Lysine 21突变对树干毕赤氏酵母木糖还原酶辅酶依赖性的影响

Mutational Research on the Role of Lysine 21 in the Pichia stipitis Xylose reductase

木糖还原酶是重组酿酒酵母工程菌利用木糖生成乙醇代谢途径中的关键酶,该关键酶在利用木糖时依赖NADPH而不是NADH是导致酿酒酵母代谢木糖生成乙醇的最终产率低的主要原因之一。为了改变树干毕赤氏酵母木糖还原酶的辅酶依赖性,对它的第21位赖基酸Lys进行了突变。利用质粒载体pET28b分别将突变后的基因K21A-XYL1、K21R-XYL1及野生基因WT-XYL1在大肠杆菌E.coliBL21(DE3)中进行表达,表达后的蛋白经His-Tag纯化柱纯化后测定酶学性质。结果表明:K21R突变子的辅酶依赖性没有改变,但K21A突变子的辅酶依赖性由NADPH完全逆转为NADH。

The xylose reductase of Pichia stipitis is one of the most important enzymes. It can be used to build up recombinant Saccharomyces cerevisiae strain for utilizing xylose and producing ethanol. Intercellular redox imbalance caused by NADPH preference over NADH for Pichia stipitis xylose reductase （PsXR） has been considered to be one of the main factors for poor ethanol productivity. Some key amino acids of PsXR, which affect the activity or coenzyme preference, were investigated in our previous study. In this study, Lys21 were rational designed for site-directed mutagenesis to alter coenzyme specificity of PsXR from NADPH and NADH into single NADH. The wild gene and mutagenesis genes were ligated into pET28b, and were transferred into E.coli BL21（DE3）. After induced by IPTG, the xylose reductases were purified. Purified mutants K21A （Lys21→Ala）, K21R（Lys21→Arg） were characterized by steady-state kinetic analysis. The results showed that the coenzyme dependence of K21A was completely reversed to NADH.