Biotechnological Products and Process Engineering

Applied Microbiology and Biotechnology

, Volume 92, Issue 3, pp 509-517

Chemo-enzymatic synthesis of polyhydroxyalkanoate (PHA) incorporating 2-hydroxybutyrate by wild-type class I PHA synthase from Ralstonia eutropha

  • Xuerong HanAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University
  • , Yasuharu SatohAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University
  • , Toshifumi SatohAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University
  • , Ken’ichiro MatsumotoAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University
  • , Toyoji KakuchiAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University
  • , Seiichi TaguchiAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University
  • , Tohru DairiAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University Email author 
  • , Masanobu MunekataAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University
  • , Kenji TajimaAffiliated withDivision of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University Email author 

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Abstract

A previously established improved two-phase reaction system has been applied to analyze the substrate specificities and polymerization activities of polyhydroxyalkanoate (PHA) synthases. We first analyzed the substrate specificity of propionate coenzyme A (CoA) transferase and found that 2-hydroxybutyrate (2HB) was converted into its CoA derivative. Then, the synthesis of PHA incorporating 2HB was achieved by a wild-type class I PHA synthase from Ralstonia eutropha. The PHA synthase stereoselectively polymerized (R)-2HB, and the maximal molar ratio of 2HB in the polymer was 9 mol%. The yields and the molecular weights of the products were decreased with the increase of the (R)-2HB concentration in the reaction mixture. The weight-average molecular weight of the polymer incorporating 9 mol% 2HB was 1.00 × 105, and a unimodal peak with polydispersity of 3.1 was observed in the GPC chart. Thermal properties of the polymer incorporating 9 mol% 2HB were analyzed by DSC and TG-DTA. T g, T m, and T d (10%) were observed at −1.1°C, 158.8°C, and 252.7°C, respectively. In general, major components of PHAs are 3-hydroxyalkanoates, and only engineered class II PHA synthases have been reported as enzymes having the ability to polymerize HA with the hydroxyl group at C2 position. Thus, this is the first report to demonstrate that wild-type class I PHA synthase was able to polymerize 2HB.

Keywords

Polyhydroxyalkanoate (PHA) Improved two-phase reaction system (iTPRS) 2-Hydroxybutyrate (2HB) Ralstonia eutropha Class I PHA synthase