嗜热细菌木糖异构酶基因xylA在酿酒酵母中的高效表达

采用PCR技术克隆得到嗜热细菌Clostridium thermohydrosulfuricum木糖异构酶(xylose isomerase XI)基因xylA,将该基因连接于酵母表达载体pMA91的磷酸甘油激酶(PGK)启动子下，得到重组质粒pBX1。通过LiAc完整细胞转化法将重组质粒转移至酿酒酵母(Saccharomyces cerevisiae)H158受体菌中，得到重组酵母转化子H612，酶活测定结果表明，成功地在酿酒酵母中得到木糖异构酶的活性表达。SDSPAGE电泳结果显示出明显的特异性表达产物带，单体分子量为43kD。由酿酒酵母重组子H612产生的木糖异构酶最高酶活条件与其在自然状态下的一致，均为85℃，pH70，在这一条件下酶的比活力为10U/mg蛋白，而在接近酵母最适生长温度的30℃和40℃时，其相对酶活分别下降37%和11%。研究结果显示在酿酒酵母中得到木糖异构酶的活性表达，为进一步在酿酒酵母菌中建立新的木糖代谢途径打下了基础。

EXPRESSION OF XYLOSE ISOMERASE GENE( xylA ) IN SACCHAROMYCES CEREVISIAE FROM CLOSTRIDIUM THERMOHYDROSULFURICUM Project of Shandong Province National Natural Fund(No.Q97DO1133)

The Clostridium thermohydrosulfuricum xylA gene encoding xylose(glucose) isomerase was cloned in the yeast expression vector pMA91 under the control of the PGK promoter, resulting in pBX-1, and transformed into Saccharomyces cerevisiae. Production of recombinant xylose isomerase was seen in the a Coomassie stained SDS-PAGE gel and the molecular mass was estimated to be 43 kD. The recombinant xylose isomerase showed the highest activity at 85 degrees C and pH7. The specific activity under these condition was 1.0 U/mg protein. At 30 degrees C and 40 degrees C, the relative activity was reduced to 3.7% and 11%, respectively, of the maximum.