易错PCR法定向进化D-海因酶的初步研究  

A Preliminary Study on the Directed Evolution of D-hydantoinase by Error-prone PCR

作　　者：孔荣[1] 钮利喜[1] 袁静明[1] 

机构地区：[1]山西大学生物技术研究所,山西太原030006

出　　处：《山西大学学报（自然科学版）》2006年第4期425-427,共3页Journal of Shanxi University(Natural Science Edition)

基　　金：山西省自然科学基金(20031042)

摘　　要：D-海因酶是生物转化D-型氨基酸的关键酶,在一定浓度M n2+存在下,经易错PCR法重复扩增,产物构建成pET-HDT重组子,并建立突变体文库.经筛选获得了内源DNA序列变异的变异株.进化酶催化底物海因水解的活性为亲本重组酶的1.3倍,催化底物对羟基苯海因水解的活性为亲本重组酶的2.4倍.D-hydantoinase （HDT） plays an important role in the bioconversion of D-amino acids. The enzyme gene was repeatedly amplified by error-prone PCR at a definite concentration of Mn^2＋ and PCR products were cloned to vector pET-3a to form the recombinant plasmid pET-HDT,followed by generating a mutant molecular library. Positive colony was selected to confirm the gene mutation by DNA sequence analysis. The mutant enzyme showed its activity as 1.3 folds as the original recombinant enzyme with DL-hydantoin as the substrate,whilst as 2.4 times with D-p-hydroxyophenylhydantoin as the substrate.

关 键 词：海因酶 易错PCR 酶活性 

分 类 号：Q814[生物学—生物工程]