Development and validation of an HPLC-based screening method to acquire polyhydroxyalkanoate synthase mutants with altered substrate specificity(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)
スポンサーリンク
概要
- 論文の詳細を見る
A rapid and convenient method for the compositional analysis of polyhydroxyalkanoate (PHA) was developed using high-performance liquid chromatography (HPLC) and alkaline sample pretreatment in a 96-well plate format. The reliability of this system was confirmed by the fact that a mutant with a D171G mutation of Aeromonas caviae PHA synthase (PhaC_<AC>), which gained higher reactivity toward 3-hydroxyhexanoate (3HHx), was selected from the D171X mutant library. Together with D171G mutant, several single mutants showing high reactivity toward 3HHx were isolated by the HPLC assay. These new mutants and double mutants combined with an N149S mutation were used to synthesize P(3-hydroxybutyrate-co-3HHx) in Ralstonia eutropha PHB~4 from soybean oil as carbon source, achieving higher levels of 3HHx fraction than the wild-type enzyme. Based on these results, the high-throughput screening system will serve as a powerful tool for exploring new and beneficial mutations responsible for regulating copolymer composition of PHA.
- 公益社団法人日本生物工学会の論文
著者
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Shimada Daisuke
Department Of Applied Physics Science University Of Tokyo
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TSUGE TAKEHARU
Department of Food Science and Technology, Faculty of Agriculture, Kyushu University
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Taguchi Seiichi
Division Of Biotechnology And Macromolecular Chemistry Graduate School Of Engineering Hokkaido Unive
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Tsuge Takeharu
Department Of Innovative And Engineered Materials Tokyo Institute Of Technology
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Watanabe Yoriko
Department Of Biology Graduate School Of Science Tohoku University
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Taguchi Seiichi
Division Of Biotechnology And Macromolecular Chemistry Graduate School Of Engineering Hokkaido Unive
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Ichinomiya Yousuke
Department of Innovative and Engineered Materials, Tokyo Institute of Technology
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Saika Azusa
Department of Innovative and Engineered Materials, Tokyo Institute of Technology
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Abe Hideki
Bioplastic Research Team, RIKEN Biomass Engineering Program
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Saika Azusa
Department Of Innovative And Engineered Materials Tokyo Institute Of Technology
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Watanabe Yoriko
Department Of Innovative And Engineered Materials Tokyo Institute Of Technology
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Ichinomiya Yousuke
Department Of Innovative And Engineered Materials Tokyo Institute Of Technology
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Abe Hideki
Bioplastic Research Team Riken Biomass Engineering Program
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Shimada Daisuke
Department Of Applied Chemistry Institute Of Science And Engineering Chuo University
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Shimada Daisuke
Department Of Innovative And Engineered Materials Tokyo Institute Of Technology
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