耐热菌D-乳酸脱氢酶突变体的可视化建模和大肠杆菌中突变体对产苯乳酸的影响
详细信息Visual modeling mutants for D-lactate dehydrogenase form aquifex aeolicus and the effect of mutants on the production of phenyl lactate acid in E.coli
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摘要: 以生物信息学为基础,分析D-乳酸脱氢酶（D-LDH）的保守氨基酸残基、活性中心氨基酸残基、蛋白质三维结构和同源建模,可视化比较建模突变体空间构象,优选最佳突变体模型。结果显示,在D-LDH的20个保守氨基酸中,4个与酶活性中心有关。比较突变体模型发现,49和297位的phe或try的苯环形成空间位阻,F49A或Y279A及F49A和Y279A双突变体可解除位阻。对已构建的三个突变体初步发酵显示,IPTG和乳糖都能诱导突变体酶在大肠杆菌中产生苯乳酸,静置培养比摇振培养产量高,用乳糖诱导时,突变体F49A（A.a D-LDH-F49A株）苯乳酸的量比野生型（A.a.D-LDH株）的高。优选可视化突变体可作为高效构建工程菌的方法之一。Abstract: Based on bioinformatics, the amino acid residues of conservative and activity center of D-lactate dehydrogenase (D-LDH) , and the three-dimensional structure model of protein was analysised.The space conformation of visualization mutant had been constructed by homology modeling, the best mutant models were selected by the calculation of the distance and angle.The results showed that the 4 amino acid residues were relevant to the activity center in 20 conservative residues of D-LDH.After the models were compared, it was found that the big molecules substrates were obstructed by the benzyl of the residues of Phenylalanine (phe) or Tyrosine (try) on the 49 and 297 position.When F49A, Y279A, F49A and Y279A were mutated, the obstacles would disappear or weaken.The three mutants constructed were made a preliminary study, the results showed that IPTG or lactose could induce mutant to produce phenyl lactic acid in E.coli.The yield of phenyl lactic acid was higher in static culture than in vibration incubator, and the one of the F49A mutant (A.a.D-LDH-F49A strains) was higher than the one of the wild type (A.a.D-LDH strains) with lactose inducing.It would be a method of constructing gene engineering strain that visualization mutants models were compared and selected.
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Keywords:
- mutant;visual;PLA;D-LDH; /
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