CN1702172A - 葡萄糖异构酶突变体 - Google Patents
葡萄糖异构酶突变体 Download PDFInfo
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Abstract
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Thermoanaerobacterium saccharolyticum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。
Description
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(Glucose isomerase，E.C.5.3.1.5，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，et al.，BiosciBiotechnol Biochem，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(Actinoplanesmissouriensis)、凝结芽孢杆菌(Bacillus coagulans)、或链霉菌(Streptomyces)。这些葡萄糖异构酶在高温(如高于65℃)下稳定性差。因此，目前工业上在60℃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Thermoanaerobacteriumsaccharolyticum、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，139：1227-1234，1993；Vieille et al.，MethodsEnzymology，330：215-24，2001；Lee et al.，Journal of General Microbiology，139：1241-1243，1993；Scriprapundh et al.，Protein Engineering，13：259-265，2000；Scriprapundh et al.，Protein Engineering，16：683-690，2003；Zeikus et 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T.saccharolyticum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，利用本领域已知的技术，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。
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)、T.saccharolyticum和链霉菌(如Streptomyces diastaticusM1033)。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出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(Pro)；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，本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。
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℃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(pH7.0)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℃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”系指来自Thermoanaerobacterium saccharolyticum ATCC49915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，139：1227-1234，1993；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位具有至少一个氨基酸的差异，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(Eur.J.Biochem.，138：9-37，1984)。
附图的简要说明：
图1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体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80℃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℃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(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1，400nM引物T2，1.5U Pfu 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 77：51-59，1989和White et al.(PCR Protocol：currentmethods and applications.Totowa，N.J.：Humana Press，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；利用引物对139FF和T2，扩增FFT2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139FR或400nM引物139FF和400nM引物T2，1.5U Pfu 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，50μM dCTP，50μMdGTP，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QIAquickExtraction 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.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-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。
按照类似步骤构建突变体MGI-W139K、MGI-W139S、MGI-W139C、MGI-W139I、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6-11。
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 77：51-59，1989)和White et al.(PCR Protocol：currentmethods and applications.Totowa，N.J.：Humana Press，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；利用引物对182AF和T2，扩增AFT2片段。扩增反应条件为：20mM Tris-HCl(pH8.8)，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，20ngpGEMT-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％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 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QIAquick Extraction 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-R182A DNA，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、MGI-R182T和MGI-R182V，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13-17。
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 et al.(Gene 77：51-59，1989)和White et al.(PCR Protocol：currentmethods and applications.Totowa，N.J.：Humana Press，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；利用引物对187SF和T2，扩增SFT2片段。扩增反应条件为：20mM Tris-HCl(pH8.8)，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187SR或400nM引物T2和400nM引物187SF，1.5U Pfu DNA聚合酶，20ngpGEMT-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20ng SFT2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-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-F187S DNA，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 et al.(Gene 77：51-59，1989)和White et al.(PCR Protocol：currentmethods and applications.Totowa，N.J.：Humana Press，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；利用引物对299QF和T2，扩增QFT2片段。扩增反应条件为：20mM Tris-HCl(pH8.8)，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，20ngpGEMT-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％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 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QIAquick Extraction 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-T299Q DNA，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。
按照类似步骤构建突变体MGI-T299I、MGI-T299Y、MGI-T299C、MGI-T299M和MGI-T299E，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Ho et al.(Gene 77：51-59，1989)和White et al.(PCR Protocol：currentmethods and applications.Totowa，N.J.：Humana Press，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：20mMTris-HCl(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μMdATP，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：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 T1FR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T1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T1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 et al.(Gene 77：51-59，1989)和White et al.(PCR Protocol：currentmethods and applications.Totowa，N.J.：Humana Press，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(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM182AF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)，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 T1FR片段，20ngFFAR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-3 DNA，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 et al.(Gene 77：51-59，1989)和White et al.(PCR Protocol：currentmethods and applications.Totowa，N.J.：Humana Press，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(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM182AF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(pH8.8)，10mM KCl，10mM(NH4)2SO4，2mMMgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM187SF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(pH8.8)，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 T1FR片段，20ngFFAR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-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-4 DNA，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1
| 目标产物名称 | 引物对 |
| 亲本 | T1：5’AGCCTAGGTTAATTAACTTTAAGAAGGAGATATACATATGAATAAATATTTTGAGA 3’T2：5’ATAAGCTCAGCGGCGCGCCTTATTCTGCAAACAAATAC 3’ |
| 突变体MGI-W139K | 139KF：5’AAGTTTTGAAAGGTACCGCAAATCTTTTCT 3’139KR：5’TGCGGTACCTTTCAAAACTTTTGTCTTGCT 3’ |
| 突变体MGI-W139S | 139SF：5’AAGTTTTGTCAGGTACCGCAAATCTTTTCT 3’139SR：5’TGCGGTACCTGACAAAACTTTTGTCTTGCT 3’ |
| 突变体MGI-W139C | 139CF：5’AAGTTTTGTGCGGTACCGCAAATCTTTTCT 3’139CR：5’TGCGGTACCGCACAAAACTTTTGTCTTGCT 3’ |
| 突变体MGI-W139I | 139IF：5’AAGTTTTGATTGGTACCGCAAATCTTTTCT 3’139IR：5’TGCGGTACCAATCAAAACTTTTGTCTTGCT 3’ |
| 突变体MGI-W139T | 139TF：5’AAGTTTTGACAGGTACCGCAAATCTTTTCT 3’139TR：5’TGCGGTACCTGTCAAAACTTTTGTCTTGCT 3 |
| 突变体MGI-W139N | 139NF：5’AAGTTTTGAACGGTACCGCAAATCTTTTCT 3’139NR：5’TGCGGTACCGTTCAAAACTTTTGTCTTGCT 3’ |
| 突变体MGI-W139F | 139FF：5’AAAAGTTTTGTTTGGTACCGCAAATCTTTTCTC 3’139FR：5′TTGCGGTACCAAACAAAACTTTTGTCTTGCTGG 3’ |
| 突变体MGI-R182P | A182PF：5’AGCTTGGCCCGGAAAACTACGTATTTTGGG 3’A182PR：5’GTAGTTTTCCGGGCCAAGCTCCTTAGTAAT 3’ |
| 突变体MGI-R182S | 182SF：5’AGCTTGGCTCAGAAAACTACGTATTTTGGG 3’182SR：5’GTAGTTTTCTGAGCCAAGCTCCTTAGTAAT 3’ |
| 突变体MGI-R182A | 182AF：5’GGAGCTTGGCGCGGAAAACTACGTATTTTGGGG 3’182AR：5′CGTAGTTTTCCGCGCCAAGCTCCTTAGTAATCT 3’ |
| 突变体MGI-R1821 | 182IF：5’AGCTTGGCATTGAAAACTACGTATTTTGGG 3’182IR：5’GTAGTTTTCAATGCCAAGCTCCTTAGTAAT 3’ |
| 突变体MGI-R182T | 182TF：5’AGCTTGGCACAGAAAACTACGTATTTTGGG 3’182TR：5’GTAGTTTTCTGTGCCAAGCTCCTTAGTAAT 3’ |
| 突变体MGI-R182V | 182VF：5’AGCTTGGCGTGGAAAACTACGTATTTTGGG 3’182VR：5’GTAGTTTTCCACGCCAAGCTCCTTAGTAAT 3’ |
| 突变体MGI-F187G | 187GF：5’ACTACGTAGGCTGGGGTGGAAGAGAAGGGT 3’187GR：5’CCACCCCAGCCTACGTAGTTTTCGCGGCCA 3’ |
| 突变体MGI-F187S | 187SF：5’ACTACGTAAGCTGGGGTGGAAGAGAAGGGT 3’187SR：5’CCACCCCAGCTTACGTAGTTTTCGCGGCCA 3’ |
| 突变体MGI-F187A | 187AF：5’ACTACGTAGCGTGGGGTGGAAGAGAAGGGT 3’187AR：5’CCACCCCACGCTACGTAGTTTTCGCGGCCA 3’ |
| 突变体MGI-F187P | 187PF：5’ACTACGTACCGTGGGGTGGAAGAGAAGGGT 3’187PR：5’CCACCCCACGGTACGTAGTTTTCGCGGCCA 3’ |
| 突变体MGI-T299I | 299IF：5’GACGCAAATATTGGCGACATGCTTTTAGGAT 3’299IR：5’CATGTCGCCAATATTTGCGTCGATTGATCCT 3’ |
| 突变体MGI-T299Y | 299YF：5’GACGCAAATTATGGCGACATGCTTTTAGGAT 3’299YR：5’CATGTCGCCATAATTTGCGTCGATTGATCCT 3’ |
| 突变体MGI-T299C | 299CF：5’GACGCAAATTGCGGCGACATGCTTTTAGGAT 3’299CR：5’CATGTCGCCGCAATTTGCGTCGATTGATCCT 3’ |
| 突变体MGI-T299M | 299MF：5’GACGCAAATATGGGCGACATGCTTTTAGGAT 3’299MR：5’CATGTCGCCCATATTTGCGTCGATTGATCCT 3’ |
| 突变体MGI-T299Q | 299QF：5’TGACGCAAATCAAGGCGACATGCTTTTGGGATG 3’299QR：5’GCATGTCGCCTTGATTTGCGTCGATTGATCCTA 3’ |
| 突变体MGI-T299E | 299EF：5’GACGCAAATGAAGGCGACATGCTTTTAGGAT 3’299ER：5’CATGTCGCCTTCATTTGCGTCGATTGATCCT 3’ |
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Lee et al.，Journal of General Microbiology，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1ml20mM磷酸钠缓冲液(pH6.5)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11：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9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-果糖通过半胱氨酸-咔唑法测定。测定方法参见Dische et al.，J.Biol.Chem，192：583-587，1951)以及Nakamura，Agr.Biol.Chem.32：701-706，1968)。用CoomassiePlusProtein Assay Reagent Kit(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11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
| 酶的名称 | 氨基酸序列的序列号 | 比活 |
| 亲本 | SEQ ID NO.：2 | 100 |
| MGI-W139S | SEQ ID NO.：7 | 392 |
| MGI-W139K | SEQ ID NO.：6 | 246 |
| MGI-W139C | SEQ ID NO.：8 | 382 |
| MGI-W139I | SEQ ID NO.：9 | 329 |
| MGI-W139T | SEQ ID NO.：10 | 254 |
| MGI-W139N | SEQ ID NO.：11 | 376 |
| MGI-W139F | SEQ ID NO.：5 | 195 |
| MGI-R182P | SEQ ID NO.：13 | 264 |
| MGI-R182S | SEQ ID NO.：14 | 327 |
| MGI-R182A | SEQ ID NO.：12 | 195 |
| MGI-R182I | SEQ ID NO.：15 | 654 |
| MGI-R182T | SEQ ID NO.：16 | 287 |
| MGI-R182V | SEQ ID NO.：17 | 617 |
| MGI-F187G | SEQ ID NO.：19 | 195 |
| MGI-F187S | SEQ ID NO.：18 | 261 |
| MGI-F187A | SEQ ID NO.：21 | 255 |
| MGI-F187P | SEQ ID NO.：20 | 325 |
| MGI-T299I | SEQ ID NO.：23 | 250 |
| MGI-T299Y | SEQ ID NO.：24 | 254 |
| MGI-T299C | SEQ ID NO.：25 | 468 |
| MGI-T299M | SEQ ID NO.：26 | 272 |
| MGI-T299Q | SEQ ID NO.：22 | 286 |
| MGI-T299E | SEQ ID NO.：27 | 338 |
| MGI-2 | SEQ ID NO.：28 | 470 |
| MGI-2AQ | SEQ ID NO.：29 | 195 |
| MGI-2FQ | SEQ ID NO.：30 | 260 |
| MGI-3 | SEQ ID NO.：31 | 512 |
| MGI-4 | SEQ ID NO.：32 | 751 |
实施例13：D-葡萄糖转化为果糖转化率之测定
主要参考Kaneko et al.，Biosci Biotechnol Biochem，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250μM之CoCl2和终浓度为5mM之MgCl2，调pH至6.5。取底物溶液B 60μl，然后加入40μl按实施例10制备的葡萄糖异构酶突变体MGI-4。80℃反应4小时后，加入100μl 20％三氯醋酸终止反应，离心(10℃，17,800g，15分钟)并收集上清液。取10μl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μBONDAPAK NH2 SS COL 3.9X300(Waters，USA)，流动相为乙腈-水(85∶15)，流速0.5毫升/分钟。探测器为ELSD 500。进样量为10μl。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200μl酶液，并加入200μl矿物油。将离心管置于80℃水浴中，0小时、2小时、4小时、8小时、16小时、32小时、72小时后分别取出一管酶液，离心(10℃，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显示亲本葡萄糖异构酶在80℃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℃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4.0小时；MGI-2的活性在80℃的半衰期为4.4小时；MGI-3的活性在80℃的半衰期为21小时；MGI-4的活性在80℃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250μM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250μM之CoCl2和终浓度为5mM之MgCl2，调pH至4.5。配制底物溶液E含1.0M的D-葡萄糖、20mM醋酸钠缓冲溶液(pH5.0)、终浓度为250μM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250μM之CoCl2和终浓度为5mM之MgCl2，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250μM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250μM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250μM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250μM之CoCl2和终浓度为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250μM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、20mM Tris-HCl缓冲溶液(pH8.5)、终浓度为250μM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、20mM Tris-HCL缓冲溶液(pH9.0)、终浓度为250μM之CoCl2和终浓度为5mM之MgCl2，调pH至9.0。取反应管11管，每管100μl反应液包括按实施例9制备的D-葡萄糖异构酶10μl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M 90μl。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-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MGI-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)、250μMCoCl2、5mM MgCl2]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10μl部分纯化的亲本葡萄糖异构酶(见实施例9)与90μl不同浓度的底物溶液N在65℃或80℃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km、饱和底物浓度下的最大反应速率Vmax与转换数Kcat。其结果如下表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
| 底物 | 65℃ | 80℃ | ||||||||||
| Km(mM) | Kcat(min-1) | Kcat/Km(mM-1min-1) | Km(mM) | Kcat(min-1) | Kcat/Km(mM-1min-1) | |||||||
| 亲本 | MGI-4 | 亲本 | MGI-4 | 亲本 | MGI-4 | 亲本 | MGI-4 | 亲本 | MGI-4 | 亲本 | MGI-4 | |
| D-葡萄糖 | 138.50 | 27.90 | 344.50 | 1009.00 | 2.49 | 36.12 | 149.40 | 51.30 | 881.10 | 2981.1 | 5.90 | 58.06 |
实施例20 固相化葡萄糖异构酶突变体MGI-4
主要参考Ge，et al.，Appl.Biochem.Biotechnol.69：57-69，1998。具体步骤是：取固相酶载体氯化聚苯乙烯乙基胺(成都化工研究所提供)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-4 8克(按实施例10所述)，室温(22℃)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-MGI-4的E.coli HB101至OD600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500ml 2％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，测得固定化细胞的活性为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(BioRight Worldwide Company Ltd.)
<120>葡萄糖异构酶突变体
<130>002FPI0007C1
<150>CN 200410042773.2
<151>2004-05-26
<160>32
<170>PatentIn version 3.2
<210>1
<211>1320
<212>DNA
<213>Thermoanaerobacterium saccharolyticum
<400>1
atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat 60
ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag 120
catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180
ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa 240
gcaagggtag aagcagcatt tgagtttttt gataagataa atgcaccttt cttctgcttc 300
catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat 360
acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgtggggt 420
accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct 480
gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt 540
ggccgcgaaa actacgtatt ttggggtgga agagaagggt acgagacgct tctcaataca 600
gatatggagt tagagcttga taactttgca agatttttgc acatggctgt tgactatgca 660
aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa 720
catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttgac 780
aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcattcca cgacttccaa 840
catgagctaa gatacgccag aataaacggt gtattaggat caattgacgc aaatacaggc 900
gacatgcttt tgggatggga tacggaccag ttccctacag atatacgcat gacaacgctt 960
gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca 1020
aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg 1080
gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac 1140
aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc 1200
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<210>2
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<213>Thermoanaerobacterium saccharolyticum
<400>2
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
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Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
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Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
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Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
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Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
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Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
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Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
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Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
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Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
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Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
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Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
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Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
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Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
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Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
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Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
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His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
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Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
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Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
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Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
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Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
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Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
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Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
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Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
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Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
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Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
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Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
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<210>3
<211>1320
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<213>Thermoanaerobacterium saccharolyticum
<220>
<221>misc_feature
<222>(415)..(417)
<223>″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>misc_feature
<222>(544)..(546)
<223>″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>misc_feature
<222>(559)..(561)
<223>″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>misc_feature
<222>(895)..(897)
<223>″n″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>3
atgaataaat attttgagaa cgtatctaaa ataaaatatg aaggaccaaa atcaaataat 60
ccttattcct ttaaatttta caatccagag gaagtaatcg atggcaagac gatggaggag 120
catctccgct tttctatagc ttattggcac acttttactg ctgatggaac agatcaattt 180
ggcaaggcta ctatgcaaag accatggaac cactacacag atcctatgga tatagcgaaa 240
gcaagggtag aagcagcatt tgagtttttt gataagataa atgcaccttt cttctgcttc 300
catgataggg atattgcccc tgaaggagat actcttagag agacaaacaa aaacttagat 360
acaatagttg ctatgataaa ggattactta aagaccagca agacaaaagt tttgnnnggt 420
accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct 480
gacgtttttg catattctgc agcgcaagtc aaaaaagccc ttgagattac taaggagctt 540
ggcnnngaaa actacgtann ntggggtgga agagaagggt acgagacgct tctcaataca 600
gatatggagt tagagcttga taactttgca agatttttgc acatggctgt tgactatgca 660
aaggaaatcg gctttgaagg tcagttcttg attgagccga agccaaagga gcctacaaaa 720
catcaatacg actttgacgt ggcaaatgta ttggcattct tgagaaaata cgaccttgac 780
aaatatttca aagtaaatat cgaagcaaac catgcgacat tggcattcca cgacttccaa 840
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gctatgtatg aagtcataaa gatgggtgga tttgacaaag gtggccttaa ctttgatgca 1020
aaagtaagac gtgcttcatt tgagccagaa gatcttttct taggtcacat agcaggaatg 1080
gatgcttttg caaaaggctt taaagttgct tacaagcttg tgaaagatgg cgtatttgac 1140
aagttcatcg aagaaagata cgcaagctac aaagaaggca ttggcgctga tattgtaagc 1200
ggtaaagctg acttcaagag ccttgaaaag tatgcattag agcacagcca gattgtaaac 1260
aaatcaggca gacaagagct attagaatca atcctaaatc agtatttgtt tgcagaataa 1320
<210>4
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<220>
<221>MISC_FEATURE
<222>(139)..(139)
<223>Xaa代表除色氨酸之外的任何氨基酸残基。
<220>
<221>MISC_FEATURE
<222>(182)..(182)
<223>Xaa代表除精氨酸之外的任何氨基酸残基。
<220>
<221>MISC_FEATURE
<222>(187)..(187)
<223>Xaa代表除苯丙氨酸之外的任何氨基酸残基。
<220>
<221>MISC_FEATURE
<222>(299)..(299)
<223>Xaa代表除苏氨酸之外的任何氨基酸残基。
<400>4
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
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Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
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Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Xaa Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Xaa Glu Asn Tyr Val Xaa Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Xaa Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>5
<211>439
<2l2>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>5
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<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 Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Lys Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>7
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>7
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ser Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>8
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>8
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Cys Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>9
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>9
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Ile Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>10
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>10
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Thr Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>11
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>11
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Asn Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>12
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>12
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>13
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>13
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Pro Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>14
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>14
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Ser Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>15
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>15
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Ile Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>16
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>16
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Thr Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>17
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>17
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Val Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>18
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>18
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>19
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>19
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Gly Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>20
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>20
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Pro Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>21
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>21
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Ala Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>22
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>22
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>23
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>23
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Ile Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>24
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>24
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Tyr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>25
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>25
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Cys Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>26
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>26
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Met Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>27
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>27
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Glu Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>28
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>28
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Thr Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>29
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>29
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Trp Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<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 Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Arg Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>31
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>31
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
Phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Phe Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
<210>32
<211>439
<212>PRT
<213>Thermoanaerobacterium saccharolyticum
<400>32
Met Asn Lys Tyr Phe Glu Asn Val Ser Lys Ile Lys Tyr Glu Gly Pro
1 5 10 15
Lys Ser Asn Asn Pro Tyr Ser Phe Lys Phe Tyr Asn Pro Glu Glu Val
20 25 30
Ile Asp Gly Lys Thr Met Glu Glu His Leu Arg Phe Ser Ile Ala Tyr
35 40 45
Trp His Thr Phe Thr Ala Asp Gly Thr Asp Gln Phe Gly Lys Ala Thr
50 55 60
Met Gln Arg Pro Trp Asn His Tyr Thr Asp Pro Met Asp Ile Ala Lys
65 70 75 80
Ala Arg Val Glu Ala Ala Phe Glu Phe Phe Asp Lys Ile Asn Ala Pro
85 90 95
phe Phe Cys Phe His Asp Arg Asp Ile Ala Pro Glu Gly Asp Thr Leu
100 105 110
Arg Glu Thr Asn Lys Asn Leu Asp Thr Ile Val Ala Met Ile Lys Asp
115 120 125
Tyr Leu Lys Thr Ser Lys Thr Lys Val Leu Phe Gly Thr Ala Asn Leu
130 135 140
Phe Ser Asn Pro Arg Phe Val His Gly Ala Ser Thr Ser Cys Asn Ala
145 150 155 160
Asp Val Phe Ala Tyr Ser Ala Ala Gln Val Lys Lys Ala Leu Glu Ile
165 170 175
Thr Lys Glu Leu Gly Ala Glu Asn Tyr Val Ser Trp Gly Gly Arg Glu
180 185 190
Gly Tyr Glu Thr Leu Leu Asn Thr Asp Met Glu Leu Glu Leu Asp Asn
195 200 205
Phe Ala Arg Phe Leu His Met Ala Val Asp Tyr Ala Lys Glu Ile Gly
210 215 220
Phe Glu Gly Gln Phe Leu Ile Glu Pro Lys Pro Lys Glu Pro Thr Lys
225 230 235 240
His Gln Tyr Asp Phe Asp Val Ala Asn Val Leu Ala Phe Leu Arg Lys
245 250 255
Tyr Asp Leu Asp Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Phe His Asp Phe Gln His Glu Leu Arg Tyr Ala Arg Ile
275 280 285
Asn Gly Val Leu Gly Ser Ile Asp Ala Asn Gln Gly Asp Met Leu Leu
290 295 300
Gly Trp Asp Thr Asp Gln Phe Pro Thr Asp Ile Arg Met Thr Thr Leu
305 310 315 320
Ala Met Tyr Glu Val Ile Lys Met Gly Gly Phe Asp Lys Gly Gly Leu
325 330 335
Asn Phe Asp Ala Lys Val Arg Arg Ala Ser Phe Glu Pro Glu Asp Leu
340 345 350
Phe Leu Gly His Ile Ala Gly Met Asp Ala Phe Ala Lys Gly Phe Lys
355 360 365
Val Ala Tyr Lys Leu Val Lys Asp Gly Val Phe Asp Lys Phe Ile Glu
370 375 380
Glu Arg Tyr Ala Ser Tyr Lys Glu Gly Ile Gly Ala Asp Ile Val Ser
385 390 395 400
Gly Lys Ala Asp Phe Lys Ser Leu Glu Lys Tyr Ala Leu Glu His Ser
405 410 415
Gln Ile Val Asn Lys Ser Gly Arg Gln Glu Leu Leu Glu Ser Ile Leu
420 425 430
Asn Gln Tyr Leu Phe Ala Glu
435
Claims (37)
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，在第139位、第182位、第187位、第299位具有至少一个突变，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，其特征在于具有说明书中所附的序列4中第139位的Xaa代表苯丙氨酸(Phe)、赖氨酸(Lys)、丝氨酸(Ser)、半胱氨酸(Cys)、异亮氨酸(Ile)、苏氨酸(Thr)、或天冬酰胺(Asn)的氨基酸序列。
4.权利要求3所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第139位的Xaa代表异亮氨酸(Ile)、丝氨酸(Ser)、半胱氨酸(Cys)、或天冬酰胺(Asn)的氨基酸序列。
5.权利要求4所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第139位的Xaa代表丝氨酸(Ser)、半胱氨酸(Cys)、或天冬酰胺(Asn)的氨基酸序列。
6.权利要求1所述的葡萄糖异构酶突变体，其中第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)。
7.权利要求6所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第182位的Xaa代表丙氨酸(Ala)、脯氨酸(Pro)、丝氨酸(Ser)、异亮氨酸(Ile)、苏氨酸(Thr)、或缬氨酸(Val)的氨基酸序列。
8.权利要求7所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第182位的Xaa代表异亮氨酸(Ile)、丝氨酸(Ser)、或缬氨酸(Val)的氨基酸序列。
9.权利要求8所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第182位的Xaa代表异亮氨酸(Ile)、或缬氨酸(Val)的氨基酸序列。
10.权利要求1所述的葡萄糖异构酶突变体，其中第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(Pro)。
11.权利要求10所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第187位的Xaa代表丝氨酸(Ser)、甘氨酸(Gly)、脯氨酸(Pro)、或丙氨酸(Ala)的氨基酸序列。
12.权利要求11所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第187位的Xaa代表脯氨酸(Pro)的氨基酸序列。
13.权利要求1所述的葡萄糖异构酶突变体，其中第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)。
14.权利要求13的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第299位的Xaa代表谷氨酰胺(Gln)、异亮氨酸(Ile)、酪氨酸(Tyr)、半胱氨酸(Cys)、蛋氨酸(Met)、或谷氨酸(Glu)的氨基酸序列。
15.权利要求14的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第299位的Xaa代表半胱氨酸(Cys)、或谷氨酸(Glu)的氨基酸序列。
16.权利要求1所述的葡萄糖异构酶突变体，其特征还在于以说明书所附的序列2为参考序列，具有至少两个选自第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、第299位的苏氨酸到其他19个天然氨基酸的突变。
17.权利要求16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)。
18.权利要求16所述的葡萄糖异构酶突变体，其中第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)。
19.权利要求16所述的葡萄糖异构酶突变体，其中第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(Pro)。
20.权利要求16所述的葡萄糖异构酶突变体，其中第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)。
21.权利要求16所述的葡萄糖异构酶突变体，其特征在于具有说明书中所附的序列4中第139位的Xaa代表苯丙氨酸(Phe)同时第182位的Xaa代表丙氨酸(Ala)的氨基酸序列(序列29)，或者序列4中第182位的Xaa代表丙氨酸(Ala)同时第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列30)、或者序列4中第139位的Xaa代表苯丙氨酸(Phe)同时第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列31)。
22.权利要求16所述的葡萄糖异构酶突变体，其特征还在于以说明书所附的序列2为参考序列，具有至少三个选自第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、第299位的苏氨酸到其他19个天然氨基酸的突变。
23.权利要求22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)。
24.权利要求22所述的葡萄糖异构酶突变体，其中第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)。
25.权利要求22所述的葡萄糖异构酶突变体，其中第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(Pro)。
26.权利要求22所述的葡萄糖异构酶突变体，其中第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)。
27.权利要求22所述的葡萄糖异构酶突变体，其特征在于具有序列4中第139位的Xaa代表苯丙氨酸(Phe)、第182位的Xaa代表丙氨酸(Ala)同时第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列32)。
28.权利要求22所述的葡萄糖异构酶突变体，特征还在于以说明书所附的序列2为参考序列，具有第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、第299位的苏氨酸到其他19个天然氨基酸的突变。
29.权利要求28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)。
30.权利要求28所述的葡萄糖异构酶突变体，其中第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)。
31.权利要求28所述的葡萄糖异构酶突变体，其中第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(Pro)。
32.权利要求28所述的葡萄糖异构酶突变体，其中第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)。
33.权利要求28所述的葡萄糖异构酶突变体，其特征在于具有序列4中第139位的Xaa代表苯丙氨酸(Phe)、第182位的Xaa代表丙氨酸(Ala)、第187位的Xaa代表丝氨酸(Ser)同时第299位的Xaa代表谷氨酰胺(Gln)的氨基酸序列(序列33)。
34.权利要求1-33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。
35.权利要求34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。
36.权利要求35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％。
37.一种DNA，其含有编码权利要求1-33任意一项所述的葡萄糖异构酶突变体的核苷酸序列。
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| CN200410047865XA CN1702172B (zh) | 2004-05-26 | 2004-06-16 | 葡萄糖异构酶突变体 |
| DE602004031308T DE602004031308D1 (de) | 2004-05-26 | 2004-07-28 | Glucoseisomerase-mutanten |
| PCT/CN2004/000876 WO2005116217A1 (en) | 2004-05-26 | 2004-07-28 | Glucose isomerase mutants |
| AT04762014T ATE497537T1 (de) | 2004-05-26 | 2004-07-28 | Glucoseisomerase-mutanten |
| US11/597,609 US7919300B2 (en) | 2004-05-26 | 2004-07-28 | Glucose isomerase mutants |
| EP04762014A EP1766026B1 (en) | 2004-05-26 | 2004-07-28 | Glucose isomerase mutants |
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| CN2008101488394A Division CN101372687B (zh) | 2004-05-26 | 2004-06-16 | 葡萄糖异构酶突变体 |
| CN2008101488337A Division CN101423828B (zh) | 2004-05-26 | 2004-06-16 | 葡萄糖异构酶突变体 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007056931A1 (fr) * | 2005-11-18 | 2007-05-24 | Bioright Worldwide Company Limited | Utilisation de mutants de la glucose isomérase |
| WO2007056930A1 (fr) * | 2005-11-18 | 2007-05-24 | Bioright Worldwide Company Limited | Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants |
| WO2007068173A1 (fr) | 2005-12-16 | 2007-06-21 | Bioright Worldwide Company Limited | Support permettant de préparer une enzyme immobilisée ou des cellules immobilisées et procédé d'utilisation de celui-ci |
| CN104293759A (zh) * | 2014-09-19 | 2015-01-21 | 浙江工业大学 | 葡萄糖异构酶基因、编码酶、载体、工程菌及应用 |
| CN104745563A (zh) * | 2015-03-05 | 2015-07-01 | 浙江工业大学 | 葡萄糖异构酶、基因、突变体、工程菌及应用 |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009109631A1 (en) * | 2008-03-07 | 2009-09-11 | Dsm Ip Assets B.V. | A pentose sugar fermenting cell |
| WO2009109630A1 (en) * | 2008-03-07 | 2009-09-11 | Dsm Ip Assets B.V. | A pentose sugar fermenting cell |
| WO2012088467A2 (en) | 2010-12-22 | 2012-06-28 | Mascoma Corporation | Genetically modified clostridium thermocellum engineered to ferment xylose |
| WO2021050342A1 (en) | 2019-09-13 | 2021-03-18 | Danisco Us Inc | Thermostable glucose isomerase variants |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE69133491T2 (de) * | 1990-01-04 | 2006-07-27 | Genencor International, Inc., Palo Alto | Glucose-Isomerase mit geändertem pH-Optimum |
| US5266475A (en) * | 1991-09-19 | 1993-11-30 | Michigan Biotechnology Institute | Glucose isomerases with improved affinity for D-glucose |
| US5656497A (en) | 1994-09-06 | 1997-08-12 | Michigan State University | Gene encoding hyperthermostable xylose isomerase from Thermotoga neapolitana |
| US7198933B2 (en) * | 2002-01-23 | 2007-04-03 | Board Of Trustees Of Michigan State University | Thermotoga neapolitana xylose isomerase polypeptides and nucleic acids encoding same |
| CN1693473A (zh) * | 2004-05-08 | 2005-11-09 | 张骏 | 果葡糖浆制备新方法与耐高温葡萄糖异构酶突变体 |
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2004
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- 2004-07-28 DE DE602004031308T patent/DE602004031308D1/de not_active Expired - Lifetime
- 2004-07-28 WO PCT/CN2004/000876 patent/WO2005116217A1/en active Application Filing
- 2004-07-28 AT AT04762014T patent/ATE497537T1/de not_active IP Right Cessation
- 2004-07-28 US US11/597,609 patent/US7919300B2/en active Active
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007056931A1 (fr) * | 2005-11-18 | 2007-05-24 | Bioright Worldwide Company Limited | Utilisation de mutants de la glucose isomérase |
| WO2007056930A1 (fr) * | 2005-11-18 | 2007-05-24 | Bioright Worldwide Company Limited | Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants |
| US7704719B2 (en) | 2005-11-18 | 2010-04-27 | Geneharbor (Hk) Technologies Ltd. | Glucose isomerase mutants, DNA thereof and use thereof |
| US7923222B2 (en) | 2005-11-18 | 2011-04-12 | Geneharbor (Hk) Technologies Ltd. | Methods of using isolated glucose isomerase |
| US8012726B2 (en) | 2005-11-18 | 2011-09-06 | Bioright Worldwide Company Limited | Method of making bioethanol by using glucose isomerase mutants |
| US8067561B2 (en) | 2005-11-18 | 2011-11-29 | Bioright Worldwide Company Limited | Isolated DNA encoding recombinant glucose isomerase |
| WO2007068173A1 (fr) | 2005-12-16 | 2007-06-21 | Bioright Worldwide Company Limited | Support permettant de préparer une enzyme immobilisée ou des cellules immobilisées et procédé d'utilisation de celui-ci |
| US8486676B2 (en) | 2005-12-16 | 2013-07-16 | Bioright Worldwide Company Limited | Carriers for enzyme or cell immobilization and immobilization method using the carriers |
| CN104293759A (zh) * | 2014-09-19 | 2015-01-21 | 浙江工业大学 | 葡萄糖异构酶基因、编码酶、载体、工程菌及应用 |
| CN104745563A (zh) * | 2015-03-05 | 2015-07-01 | 浙江工业大学 | 葡萄糖异构酶、基因、突变体、工程菌及应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| US20080113415A1 (en) | 2008-05-15 |
| EP1766026A1 (en) | 2007-03-28 |
| EP1766026B1 (en) | 2011-02-02 |
| EP1766026A4 (en) | 2007-10-10 |
| US7919300B2 (en) | 2011-04-05 |
| ATE497537T1 (de) | 2011-02-15 |
| CN1702172B (zh) | 2011-12-07 |
| DE602004031308D1 (de) | 2011-03-17 |
| WO2005116217A1 (en) | 2005-12-08 |
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