毕赤酵母木糖还原酶定点突变改善其对双辅酶的亲和力

Modification of Dual-coenzyme Affinity of Xylose Reductase from Pichia stipitis by Site-directed Mutagenesis

通过毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)基因定点突变,获得NADH高亲和力的毕赤酵母木糖还原酶(PsXR),改善了辅酶不同而导致的酿酒酵母胞内氧化还原失衡.同时克隆了PsXR编码基因,通过BLAST工具进行同源性搜索,并用生物软件进行序列比对和结构分析,确定突变位点.用融合PCR方法进行定点突变,并在大肠杆菌表达系统中进行融合表达,且对表达产物进行HIS-TAG亲和纯化,分光光度法检测酶活性,计算比活力.本研究成功获得突变XR编码基因,并收集了纯化的突变蛋白.酶活性检测和比活力计算显示,3种突变酶对2种辅酶的亲和力在一定程度上都发生了变化.与未突变的PsXR相比,3种突变酶对辅酶NADPH的亲和力均显著下降,突变酶M3对辅酶NADH的亲和力未发生变化,而突变酶M1和M4对辅酶NADH的亲和力显著升高,其中突变酶M1对NADH的亲和力明显提高,对NADPH的亲和力明显下降,其活性主要依赖辅酶NADH,提示K270R位点在XR与辅酶结合中起关键作用.

In this study, we obtained the NADH-preferring xylose reductase from Pichia stipitis （PsXR） and introduced site-directed mutations designed to correct the intracellular redox imbalance caused by coenzyme differences in Saccharomyces cerevisiae. The mutation sites in cloned PsXR gene were identified by BLAST homogenous matching and molecular structure analysis. The mutations were introduced by nested PCR and the mutant proteins were expressed in E. coli then purified with a HIS-TAG column. The specific enzyme activity of the mutant proteins was assayed by spectrophotometry. The results showed that three of the mutants （M1, M3 and M4） differed in the enzyme activity, specific activity and the coenzyme affinity to NADPH or NADH. Comparing to wild-type PsXR, coenzyme affinity to NADPH was remarkably decreased in all three mutants. The coenzyme affinity to NADH in M1 and M4 was increased significantly, but changed mildly in M3. M1 showed the most remarkably increase of NADH-preferring activity and decrease of NADPH-preferring activity, demonstrating a maximum dependency to NADH. This implies that the mutation site K270R in M1 plays an important role in determining the coenzyme preference of PsXR.