Taguchi S, Maehara A, Takase K, Nakahara M, Nakamura H, Doi Y
Polymer Chemistry Laboratory, RIKEN Institute, 2-1 Hirosawa, Saitama 351-0198, Wako-shi, Japan.
FEMS Microbiol Lett. 2001 Apr 20;198(1):65-71. doi: 10.1111/j.1574-6968.2001.tb10620.x.
Polymerase is a central enzyme involved in the biosynthesis of polyhydroxybutyrate (PHB), a well-known bacterial biodegradable polyester. In this study, we have established an in vivo assay system to analyze mutational effects of Ralstonia eutropha polymerase (termed PhbC(Re)) on the level of PHB accumulation in recombinant strains of Escherichia coli. This in vitro evolution system consists of a polymerase chain reaction-mediated random mutagenesis and two assay procedures, a plate assay using a PHB-staining dye and a high-pressure liquid chromatographic assay based on the converting reaction from PHB to crotonic acid. The distribution pattern of the PHB accumulation level of the mutant population using 378 clones arbitrarily selected, suggested that the present level of PhbC(Re) is high and well-optimized. It is noteworthy that many of the amino acid substitutions affecting the PHB accumulation occurred in the conserved positions or regions within an 'alpha/beta hydrolase fold' which is commonly found among hydrolytic enzymes. From a good correlation with the level of PHB accumulation, an activity estimation of the PhbC(Re) would be efficiently achieved by monitoring the level of PHB accumulation using the in vivo assay system established here.
聚合酶是参与聚羟基丁酸酯（PHB）生物合成的核心酶，PHB是一种著名的细菌可生物降解聚酯。在本研究中，我们建立了一种体内分析系统，以分析真养产碱菌聚合酶（称为PhbC(Re)）对大肠杆菌重组菌株中PHB积累水平的突变效应。这种体外进化系统由聚合酶链反应介导的随机诱变和两种分析程序组成，一种是使用PHB染色染料的平板分析，另一种是基于PHB向巴豆酸转化反应的高压液相色谱分析。使用任意选择的378个克隆对突变群体的PHB积累水平分布模式表明，目前PhbC(Re)的水平很高且优化良好。值得注意的是，许多影响PHB积累的氨基酸替换发生在“α/β水解酶折叠”内的保守位置或区域，这种结构常见于水解酶中。鉴于与PHB积累水平具有良好的相关性，通过使用此处建立的体内分析系统监测PHB积累水平，可以有效地实现对PhbC(Re)活性的估计。
