Applied Microbiology and Biotechnology

, Volume 94, Issue 2, pp 365–376

In vitro synthesis of polyhydroxyalkanoate (PHA) incorporating lactate (LA) with a block sequence by using a newly engineered thermostable PHA synthase from Pseudomonas sp. SG4502 with acquired LA-polymerizing activity

  • Kenji Tajima
  • Xuerong Han
  • Yasuharu Satoh
  • Ayako Ishii
  • Yuji Araki
  • Masanobu Munekata
  • Seiichi Taguchi
Biotechnological products and process engineering

DOI: 10.1007/s00253-011-3840-z

Cite this article as:
Tajima, K., Han, X., Satoh, Y. et al. Appl Microbiol Biotechnol (2012) 94: 365. doi:10.1007/s00253-011-3840-z

Abstract

Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 °C, as a source of thermostable enzymes. In this study, we cloned a pha locus from the bacterium and identified two genes encoding PHA synthases (PhaC1SG and PhaC2SG). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1SG to evaluate the potential of the resulting protein as a “thermostable LPE”. The mutated PhaC1SG [PhaC1SG(STQK)] showed high thermal stability in synthesizing P(LA-co-3HB) in an in vitro reaction system under a range of high temperatures. Requirement of 3HBCoA as a priming unit for LA polymerization by the LPE has been suggested in both of the in vitro and in vivo experiments. Based on the finding, the PhaC1SG(STQK)-mediated synthesis of a LA-based copolymer with a block sequence was achieved in the in vitro system by sequential feeding of the corresponding two substrates. This in vitro reaction system using the thermostable LPE provides us with a versatile way to synthesize the various types of LA-based copolymers with desired sequence patterns, random or block, depending on the way of supplying hydroxyalkanoates (mixed or sequential feeding).

Keywords

Polyhydroxyalkanoate (PHA) Thermal stability Block copolymer LA-polymerizing enzyme (LPE) In vitro polymerization Pseudomonas sp. SG4502 

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Kenji Tajima
    • 1
  • Xuerong Han
    • 1
  • Yasuharu Satoh
    • 1
  • Ayako Ishii
    • 1
  • Yuji Araki
    • 1
  • Masanobu Munekata
    • 1
  • Seiichi Taguchi
    • 1
  1. 1.Division of Biotechnology and Macromolecular Chemistry, Faculty of EngineeringHokkaido UniversitySapporoJapan

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