Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase
Site-directed saturation mutagenesis at residue F420 and recombination with another beneficial mutation of Ralstonia eutropha polyhydroxyalkanoate synthase
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DOI：
10.1007/s10529-005-5186-z
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发表时间：
2005-05-01
影响因子：
2.7
通讯作者：
Doi, Y
中科院分区：
文献类型：
--
作者：
Normi, YM;Hiraishi, T;Doi, Y
F420 S取代增强了真养罗尔斯通氏菌PHA合酶（PhaC（Re））的比活性。我们已经对PhaC（Re）的F420进行了定点饱和诱变，并且在F420突变体中，F420 S突变体产生了最高的聚（3-羟基丁酸酯）（PHB）含量。体外活性测定表明，F420 S酶与野生型酶相比，其滞后期显著减少。通过F420 S突变与G4 D突变的组合实现了对PHB积累的增强，这赋予了高的PHB含量和体内高浓度的PhaC（Re）酶。G4 D/F420 S突变体的PHB含量和体内PhaC（Re）酶浓度高于F420 S突变体，而双突变体的PHB聚合物的分子量与F420 S突变体的相似。
The F420S substitution enhances the specific activity of Ralstonia eutropha PHA synthase (PhaC(Re)). We have now carried out site-directed saturation mutagenesis of F420 of PhaC(Re) and, amongst the F420 mutants, the F420S mutant gave the highest poly(3-hydroxybutyrate) (PHB) content. In vitro activity assay showed that the F420S enzyme had a significant decrease in its lag phase compared to that of the wild-type enzyme. Enhancement of PHB accumulation was achieved by combination of the F420S mutation with a G4D mutation, which conferred high PHB content and high in vivo concentration of PhaC(Re) enzyme. The G4D/F420S mutant gave a higher PHB content and in vivo concentration of PhaC(Re) enzyme than the F420S mutant, while the molecular weight of the PHB polymer of the double mutant was similar to that of the F420S mutant.