Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 94, Issue 2, Pages 365-376Publisher
SPRINGER
DOI: 10.1007/s00253-011-3840-z
Keywords
Polyhydroxyalkanoate (PHA); Thermal stability; Block copolymer; LA-polymerizing enzyme (LPE); In vitro polymerization; Pseudomonas sp SG4502
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Funding
- Global COE Program [B01]
- Ministry of Education, Culture, Sports, Science, and Technology, Japan [21310060, 21760632]
- Regional Innovation Cluster Program (Global Type)
- Northern Advancement Center for Science & Technology (NOASTEC)
- Grants-in-Aid for Scientific Research [21310060, 24651090, 23310059, 21760632] Funding Source: KAKEN
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Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55 A degrees 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 (PhaC1(SG) and PhaC2(SG)). Two mutations, Ser324Thr and Gln480Lys, corresponding to those of a lactate (LA)-polymerizing enzyme (LPE) from mesophilic Pseudomonas sp. 61-3 were introduced into PhaC1(SG) to evaluate the potential of the resulting protein as a thermostable LPE. The mutated PhaC1(SG) [PhaC1(SG)(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 PhaC1(SG)(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).
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