CAJ | 학술논문

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。
본연구통과설계간병인물급문헌삼고,PCR 극륭득도기열염양균균주 xyl-d 적SADH,병이용정점유변기술장해매적198위감안산(Gly)돌변위정안산(Arg)。장야생형화돌변기인련접도원핵표체재체pET28a,IPTG유도표체,재경얼주순화,연후비교료량개단백분별이 NAD/NADH,NADP/NADPH 위보인자、이병순혹이정철위저물、55℃조건하적매활。발현돌변후단백적총체최화활성상대우야생형유소하강,기중돌변단백대 NAD/NADH 적친화성분별하강료약8배화6배,이차대NADPH 적친화성하강야비상명현,단대NADP적친화성변화각불대。
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.