PMID: 11325555Apr 28, 2001Paper

Analysis of mutational effects of a polyhydroxybutyrate (PHB) polymerase on bacterial PHB accumulation using an in vivo assay system

FEMS Microbiology Letters
Seiichi TaguchiYoshiharu Doi

Abstract

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 syste...Continue Reading

References

Apr 1, 1992·Protein Engineering·D L OllisJ Schrag
Sep 7, 1987·Journal of Theoretical Biology·S Kauffman, S Levin
Dec 1, 1996·Applied and Environmental Microbiology·S TaguchiH Momose
Feb 17, 1998·Applied and Environmental Microbiology·S TaguchiH Momose
Mar 6, 1999·Microbiology and Molecular Biology Reviews : MMBR·L L Madison, G W Huisman
Jul 23, 1999·International Journal of Biological Macromolecules·B H Rehm, A Steinbüchel
Sep 30, 1999·Journal of Biochemistry·S TaguchiH Momose
Dec 11, 1999·Nucleic Acids Research·A BatemanE L Sonnhammer

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Citations

May 15, 2013·Applied Biochemistry and Biotechnology·Johanna Katherine BocanegraJose Gregório Cabrera Gomez
Nov 6, 2004·Applied and Environmental Microbiology·Nuttawee NiamsiriCarl A Batt
Oct 16, 2008·The Biochemical Journal·Marina C TheodorouDimitrios A Kyriakidis
Dec 5, 2018·Applied Microbiology and Biotechnology·Min Fey ChekSeiichi Taguchi
May 31, 2019·Frontiers in Microbiology·Chee Kent LimPatrick K H Lee
Apr 7, 2020·Advanced Materials·So Young ChoiSang Yup Lee
Nov 24, 2006·Applied Microbiology and Biotechnology·Christopher T Nomura, Seiichi Taguchi

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