催化吲哚生成靛蓝的细胞色素P450BM-3定向进化研究  

Cytochrome P450BM-3 Mutants With Improved Catalytic Properties of Hydroxylating Indole to Indigo by Error-prone PCR*

作　　者：李红梅[1] 梅乐和[1] URLACHER VLADA SCHMID ROLF D 

机构地区：[1]浙江大学化学工程与生物工程系,杭州310027 [2]Institute of Technical Biochemistry University of Stuttgart Stuttgart,Germany

出　　处：《生物化学与生物物理进展》2005年第7期630-635,共6页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金资助项目(20176050);CSC-DAAD国家留学基金中德合作科技人员交流PPP项目;浙江省科技计划项目(2004c33036);国家教育部留学回国人员基金;浙江省留学回国人员基金~~

摘　　要：以催化吲哚产生的靛蓝在630nm处具有特殊的吸收峰为高通量筛选指标,将来源于Bacillusmegaterium的细胞色素P450BM-3单加氧酶的基因序列用易错聚合酶链式反应进行定向进化,通过多轮突变,在原有的能产靛蓝的高活力突变酶的基础上成功获得了三个高于亲本酶的突变酶,突变酶的酶活分别是亲本酶的6.6倍(hml001),9.6倍(hml002)和5.3倍(hml003),并对突变酶的动力学参数进行了分析.突变酶DNA测序的结果表明,hml001含有一个有义氨基酸置换I39V,hml002含有三个有义氨基酸置换D168N,A225V,K440N,hml003含有一个有义氨基酸置换E435D,这些突变位点有些远离底物结合部位,有些位于底物结合部位.Monooxygenase domain of cytochrome P450BM-3 from Bacillus megaterium was evolved by error-prone PCR. Three mutants (D168N, A225V, K440N; E4351); 139V) with higher hydroxylating activity than the parent type P450BM-3((A74G, F87V, L188Q)) were obtained, coupled with a sensitive screening method of absorption of hydroxylating indole to indigo at 630mn. The catalytic activities of three mutants were 6.6(hm1001), 9.6(hm1002), 5.3 (hm1003) fold higher than that of the parent type P450BM-3 respectively. The kinetic analysis revealed that the mutant enzymes exhibit a higher substrate binding ability and catalytic efficiency than the parent enzyme. DNA sequence indicated that hm1001 and hm1003 cover one amino acid substitution (139V and E435D, respectively), hm1002 contains three amino acid substitutions (D168N, A225V, K440N).

关 键 词：细胞色素P450BM-3 定向进化 靛蓝 易错聚合酶链式反应 催化活性 

分 类 号：Q7[生物学—分子生物学]