[1]李梓君,方柏山,杨仲丽,等.应用易错PCR定向进化甘油脱氢酶[J].华侨大学学报(自然科学版),2010,31(6):661-666.[doi:10.11830/ISSN.1000-5013.2010.06.0661]
　LI Zi-jun,FANG Bai-shan,YANG Zhong-li,et al.Directed Evolution of Glycerol Dehydrogenase by Error Prone PCR[J].Journal of Huaqiao University(Natural Science),2010,31(6):661-666.[doi:10.11830/ISSN.1000-5013.2010.06.0661]
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应用易错PCR定向进化甘油脱氢酶()
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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6期
页码:
661-666
栏目:
出版日期:
2010-11-20

文章信息/Info

Title:
Directed Evolution of Glycerol Dehydrogenase by Error Prone PCR
文章编号:
1000-5013(2010)06-0661-06
作者:
李梓君; 方柏山; 杨仲丽; 刘嘉
华侨大学工业生物技术福建省高等学校重点实验室
Author(s):
LI Zi-jun;  FANG Bai-shan;  YANG Zhong-li;  LIU Jia
Key Laboratory of Industrial Biotechnology Fujian Province, Huaqiao University, Quanzhou 362021, China
关键词:
甘油脱氢酶;  克雷伯杆菌;  定向进化;  易错聚合酶链式反应
Keywords:
glycerol dehydrogenase;  Klebsiella pneumoniae;  directed evolution;  error-prone polymerase chain reaction
分类号:
Q814
DOI:
10.11830/ISSN.1000-5013.2010.06.0661
文献标志码:
A
摘要:
经过连续易错聚合酶链式反应(Error-Prone PCR)向克雷伯杆菌(Klebsiella pneumoniae)甘油脱氢酶基因中引入突变,并构建突变文库.依据突变体催化番红O显色的特性,采用琼脂平板法和96微孔板法相结合的高通量筛选方法,成功获得突变体gldA-74,其酶活力是亲本重组酶的2.73倍.通过软件对突变体gldA-74酶基因和亲本重组酶基因进行分析对比,发现突变体gldA-74酶基因发生了一处点突变(A964T),该突变使一处氨基酸被取代.将亲本重组酶和进化酶的高效表达产物经Ni柱纯化后,酶学性质的测定结果表明,甘油的Km值由0.58 mmol.L-1升高至0.63 mmol.L-1,而NAD的Km值由0.74 mmol.L-1升高至0.77mmol.L-1,并且酶的最适pH值由原来的11.5升高至12.5,而最适温度由65℃降至55℃.
Abstract:
The GDH enzyme gene from Klebsiella pneumoniae was repeatedly amplified by two sequential error-prone polymerase chain reaction(PCR),and the gene library was built.The mutant can catalyze safranin O to show colors,and on the base of this property,the gldA-74 mutant was obtained by high throughput screening method using active agar plates in combination with 96-well microplates.The activity of the gldA-74 mutant was showed 2.73 fold of that of the parent recombinase.Gene analysis of the gldA-74 mutant indicated that the mutant enzyme had a point mutation(A964T) which resulted in an amino acid being replaced.Then,the highly expressed productions of recombinase GDH and evolved GDH were purified by Ni-NTA and the enzymatic properties were determined.The results showed that the Km of GDH increased from 0.58 mmol·L-1 to 0.63 mmol·L-1,and the Km of NAD increased as well,from 0.71 mmol·L-1 to 0.77 mmol·L-1.The optimum pH of mutant enzyme also increased from 11.5 to 12.5 while the optimum temperature decreased from 65 ℃ to 55 ℃.

参考文献/References:

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备注/Memo

备注/Memo:
国家重点基础研究发展(973)计划项目(2006AA020103); 国家自然科学基金资助项目(20676048)
更新日期/Last Update: 2014-03-23