可视化突变建模、定点突变和表达超耐热菌D-乳酸脱氢酶

Visualization of Mutation to Structure Homologous Modeling,Site-Directed Mutagenesis and Express D-Lactate Dehydrogenase from Aquifex aeolicus

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。

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.