定点突变提高氏木霉木聚糖酶（XYNII）的稳定性.PDFVIP

生 物 工 程 学 报 Chin J Biotech 2010, May 25; 26(5): 623−629 Chinese Journal of Biotechnology ISSN 1000-3061 cjb@ ©2010 CJB, All rights reserved. 工业生物技术 定点突变提高里氏木霉木聚糖酶 (XYN II) 的稳定性 韩承业，余世袁，欧阳嘉，李鑫，周娟，许艳 南京林业大学 林木遗传与生物技术省部共建教育部重点实验室，南京 210037 摘 要: 通过定点突变的方法，在来源于里氏木霉 Trichderma reesei 的木聚糖酶XYN II 的N-末端两个 β折叠片层间添 加二硫键，以提高木聚糖酶的稳定性。原酶XYN-OU 和突变酶XYN-HA12 (T2C 、T28C 和S156F) 分别在毕赤酵母中 分泌表达，突变酶与原酶纯化后进行酶学性质比较。结果表明：突变酶最适反应温度由50 ℃提高到 60 ℃；在70 ℃的半 衰期由 1 min 提高到 14 min；最适反应pH 为5.0 ，与原酶保持一致，但是在50 ℃、30 min 条件下的pH 稳定范围由4.0~9.0 扩展到3.0~10.0 。对木聚糖酶分子改良的结果反映出在 β 片层间添加二硫键可以有效改善酶在较高温度下三维结构的刚 性，提高热稳定性。 关键词: 定点突变，木聚糖酶，热稳定性，里氏木霉 Enhancing stability of Trichoderma reesei xylanase (XYN II) by site-directed mutagenesis Chengye Han, Shiyuan Yu, Jia Ouyang, Xin Li, Juan Zhou, and Yan Xu Key Laboratory of Forest Genetics Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China Abstract: We engineered a disulphide bridge between two adjacent double-layered β-sheet at the N-terminal region of Trichoderma reesei endo-1,4-β-xylanase II(XYN II) by site-directed mutagenesis. The native xylanase XYN-OU and the mutated xylanase XYN-HA12 (T2C, T28C and S156F) were separately expressed in Pichia pastoris . Both xylanases were purified and characterized. The optimum temperature of XYN-HA12 was increased from 50°C to 60°C, relative to XYN-OU. At 70°C, the halftime of inactivation for XYN-OU and XYN-HA12 were 1 min and 14 min, respectively. Th