xyl-d二型乙醇脱氢酶(SADH)G198R突变体的研究
The study onxyl-d second alcohol dehydrogenase (SADH) G198R mutant
作者：韩俊(四川大学生命科学学院生物资源与生态环境教育部重点实验室)；刘志斌(四川大学生命科学学院生物资源与生态环境教育部重点实验室)；马诗淳(四川大学生命科学学院生物资源与生态环境教育部重点实验室)；杨毅(四川大学生命科学学院生物资源与生态环境教育部重点实验室)
Author：HAN Jun(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；LIU Zhi-Bin(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；MA Shi-Chun(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；YANG Yi(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)
收稿日期：2013-09-04 年卷（期）页码：2015,52(1):187-192
期刊名称：四川大学学报: 自然科学版
Journal Name：Journal of Sichuan University (Natural Science Edition)
关键字：SADH; PCR定点诱变技术; xyl-d
Key words：Second alcohol dehydrogenase; PCR site directed mutagenesis techniques; xyl-d
基金项目：国家自然科学基金(No. 31171586); 国家863计划（2012AA022204）
中文摘要
本研究通过设计简并引物及文献参考, PCR克隆得到嗜热厌氧菌菌株xyl d的SADH, 并利用定点诱变技术将该酶的198位苷氨酸(Gly)突变为精氨酸(Arg). 将野生型和突变基因连接到原核表达载体pET28a, IPTG诱导表达, 再经镍柱纯化, 然后比较了两个蛋白分别以NAD/NADH, NADP/NADPH为辅因子、异丙醇或异丁醛为底物、55 ℃条件下的酶活. 发现突变后蛋白的总体催化活性相对于野生型有所下降, 其中突变蛋白对NAD/NADH的亲和性分别下降了约8倍和6倍, 而且对NADPH的亲和性下降也非常明显, 但对NADP的亲和性变化却不大.
英文摘要
The SADH of xyl d had been cloned by designing degenerate primer , and alerted Gly198 to Arg by PCR site directed mutagenesis techniques .The gene of the wild type and the mutant were linked to the prokaryotic expression carrier pET28a, then induced by IPTG and purified out by Nickel column. The catalytic activity of the two proteins were detected and compared when NAD / NADH, NADP / NADPH as coenzyme factors, isopropanol or isobutyraldehyde as substrate and the temperature was 55 ℃. It was found that the overall catalytic activity of the mutant enzyme was decreased. And the mutant protein affinity for NAD / NADH were respectively decreased to about 8 fold and 6 fold. The affinity for NADPH also decreased obviously. But The affinity for NADP changed a little.
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