xyl-d二型乙醇脱氢酶(SADH)G198R？ね槐涮宓难芯？
The study onxyl-d second alcohol dehydrogenase (SADH) G198R mutant

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.
The SADH of xyl d had been cloned by designing degenerate primer , and alerted Gly？？？？198？？ 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