酿酒酵母中的嗜热细菌木糖异构酶活性表达及木糖代谢研究

Active Expression of Xylose Isomerase from Thermobifida fusca in Saccharomyces Cerevisiae and Its Influence on Xylose Metabolism

关键词:  木糖代谢  木糖异构酶  嗜热菌放线菌  酿酒酵母

Key words:  xylose metabolize  xylose isomerase  Thermobifida fusca  Saccharomyces cerevisiae

摘要:

将来自嗜热放线细菌Thermobifida fusca的木糖异构酶(xylose isomerase,XI)基因xylA,连接于酵母表达载体pYES2的半乳糖诱导启动子(PGAL)下,得到重组质粒pYES2-xylA,用其转化酵母菌INVSc1,测定重组酵母菌株INVSc1-xylA的木糖异构酶活性,并对重组酵母菌株进行木糖葡萄糖共发酵试验,探讨在酿酒酵母中建立新的生产乙醇木糖代射途径。结果木糖异构酶在75℃,pH值6.8的酶活力最高,在酿酒酵母中成功地获得活性表达,并且SDS-PAGE电泳有明显的特异性表达产物带,单体分子量为43kD。INVSc1-xylA在木糖葡萄糖共发酵实验中消耗木糖和产生乙醇分别比对照菌提高53.8%和36%,提高了其生产乙醇的能力。

Abstract:

The xylose isomerase gene xylA of Thermobifida fusca was successfully cloned in the yeast expression vector pYES2 to construct recomninant that consume xylose to produce ethanol.The fused gene was under the control of the galactose-ind-ucible promoter (PGAL).The recombinant plasmid was transformed into the yeast stain Saccharomyces cerevisiae INVSc1 by the lithium acetate method.The recombinant strain named INVSc1/pYES2-xylA was abtained.The recombinant xylose isomerase activity was determined by the modified glucose oxidase assay and showed the highest activity at 75℃ and pH6.8.Production of recombinant xylose isomerase was seen in the Coomassie stained SDS-PAGE gel and the molecular mass was estimated to be 43kD.Glucose and xylose co-fermentation by INVSc1/pYES2-xylA were carried out, the consumption of xylose and the production of ethanol were 53.8% and 36% higer than the control strain INVSc1/pYES2.