[1]刘治江,贺淹才,李红然,等.采用易错PCR对粘质沙雷氏菌几丁质酶C进行定向进化[J].华侨大学学报(自然科学版),2010,31(1):58-64.[doi:10.11830/ISSN.1000-5013.2010.01.0058]
　LIU Zhi-jiang,HE Yan-cai,LI Hong-ran,et al.Directed Evolution of Chitinase C from Serratia marcescens by Error-Prone PCR[J].Journal of Huaqiao University(Natural Science),2010,31(1):58-64.[doi:10.11830/ISSN.1000-5013.2010.01.0058]
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采用易错PCR对粘质沙雷氏菌几丁质酶C进行定向进化()
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《华侨大学学报（自然科学版）》[ISSN:1000-5013/CN:35-1079/N]

卷:
第31卷
期数:
2010年第1期
页码:
58-64
栏目:
出版日期:
2010-01-20

文章信息/Info

Title:
Directed Evolution of Chitinase C from Serratia marcescens by Error-Prone PCR
文章编号:
1000-5013(2010)01-0058-07
作者:
刘治江; 贺淹才; 李红然; 刘嘉; 施腾鑫
华侨大学工业生物技术福建省高等学校重点实验室
Author(s):
LIU Zhi-jiang;  HE Yan-cai;  LI Hong-ran;  LIU Jia;  SHI Teng-xin
Key Laboratory of Industriql Biotchnology of Fujian Province University, Huaqiao University, Quanzhou 362021, China
关键词:
粘质沙雷氏菌;  几丁质酶C;  分子定向进化;  易错聚合酶链反应;  包涵体
Keywords:
Serratia marcescens;  chitinase C;  molecular directed evolution;  error-prone polymerase chain reaction;  inclusion bodies
分类号:
Q786
DOI:
10.11830/ISSN.1000-5013.2010.01.0058
文献标志码:
A
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.
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.

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备注/Memo

备注/Memo:
福建省自然科学基金资助项目(C04010011)
更新日期/Last Update: 2014-03-23