Normi Yahaya M, Hiraishi Tomohiro, Taguchi Seiichi, Abe Hideki, Sudesh Kumar, Najimudin Nazalan, Doi Yoshiharu
Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
Macromol Biosci. 2005 Mar 15;5(3):197-206. doi: 10.1002/mabi.200400181.
Modification of the type I polyhydroxyalkanoate synthase of Ralstonia eutropha (PhaC(Re)) was performed through systematic in vitro evolution in order to obtain improved PhaC(Re) having an enhanced activity of poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli. For the first time, a beneficial G4D N-terminal mutation important for the enhancement of both PHB content in dry cells and PhaC(Re) level in vivo was identified. Site-directed saturation mutagenesis at the G4 position enabled us to identify other mutations conferring similar enhanced characteristics. In addition, the PHB homopolymer synthesized by most G4X single mutants also had higher molecular weights than that of the wild-type. In vitro enzymatic assays of purified G4D mutant PhaC(Re) revealed that the mutant enzyme exhibited slightly lower activity and reaction efficiency compared to the wild-type enzyme. [diagram in text].
为了获得在重组大肠杆菌中具有增强的聚（3-羟基丁酸酯）（PHB）合成活性的改良型真养产碱菌（Ralstonia eutropha）I型聚羟基脂肪酸酯合酶（PhaC(Re)），通过系统的体外定向进化对其进行了改造。首次鉴定出一个对提高干细胞中PHB含量和体内PhaC(Re)水平都很重要的有益G4D N端突变。在G4位置进行定点饱和诱变使我们能够鉴定出赋予类似增强特性的其他突变。此外，大多数G4X单突变体合成的PHB均聚物的分子量也高于野生型。对纯化的G4D突变体PhaC(Re)进行的体外酶活性测定表明，与野生型酶相比，突变体酶的活性和反应效率略低。[文中有图]
