Lysine21突变对树干毕赤氏酵母木糖还原酶辅酶依赖性的影响.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2008, June 25; 24(6): 1108- 1111 journals.im.ac.cn Chinese Journal of Biotechnology ISSN 1000-3061 cjb@im.ac.cn © 2008 Institute of Microbiology, CAS CSM, All rights reserved 研究报告 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 曾琦锴, 杜红丽, 翟志臣, 林小琼, 林影 华南理工大学生物科学与工程学院 广州 510640 摘 要: 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. coli BL21(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 。 关键词: 木糖还原酶, 定点突变, 辅酶依赖性 Mutational Research on the Role of Lysine 21 in the Pichia stipitis Xylose reductase Qikai Zeng, Hongli Du, Zhichen Zhai, Xiaoqiong Lin, and Ying Lin School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China Abstract: 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 int