CN1966679B - 葡萄糖异构酶突变体、其应用以及编码该突变体的dna - Google Patents
葡萄糖异构酶突变体、其应用以及编码该突变体的dna Download PDFInfo
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Abstract
本发明披露应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(Xylose isomerase)是戊糖发酵途径的关键酶。该酶是最重要的工业酶制剂之一(Kaneko，et al.，Biosci Biotechnol 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(Actinoplanes missouriensis)、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重量％。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Thermoanaerobacterium saccharolyticum(下文称T.saccharolyticum)、Thermotoga neapolitana等数种耐高温菌中分离并研究了耐高温葡萄糖异构酶(见Lee et al.，Journal of GeneralMicrobiology，87：1227-1234，1993；Vieille et al.，MethodsEnzymology，330：215-24，2001；Lee et al.，Journal of GeneralMicrobiology，87：1241-1243，1993；Scriprapundh et al.，ProteinEngineering，13：259-265，2000；Scriprapundh et al.，ProteinEngineering，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，目前仍存在对高活力、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重量％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重量％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，先利用分子生物学技术，构建含有亲本葡萄糖异构酶基因的载体质粒，然后设定定点突变的位点以及突变后的氨基酸，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。从阳性克隆中提取质粒DNA，进行DNA序列测定分析，以确定引入的突变。最后，以D-葡萄糖为底物测定具有所设定位点突变的葡萄糖异构酶的酶活力，将该酶活力与亲本的酶活力相比较，从而筛选出具有比亲本高的葡萄糖异构酶催化活性的本发明的葡萄糖异构酶突变体。
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、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，以说明书中所附的序列2为参考序列，其第139位突变为苯丙氨酸(Phe)、第182位突变为丙氨酸(Ala)、第187位突变为丝氨酸(Ser)突变以及第299位突变为谷氨酰胺(Gln)，其还同时在第87位、第217位、第260位、第276位具有至少一个突变，并且以D-葡萄糖为底物其具有比所述亲本高的葡萄糖异构酶催化活性。优选的是，所述第87位为甲硫氨酸(Met)或亮氨酸(Leu)；所述第217位为精氨酸(Arg)、或色氨酸(Trp)、或甘氨酸(Gly)；所述第260位为谷氨酸(Glu)或丙氨酸(Ala)；和/或所述第276位为甘氨酸(Gly)或苏氨酸(Thr)。序列表中的SEQ ID NO.：4显示了一种本发明的葡萄糖异构酶突变体的氨基酸序列，其中的Xaa代表突变位点的氨基酸。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MGI4-F87L、MGI4-F87M、MGI4-V217R、MGI4-V217W、MGI4-D260E、MGI4-F276G、MGI4-24、MGI4-25、MGI4-34、MGI4-35的氨基酸序列。
这些突变体具有高的催化活性。例如，在本发明获得的一系列突变体中，一个具有七个点突变的突变体MGI4-34的比活性较亲本高769％，且在80℃反应26小时后仍保持50％或以上的活力。另一个具有七个点突变的突变体MGI4-35的比活性较亲本高727％，且在80℃反应27小时后仍保持50％或以上的活力。
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重量％的果葡糖浆，或用于生产果糖含量低于55重量％的果葡糖浆。本发明的葡萄糖异构酶突变体可用于生产果糖含量高于90重量％以上的结晶果糖。
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T.saccharolyticumATCC 49915的葡萄糖异构酶，其核苷酸序列如序列1所示，氨基酸序列如序列2所示。本发明中亲本基因的核苷酸序列与公布的T.saccharolyticum葡萄糖异构酶的基因序列(Lee et al.，Journal ofGeneral Microbiology，139：1227-1234，1993；GenBank L09699)相比有两个核苷酸的差异，即与基因库的核苷酸序列(GenBank L09699)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，是指说明书中所附的序列1，当其为氨基酸序列时，是指说明书中所附的序列2。在将参考序列和其它突变的葡萄糖异构酶序列进行排序比较时，可以手工进行，也可以用计算机进行(目前有许多可供利用的计算机软件，例如CLUSTALW，AMAS，DIALIGN程序等)。
本申请文本中所用的术语“位点”或“第x位”是指当本发明的葡萄糖异构酶突变体的序列与参考序列的排序对比达最大同源性时，被比较的葡萄糖异构酶突变体的各核苷酸或氨基酸所对应的参考序列中的核苷酸或氨基酸位置。
本申请文本所用的术语“葡萄糖异构酶突变体”是指一种以说明书中所附序列2所示氨基酸序列为参考序列，其第139位突变为苯丙氨酸(Phe)、第182位突变为丙氨酸(Ala)、第187位突变为丝氨酸(Ser)突变以及第299位突变为谷氨酰胺(Gln)，同时在第87位、第217位、第260位、第276位具有至少一个突变，并且以D-葡萄糖为底物生成果糖时其具有比亲本高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)。
附图说明
图1.亲本葡萄糖异构酶与葡萄糖异构酶突变体在80℃的热稳定性图谱。其中MGI4-34和MGI4-35代表具有七个突变位点的葡萄糖异构酶突变体，具体参见实施例12。
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
根据基因库公布的基因序列(GenBank L09699)设计引物T1和T2(见表1)。用引物对T1和T2从T.saccharolyticum ATCC 49915(购自ATCC，USA)中扩增葡萄糖异构酶亲本基因。
扩增条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1，400nM引物T2，1.5U TaqDNA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。与基因库公布的核苷酸序列(GenBank L09699)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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对139FF和139FR(见表1)，将亲本氨基酸序列中第139位点的Trp(W)突变为Phe(F)，获得突变体MGI-W139F。引物对T1和T2见实施例1。
利用引物对T1和139FR，扩增T1FR片段，引物对139FF和T2，扩增FFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM 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％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1FR片段与20ngFFT2片段，用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI-W139F。将MGI-W139F与载体pGEMT-Easy连接，得质粒pGEMT-MGI-W139F。将质粒pGEMT-MGI-W139F转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI-W139F DNA，经DNA测序确定引入的点突变无误。
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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对182AF和182AR(见表1)，将亲本氨基酸序列中第182位点的Arg(R)突变为Ala(A)，获得突变体MGI-R182A。引物T1和T2见实施例1。
利用引物对T1和182AR，扩增T1AR片段，引物对182AF和T2，扩增AFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物182AR或400nM引物182AF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction 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(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 PfuDNA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。
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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对187SF和187SR(见表1)，将亲本氨基酸序列中第187位点的Phe(F)突变为Ser(S)，获得突变体MGI-F187S。引物T1和T2见实施例1。
利用引物对T1和187SR，扩增T1SR片段，引物对187SF和T2，扩增SFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM 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，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QIAquickExtraction 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 PfuDNA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。
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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-TS(见实施例1)为模板，设计引物对299QF和299QR(见表1)，将亲本氨基酸序列中第299位点的Thr(T)突变为Gln(Q)，获得突变体MGI-T299Q。引物T1和T2见实施例1。
利用引物对T1和299QR，扩增T1QR片段，引物对299QF和T2，扩增QFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物299QR或400nM引物299QF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction 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(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 PfuDNA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QIAquiek 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。
实施例6：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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
按实施例2扩增和回收T1FR片段，按实施例5扩增和回收QFT2片段。用引物对139FF(见表1)和182AR(见表1)扩增FFAR片段。FFAR片段扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 139FF和400nM 182AR，1.5U PfuDNA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QIAquickExtraction Gel Kit回收，得到FFAR片段。用引物对182AF(见表1)和187SR(见表1)扩增AFSR片段。AFSR片段扩增反应条件为：20mMTris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM 182AF和400nM 187SR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到AFSR片段。用引物对187SF(见表1)和299QR(见表1)扩增SFQR片段。SFQR片段扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM 187SF和400nM 299QR，1.5U Pfu DNA聚合酶，20ng pGEMT-TS，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到SFQR片段。然后扩增全长基因。扩增反应条件为：20mM Tris-HCl(pH 8.8)，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 PfuDNA聚合酶，20ng T1FR片段，20ng FFAR片段，20ng AFSR片段，20ng SFQR片段和20ng QFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI-4。将MGI-4与载体pGEMT-Easy连接，得质粒pGEMT-MGI-4。将质粒pGEMT-MGI-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-4DNA，经DNA测序确定引入的点突变无误。MGI-4序列含有W139F、R182A、F187S和T299Q的四突变。
实施例7：葡萄糖异构酶五突变组合MGI4-F87L和MGI4-F87M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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例6)为模板，设计引物对87LF和87LR(见表1)，将MGI-4氨基酸序列中第87位点的Phe(F)突变为Leu(L)，获得突变体MGI4-F87L。引物对T1和T2见表1。
用引物对T1和87LR，扩增T1LR片段，用引物对87LF和T2，扩增LFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，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87LR或400nM引物87LF和400nM引物T2，1.5U Pfu DNA聚合酶(Promega，USA)，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit(QIAGEN，German)回收，得到T1LR片段和LFT2片段。然后扩增全长基因。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段与20ngLFT2片段，用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-F87L。将MGI4-F87L与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-F87L。将质粒pGEMT-MGI4-F87L转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-F87L DNA，经DNA测序确定引入的点突变无误。所得突变体序列含有F87L、W139F、R182A、F187S和T299Q五个突变。MGI4-F87L氨基酸序列见序列表序列5。
按照类似步构建突变体MGI4-F87M，所用引物见表1。MGI4-F87M突变体序列含有F87M、W139F、R182A、F187S和T299Q五个突变。所得突变体氨基酸序列见序列表序列6。
实施例8：葡萄糖异构酶五突变组合MGI4-V217R和MGI4-V217W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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例6)为模板，设计引物对217RF和217RR(见表1)，将MGI-4氨基酸序列中第217位点的Val(V)突变为Arg(R)，获得突变体MGI4-V217R。引物T1和T2见实施例1。
用引物对T1和217RR，扩增T1RR片段，用引物对217RF和T2，扩增RFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2S04，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物217RR或400nM引物217RF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到T1RR片段和RFT2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 PfuDNA聚合酶，20ng T1RR片段与20ng RFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-V217R。将MGI4-V217R与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-V217R。将质粒pGEMT-MGI4-V217R转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-V217R DNA，经DNA测序确定引入的点突变无误。所得突变体序列含有W139F、R182A、F187S、V217R和T299Q五个突变。MGI4-V217R的氨基酸序列见序列表序列7。
按照类似步构建突变体MGI4-V217W，所用引物见表1。MGI4-V217W突变体序列含有W139F、R182A、F187S、V217W和T299Q五个突变。所得突变体的氨基酸序列见序列表序列8。
实施例9：葡萄糖异构酶五突变组合MGI4-D260E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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例6)为模板，设计引物对260EF和260ER(见表1)，将MGI-4氨基酸序列中第260位点的Asp(D)突变为Glu(E)，获得突变体MGI4-D260E。引物T1和T2见表1。
用引物对T1和260ER，扩增T1ER片段，用引物对260EF和T2，扩增EFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物260ER或400nM引物T2和400nM引物260EF，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到T1ER片段和EFT2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 PfuDNA聚合酶，20ng T1ER片段与20ng EFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-D260E。将MGI4-D260E与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-D260E。将质粒pGEMT-MGI4-D260E转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-D260E DNA，经DNA测序确定引入的点突变无误。所得突变体序列含有W139F、R182A、F187S、D260E和T299Q五个突变。MGI4-D260E的氨基酸序列见序列表序列9。
实施例10：葡萄糖异构酶五突变组合MGI4-F276G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：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
以质粒pGEMT-MGI-4(见实施例6)为模板，设计引物对276GF和276GR(见表1)，将MGI-4氨基酸序列中第276位点的Phe(F)突变为Gly(G)，获得突变体MGI4-F276G。引物T1和T2见表1。
用引物对T1和276GR，扩增T1GR片段，用引物对276GF和T2，扩增GFT2片段。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mMKCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM引物276GR或400nM引物276GF和400nM引物T2，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到T1GR片段和GFT2片段。然后扩增全长基因。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U PfuDNA聚合酶，20ng T1GR片段与20ng GFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-F276G。将MGI4-F276G与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-F276G。将质粒pGEMT-MGI4-F276G转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-F276G DNA，经DNA测序确定引入的点突变无误。所得突变体序列含有W139F、R182A、F187S、F276G和T299Q五个突变。MGI4-F276G的氨基酸序列见序列表序列10。
实施例11：葡萄糖异构酶六突变组合MGI4-24和MGI4-25之构建
定点突变技术参考Ho et al.(Gene 77：51-59，1989)和White et al.(PCR Protocol：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
按实施例7扩增和回收T1LR片段。用引物对87LF和260AR(见表1)扩增和回收LFAR片段。LFAR片段扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM87LF和400nM 260AR，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到LFAR片段。用引物对260AF和T2(见表1)扩增和回收AFT2片段。AFT2片段扩增反应条件为：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 260AF和400nM T2，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquickExtraction Gel Kit回收，得到AFT2片段。然后扩增全长基因。扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μM dTTP，50μMdCTP，50μM dGTP，400nM引物T1和400nM T2，1.5U Pfu DNA聚合酶，20ng T1LR片段，20ng LFAR片段和20ng AFT2片段，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick DNA回收试剂盒，得到全长突变基因MGI4-24。将MGI4-24与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-24。将质粒pGEMT-MGI4-24转入感受态细菌细胞E.coliHB 101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-24DNA，经DNA测序确定引入的点突变无误。MGI4-24的氨基酸序列见序列表序列11。所得突变体序列含有F87L、W139F、R182A、F187S、D260A和T299Q六个突变。
按照类似步骤构建突变体MGI4-25。突变体MGI4-25所用引物对为T1和87LR、87LF和276TR、276TF和T2(均见表1)，所得突变体序列含有F87L、W139F、R182A、F187S、F276T和T299Q六个突变。所得突变体的氨基酸序列见序列表序列12。
实施例12：葡萄糖异构酶七突变组合MGI4-34和MGI4-35之构建
定点突变技术参考Ho et al.(Gene 77：51-59，1989)和White et al.(PCR Protocol：current methods and applications.Totowa，N.J.：HumanaPress，1993)之描述。
按实施例7扩增和回收T1LR片段。用引物对87LF和217GR(见表1)扩增和回收LFGR片段。LFGR片段扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％TritonX-100，50μM dATP，50μM dTTP，50μM dCTP，50μM dGTP，400nM87LF和400nM 217GR，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到LFGR片段。用引物对217GF和276TR(见表1)扩增和回收GFTR片段。GFTR片段扩增反应条件为：20mM Tris-HCl(pH 8.8)，10mM KCl，10mM(NH4)2SO4，2mM MgSO4，0.1％Triton X-100，50μM dATP，50μMdTTP，50μM dCTP，50μM dGTP，400nM 217GF和400nM 276TR，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到GFTR片段。用引物对276TF和T2(见表1)扩增和回收TFT2片段。TFT2片段扩增反应条件为：20mMTris-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 276TF和400nM T2，1.5U Pfu DNA聚合酶，20ng pGEMT-MGI-4，再用无菌水调反应体积至50μl。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到TFT2片段。然后扩增全长基因。扩增反应条件为：20mM Tris-HCl(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 T1LR片段，20ng LFGR片段，20ng GFTR片段和20ng TFT2片段，再用无菌水调反应体积至50ul。PCR扩增反应程序为：95℃3分钟，35圈循环：95℃50秒、52℃30秒和72℃3分钟，最后72℃5分钟。经1％琼脂糖胶电泳分离并用QIAquick Extraction Gel Kit回收，得到全长突变基因MGI4-34。将MGI4-34与载体pGEMT-Easy连接，得质粒pGEMT-MGI4-34。将质粒pGEMT-MGI4-34转入感受态细菌细胞E.coli HB101，在1％MacConkey平板(含1％D-木糖和50mg/L氨苄青霉素)上筛选出具葡萄糖异构酶活性的克隆。从克隆中提取质粒pGEMT-MGI4-34DNA，经DNA测序确定引入的点突变无误。MGI4-34序列含有F87L、W139F、R182A、F187S、V217G、F276T和T299Q七个突变。MGI4-34的氨基酸序列见序列表序列13。
按照类似步骤构建七突变体MGI4-35。突变体MGI4-35所用引物对为T1和87LR、87LF和217GR、217GF和260AR、260AF和T2(均见表1)，所得突变体序列含有F87L、W139F、R182A、F187S、V217G、D260A和T299Q七突变。所得突变体的氨基酸序列见序列表序列14。
扩增亲本葡萄糖异构酶(亲本)与实施例1-12之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-W139F | 139FF：5’AAAAGTTTTGTTTGGTACCGCAAATCTTTTCTC 3’139FR：5’TTGCGGTACCAAACAAAACTTTTGTCTTGCTGG 3’ |
| 突变体MGI-R182A | 182AF：5’GGAGCTTGGCGCGGAAAACTACGTATTTTGGGG 3’182AR：5’CGTAGTTTTCCGCGCCAAGCTCCTTAGTAATCT 3’ |
| 突变体MGI-F187S | 187SF：5’ACTACGTAAGCTGGGGTGGAAGAGAAGGGT 3’187SR：5’CCACCCCAGCTTACGTAGTTTTCGCGGCCA 3’ |
| 突变体MGI-T299Q | 299QF：5’TGACGCAAATCAAGGCGACATGCTTTTGGGATG 3’299QR：5’GCATGTCGCCTTGATTTGCGTCGATTGATCCTA 3’ |
| 突变体MGI4-F87L | 87LF：5’GAAGCAGCACTGGAGTTTTTTGATAAGATAA 3’87LR：5’AAAAACTCCAGTGCTGCTTCTACCCTTGCTTTC 3’ |
| 突变体MGI4-F87M | 87MF：5’GAAGCAGCAATGGAGTTTTTTGATAAGATAA 3’87MR：5’AAAAACTCCATTGCTGCTTCTACCCTTGCTTTC 3’ |
| 突变体MGI4-V217G | 217GF：5’ACATGGCTGGCGACTATGCAAAGGAAATCG 3’217GR：5’GCATAGTCGCCAGCCATGTGCAAAAATCTT 3’ |
| 突变体MGI4-V217W | 217WF：5’ACATGGCTTGGGACTATGCAAAGGAAATCG 3’217WR：5’GCATAGTCCCAAGCCATGTGCAAAAATCTT 3’ |
| 突变体MGI4-V217T | 217TF：5’ACATGGCTACCGACTATGCAAAGGAAATCG 3’217TR：5’GCATAGTCGGTAGCCATGTGCAAAAATCTT 3’ |
| 突变体MGI4-D260E | 260EF：5’ACGACCTTGAAAAATATTTCAAAGTAAATA 3’260ER：5’AAATATTTTTCAAGGTCGTATTTTCTCAAG 3’ |
| 突变体MGI4-D260A | 260AF：5’ACGACCTTGCGAAATATTTCAAAGTAAATA 3’260AR：5’AAATATTTCGCAAGGTCGTATTTTCTCAAG 3’ |
| 突变体MGI4-F276G | 276GF：5’ACATTGGCAGGCCACGACTTCCAACATGAGC 3’276GR：5’GAAGTCGTGGCCTGCCAATGTCGCATGGTTT 3’ |
| 突变体MGI4-F276T | 276TF：5’ACATTGGCAACCCACGACTTCCAACATGAGC 3’276TR：5’GAAGTCGTGGGTTGCCAATGTCGCATGGTTT 3’ |
实施例13：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 GeneralMicrobiology，87：1227-1234(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1ml 20mM磷酸钠缓冲液(pH 6.5)中，加CoCl2和MgCl2至终浓度分别为250μM和5mM。然后用超声波裂解细菌细胞。离心(10℃，17800g，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℃，17800g，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14：葡萄糖异构酶突变体的提取与纯化
葡萄糖异构酶突变体MGI4-35的提取与纯化与实施例13同，其中所用质粒为pGEMT-MGI4-35。其它葡萄糖异构酶突变体也照实施例13所述提取与纯化。
实施例15：亲本葡萄糖异构酶活性的测定
底物溶液A含1.0M的D-葡萄糖、20mM磷酸钠、250μM的CoCl2和5mM的MgCl2，pH6.5。取底物溶液A 90μl，然后加入10μl按实施例1和13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Coomassie
Plus ProteinAssay 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-葡萄糖为果糖所需的酶量。表2显示亲本葡萄糖异构酶的相对比活性。
实施例16：葡萄糖异构酶突变体活性的测定
葡萄糖异构酶突变体活性测定与实施例15同。表2显示各葡萄糖异构酶突变体与亲本葡萄糖异构酶(亲本)比活性的差异。
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 | 相对比活性 |
| 亲本 | SEQ ID NO.：2 | 100 |
| MGI4-F87L | SEQ ID NO.：5 | 271 |
| MGI4-F87M | SEQ ID NO.：6 | 346 |
| MGI4-V217R | SEQ ID NO.：7 | 609 |
| MGI4-V217W | SEQ ID NO.：8 | 151 |
| MGI4-D260E | SEQ ID NO.：9 | 560 |
| MGI4-F276G | SEQ ID NO.：10 | 365 |
| MGI4-24 | SEQ ID NO.：11 | 501 |
| MGI4-25 | SEQ ID NO.：12 | 652 |
| MGI4-34 | SEQ ID NO.：13 | 869 |
| MGI4-35 | SEQ ID NO.：14 | 827 |
实施例17：亲本葡萄糖异构酶热稳定性测定
将按实施例13获得的部分纯化的亲本葡萄糖异构酶置于4管1.5ml离心管中。每管离心管分别加入200μl酶溶液，并加入200μl矿物油。将离心管置于80℃水浴中，0小时、2小时、6小时、27小时后分别取出一管酶液，离心(10℃，17800g，20分钟)后，取上清液，按实施例15来测定葡萄糖异构酶残余蛋白的比活性。图1显示亲本葡萄糖异构酶在80℃的热稳定性。
实施例18：葡萄糖异构酶突变体热稳定性测定
葡萄糖异构酶突变体MGI4-34(见实施例12和14)或MGI4-35(见实施例12和14)热稳定性测定与实施例17同。图1显示葡萄糖异构酶突变体MGI4-34或MGI4-35在80℃的热稳定性。如图所示，亲本葡萄糖异构酶的活性在80℃的半衰期为4.1小时；MGI4-34的活性在80℃的半衰期为26小时；MGI4-35的活性在80℃的半衰期大于27小时。
实施例19：固相化葡萄糖异构酶突变体MGI4-35
主要参考Ge，et al.，Appl.Biochem.Biotechnol.69：57-69，1998。具体步是：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(pH 8.0)1升和部分提纯之葡萄糖异构酶突变体MGI4-35(按实施例12和14所述制备)8克，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16所述，其中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20：固定化含葡萄糖异构酶突变体MGI4-35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-MGI4-35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16所述，其中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(Glucose lsomerase Mutants)
<130>F1-050437-59
<160>14
<170>PatentIn version 3.2
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<213>Thermoanaerobacterium saccharolyticum
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accgcaaatc ttttctccaa tccgagattt gtacatggtg catcaacatc ctgcaatgct 480
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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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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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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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<221>MISC_FEATURE
<222>(276)..(276)
<223>Xaa represents any other amino acid residue other than phenylalanine.
<400>4
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 Xaa 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 Xaa 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 Xaa Lys Tyr Phe Lys Val Asn Ile Glu Ala Asn His Ala
260 265 270
Thr Leu Ala Xaa 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>5
<211>439
<212>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 Leu 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
<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 Met 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
<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 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 Arg 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>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 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 Trp 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>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 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 Glu 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>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 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 Gly 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>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 Leu 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 Ala 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>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 Leu 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 Thr 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>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 Leu 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 Gly 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 Thr 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>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 Leu 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 Gly 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 Ala 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 (27)
1.一种葡萄糖异构酶突变体，其中在说明书中所附的序列2的基础上，其第139位突变为苯丙氨酸(Phe)、第182位突变为丙氨酸(Ala)、第187位突变为丝氨酸(Ser)突变以及第299位突变为谷氨酰胺(Gln)，其特征在于同时具有选自第87位、第217位、第260位、第276位的至少一个突变，并且以D-葡萄糖为底物其具有比亲本高的葡萄糖异构酶催化活性，其中所述亲本为来自T.saccharolyticumATCC 49915的葡萄糖异构酶。
2.权利要求1所述的葡萄糖异构酶突变体，其中第87位突变为甲硫氨酸(Met)或亮氨酸(Leu)。
3.权利要求2所述的葡萄糖异构酶突变体，其氨基酸序列为序列表中序列5或序列6所示的氨基酸序列。
4.权利要求1所述的葡萄糖异构酶突变体，其中第217位的氨基酸突变为精氨酸(Arg)、或色氨酸(Trp)、或甘氨酸(Gly)。
5.权利要求4所述的葡萄糖异构酶突变体，其氨基酸序列为序列表中序列7或8所示的氨基酸序列。
6.权利要求1所述的葡萄糖异构酶突变体，其中第260位的氨基酸突变为谷氨酸(Glu)或丙氨酸(Ala)。
7.权利要求6所述的葡萄糖异构酶突变体，其氨基酸序列为序列表中序列9所示的氨基酸序列。
8.权利要求1所述的葡萄糖异构酶突变体，其中第276位的氨基酸为甘氨酸(Gly)或苏氨酸(Thr)。
9.权利要求8所述的的葡萄糖异构酶突变体，其氨基酸序列为说明书中所附序列表中序列10所示的氨基酸序列。
10.权利要求1所述的葡萄糖异构酶突变体，其具有至少两个选自第87位、第217位、第260位、第276位的突变。
11.权利要求10所述的葡萄糖异构酶突变体，其中第87位为甲硫氨酸(Met)或亮氨酸(Leu)。
12.权利要求10所述的葡萄糖异构酶突变体，其中第217位为精氨酸(Arg)、或色氨酸(Trp)、或甘氨酸(Gly)。
13.权利要求10所述的葡萄糖异构酶突变体，其中第260位为谷氨酸(Glu)或丙氨酸(Ala)。
14.权利要求10所述的葡萄糖异构酶突变体，其中第276位为甘氨酸(Gly)或苏氨酸(Thr)。
15.权利要求10所述的葡萄糖异构酶突变体，其氨基酸序列为序列表中序列11所示的氨基酸序列。
16.权利要求10所述的葡萄糖异构酶突变体，其氨基酸序列为序列表中序列12所示的氨基酸序列。
17.权利要求1所述的葡萄糖异构酶突变体，其具有至少三个选自第87位、第217位、第260位、第276位的突变。
18.权利要求17所述的葡萄糖异构酶突变体，其中第87位为甲硫氨酸(Met)或亮氨酸(Leu)。
19.权利要求17所述的葡萄糖异构酶突变体，其中第217位为精氨酸(Arg)、或色氨酸(Trp)、或甘氨酸(Gly)。
20.权利要求17所述的葡萄糖异构酶突变体，其中第260位为谷氨酸(Glu)或丙氨酸(Ala)。
21.权利要求17所述的葡萄糖异构酶突变体，其中第276位为甘氨酸(Gly)或苏氨酸(Thr)。
22.权利要求17所述的葡萄糖异构酶突变体，其氨基酸序列为序列表中序列13所示的氨基酸序列。
23.权利要求17所述的葡萄糖异构酶突变体，其氨基酸序列为序列表中序列14所示的氨基酸序列。
24.权利要求1-23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。
25.权利要求24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。
26.权利要求25所述的应用，其中所述的果葡糖浆的果糖含量为等于或高于55重量％。
27.一种DNA，其核苷酸序列为编码权利要求1-23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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| CN2005101236065A CN1966679B (zh) | 2005-11-18 | 2005-11-18 | 葡萄糖异构酶突变体、其应用以及编码该突变体的dna |
| EP06805105A EP1956083B1 (en) | 2005-11-18 | 2006-10-30 | Glucose isomerase mutants, the use thereof and the dnas encoding the same |
| US12/093,859 US7704719B2 (en) | 2005-11-18 | 2006-10-30 | Glucose isomerase mutants, DNA thereof and use thereof |
| PCT/CN2006/002901 WO2007056930A1 (fr) | 2005-11-18 | 2006-10-30 | Mutants de glucose isomerase, utilisation mutants et adn codant pour ces mutants |
| JP2008540429A JP4955692B2 (ja) | 2005-11-18 | 2006-10-30 | グルコースイソメラーゼ変異体、そのｄｎａ及びその使用 |
| DE602006019135T DE602006019135D1 (de) | 2005-11-18 | 2006-10-30 | Glucoseisomerase-mutanten, deren verwendung und die dafür codierenden dnas |
| AT06805105T ATE492634T1 (de) | 2005-11-18 | 2006-10-30 | Glucoseisomerase-mutanten, deren verwendung und die dafür codierenden dnas |
| US12/723,833 US7923222B2 (en) | 2005-11-18 | 2010-03-15 | Methods of using isolated glucose isomerase |
| US12/723,918 US8067561B2 (en) | 2005-11-18 | 2010-03-15 | Isolated DNA encoding recombinant glucose isomerase |
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| CN2005101236065A CN1966679B (zh) | 2005-11-18 | 2005-11-18 | 葡萄糖异构酶突变体、其应用以及编码该突变体的dna |
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| CN1966679B true CN1966679B (zh) | 2011-08-17 |
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| US (3) | US7704719B2 (zh) |
| EP (1) | EP1956083B1 (zh) |
| JP (1) | JP4955692B2 (zh) |
| CN (1) | CN1966679B (zh) |
| AT (1) | ATE492634T1 (zh) |
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| WO2012088467A2 (en) | 2010-12-22 | 2012-06-28 | Mascoma Corporation | Genetically modified clostridium thermocellum engineered to ferment xylose |
| CN102443578B (zh) * | 2011-12-08 | 2013-10-16 | 江南大学 | 一种葡萄糖异构酶突变体及其应用 |
| 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 |
| EP3619306A1 (en) | 2017-05-05 | 2020-03-11 | c-LEcta GmbH | Glucose isomerase |
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| EP0351029B1 (en) | 1988-07-15 | 2002-03-06 | Genencor International, Inc. | Novel glucose isomerase enzymes and their use |
| FI102543B1 (fi) | 1990-01-04 | 1998-12-31 | Genencor Int | Mutantti glukoosi-isomeraasit |
| US5656497A (en) | 1994-09-06 | 1997-08-12 | Michigan State University | Gene encoding hyperthermostable xylose isomerase from Thermotoga neapolitana |
| CN1088108C (zh) * | 1997-09-30 | 2002-07-24 | 中国科学技术大学 | 一种葡萄糖异构酶的247位单突变体酶和138,247位双突变体酶及其构建方法 |
| SE9901298D0 (sv) * | 1999-04-09 | 1999-04-09 | Forskarpatent I Syd Ab | Xylose isomerase with improved kinetic properties |
| JP2000333684A (ja) * | 1999-05-26 | 2000-12-05 | Novo Nordisk As | グルコースイソメラーゼ活性を有するポリペプチド及びそれをコードする核酸 |
| 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 |
| CN1693476A (zh) * | 2004-05-08 | 2005-11-09 | 张骏 | 一种精液果糖测定新方法及用于该方法的葡萄糖异构酶突变体 |
| CN1702172B (zh) * | 2004-05-26 | 2011-12-07 | 百瑞全球有限公司 | 葡萄糖异构酶突变体 |
| CN1743454A (zh) | 2004-09-02 | 2006-03-08 | 百瑞全球有限公司 | 葡萄糖异构酶突变体及其应用 |
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Also Published As
| Publication number | Publication date |
|---|---|
| EP1956083A4 (en) | 2009-02-25 |
| CN1966679A (zh) | 2007-05-23 |
| US8067561B2 (en) | 2011-11-29 |
| EP1956083B1 (en) | 2010-12-22 |
| ATE492634T1 (de) | 2011-01-15 |
| JP4955692B2 (ja) | 2012-06-20 |
| US20100228016A1 (en) | 2010-09-09 |
| US7704719B2 (en) | 2010-04-27 |
| US7923222B2 (en) | 2011-04-12 |
| WO2007056930A1 (fr) | 2007-05-24 |
| DE602006019135D1 (de) | 2011-02-03 |
| US20080305529A1 (en) | 2008-12-11 |
| JP2009515536A (ja) | 2009-04-16 |
| US20100227365A1 (en) | 2010-09-09 |
| EP1956083A1 (en) | 2008-08-13 |
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