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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2012, Vol. 32 Issue (05): 73-78    
技术与方法     
可视化突变建模、定点突变和表达超耐热菌D-乳酸脱氢酶
田晋红1,2,3, 刘琦2, 战丽萍2, 周泽扬1,4
1. 西南大学蚕学与系统生物学研究所 重庆 400716;
2. 西南大学药学院 重庆 400716;
3. 重庆市兽药工程技术研究中心 重庆 400716;
4. 重庆师范大学生命科学学院 重庆 400047
Visualization of Mutation to Structure Homologous Modeling, Site-Directed Mutagenesis and Express D-Lactate Dehydrogenase from Aquifex aeolicus
TIAN Jin-hong1,2,3, LIU Qi2, ZHAN Li-ping2, ZHOU Ze-yang1,4
1. Institute of Sericulture and Systems Biology,Southwest University,Chongqing 400716,China;
2. College of Pharmaceutical Sciences,Southwest University,Chongqing 400716,China;
3. Chongqing Engineering Technology Research Centre of Veterinary Drugs,Chongqing 400716,China;
4. College of Life Science,Chongqing Normal University,Chongqing 400047,China
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摘要：

目的:构建产天然防腐剂苯乳酸的工程菌。方法:分析超耐热菌(Aquifex aeolicus,A.aeolicus)D-乳酸脱氢酶(D-LDH)的三维构象,并与构建的可视化突变体三维模型进行对比,通过比较酶活性中心氨基酸残基与底物的空间构象,优选最佳模型进行定点突变、克隆、表达和苯乳酸发酵实验。结果:优选到F49A和Y297S两个单突变模型和一个F49A/Y297S双突变模型,分别进行定点突变和工程菌构建,三个突变工程菌均能发酵产生苯乳酸。结论:可视化定点突变乳酸脱氢酶可作为构建高产苯乳酸工程菌的有效方法。
关键词： 可视化;  突变;  苯乳酸;  乳酸脱氢酶    
Abstract:

Objective to construct high yields recombinant E. coli strains producing phenyl lactic acid(PLA). Methods Analyze the X-ray structure of active site amino acid residues of D-LDH from Aquifex aeolicus, and compare it with mutant D-LDH of the homologous model. The optimized 3D structures of the mutant enzyme were elected by analyzing the space conformation of the amino acid residues in substrate binding domain of the mutant D-LDH. The genes of enzyme were site-directed mutated, cloned and expressed. Results F49A or Y297S mutants were finally elected for single-site mutation models, F49A/Y297S mutant was elected for double -site mutations, and three recombinant E. coli strains were proved for PLA production. Conclusion The method of mutation visualization is an effective method for construction of high yield gene engineering bacterium producing PLA.
Key words: Visualization    Mutant    PLA D-LDH
收稿日期: 2012-01-31 出版日期: 2012-05-25
ZTFLH:  Q812  
基金资助:

重庆市科技创新能力建设资助项目(CSTC,2009CB1010)
通讯作者: 周泽扬     E-mail: zyzhou@cqnu.edu.cn
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引用本文:

田晋红, 刘琦, 战丽萍, 周泽扬. 可视化突变建模、定点突变和表达超耐热菌D-乳酸脱氢酶[J]. 中国生物工程杂志, 2012, 32(05): 73-78.

TIAN Jin-hong, LIU Qi, ZHAN Li-ping, ZHOU Ze-yang. Visualization of Mutation to Structure Homologous Modeling, Site-Directed Mutagenesis and Express D-Lactate Dehydrogenase from Aquifex aeolicus. China Biotechnology, 2012, 32(05): 73-78.

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https://manu60.magtech.com.cn/biotech/CN/    或    https://manu60.magtech.com.cn/biotech/CN/Y2012/V32/I05/73


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