsnGinGlyAspMetl_euLeu290295300GlyTrpAspThrAspGinPheProThrAsplieArgMetThrThrLeu305310315320AlaMetTyrGluValliel_ysMetGlyGlyPheAspLysGlyGlyLeu325330335AsnPheAspAlaLysValArgArgAlaSerPheGluProGluAspLeu340345350PheLeuGlyHis355leAlaGlyMetAspAlaPheAlaLysGlyPhel_ys360365<image>imageseeoriginaldocumentpage64</image>权利要求1.一种葡萄糖异构酶突变体，其特征在于以序列表中的序列2为参考序列，具有在第139位的单突变，并且以D-葡萄糖为底物其具有比亲本高出至少50％的葡萄糖异构酶催化活性。2.权利要求1所述的葡萄糖异构酶突变体,其中第139位的色氨酸突变为赖氨酸(Lys)、或丝氨酸(Ser)、或半胱氨酸(Cys)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe)。3.权利要求2所述的葡萄糖异构酶突变体,其中第139位的色氨酸突变为异亮氨酸(Ile)、丝氨酸(Ser)、半胱氨酸(Cys)、或天冬酰胺(Asn)。4.权利要求3所述的葡萄糖异构酶突变体,其中第139位的色氨酸突变为丝氨酸(Ser)、半胱氨酸(Cys)、或天冬酰胺(Asn)。5.—种葡萄糖异构酶突变体，其特征在于以序列表中的序列2为参考序列，具有在第139位和182位的双突变，并且以D-葡萄糖为底物其具有比亲本高出至少50%的葡萄糖异构酶催化活性。6.权利要求5所述的葡萄糖异构酶突变体，其中第139位的色氨酸突变为赖氨酸(Lys)、或丝氨酸(Ser)、或半胱氨酸(Cys)、或异亮氨酸(Ile)、或苏氨酸(Thr)、或天冬酰胺(Asn)、或苯丙氨酸(Phe);同时第182位的精氨酸突变为脯氨酸(Pro)、或丝氨酸(Ser)、或丙氨酸(Ala)、或异亮氨酸(Ile)、或苏氮酸(Thr)、或缬氨酸(Val)。7.权利要求6所述的葡萄糖异构酶突变体，其中第139位的色氨酸突变为苯丙氨酸(Phe)同时第182位的精氨酸突变为丙氨酸(Ala)。8.权利要求1-7任意一项所述的葡萄糖异构酶突变体的应用,其中所述的葡萄糖异构酶突变体用于转化D-葡萄糖至果糖。9.权利要求8所述的葡萄糖异构酶突变体的应用,其中所述的应用为制备果葡糖浆。10.权利要求9所述的应用，其中所述的果葡糖浆的果糖含量为至少55重量％。11.一种DNA，其含有编码权利要求l-7任意一项所述的葡萄糖异构酶突变体的核苷酸序列。全文摘要本发明披露利用基因突变技术改良的一系列耐热高催化活性Thermoanaerobacteriumsaccharolyticum葡萄糖异构酶突变体。这些葡萄糖异构酶突变体可用于直接产生果糖含量等于或高于55重量％的果葡糖浆，或用于产生果糖含量低于55重量％的果葡糖浆。文档编号C12N9/92GK101423828SQ20081014883公开日2009年5月6日申请日期2004年6月16日优先权日2004年5月26日发明者傅荣昭,刘兆明,冬沈,骏王,金彩科,陈军明申请人:百瑞全球有限公司