【摘要】 以重组E.coliBL21(DE3)pLysS/pET22b-Chi C为亲本,采用易错聚合酶链反应(PCR)技术,对粘质沙雷氏菌几丁质酶C基因(Chi C)进行定向进化研究.经过两轮易错PCR后,建立突变体文库,进行平板初筛、包涵体复性及酶活检测复筛,获得一酶活较高的突变酶(Mut-Chi C),其催化活性为亲本重组酶的1.9倍,比活力为出发菌酶活的3.3倍.对突变酶的酶学性质进行初步研究,其最适pH值为5.0,最适温度为60℃,而出发菌该酶的最适温度为40℃.进化酶基因经DNA测序并通过软件与亲本型酶基因进行分析比对,表明突变酶Mut-Chi C基因发生点突变,使4处氨基酸被取代,且均为有义突变.更多还原

【Abstract】 Molecular directed evolution to Serratia marcescens chitinase gene Chi C was conducted by error prone polymerase chain reaction(PCR).After two sequential error prone PCR,a mutagenesis gene library was generated,and then by plate screening,inclusion body renaturing,enzyme activity measuring and the repeat screening,one mutant(Mut-Chi C) with higher enzyme activity was obtained.Its enzyme activity was increase as 1.9 times high as the original wild type,and the specific activity was found 3.3 times of the original.The enzymic characters of the Mut-Chi C were determine.The results showed that the optimal pH value and temperature of Mut-Chi C were 5.0 and 60 ℃ respectively,while the optimal temperature of the original wild type is 40 ℃.This kind of chitinase that has a higher optimum temperature may have practical significance.The Mut-Chi C DNA was sequenced,and both the mutantin gene analysis and the comparison with the wild type were determine by intent software,the results demonstrate that the mutant enzyme had four mutations,each has a amino acid substitution,and all are sense mutations.更多还原