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
| Autor: | Xuerong Han, Masanobu Munekata, Kenji Tajima, Yuji Araki, Seiichi Taguchi, Yasuharu Satoh, Ayako Ishii |
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| Rok vydání: | 2012 |
| Předmět: |
Hot Temperature
Block copolymer Applied Microbiology and Biotechnology Polyhydroxyalkanoates LA-polymerizing enzyme (LPE) In vivo Pseudomonas Enzyme Stability Point Mutation Polyhydroxyalkanoate (PHA) Lactic Acid Cloning Molecular Gene chemistry.chemical_classification biology Thermal stability General Medicine biology.organism_classification Recombinant Proteins In vitro Enzyme Amino Acid Substitution chemistry Biochemistry In vitro polymerization Mutant Proteins Pseudomonas sp. SG4502 Acyltransferases Bacteria Biotechnology Mesophile |
| Zdroj: | Applied Microbiology and Biotechnology. 94:365-376 |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | Recently, we succeeded in isolating a thermotolerant bacterium, Pseudomonas sp. SG4502, which is capable of accumulating polyhydroxyalkanoate (PHA) even at 55℃, 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). |
| Databáze: | OpenAIRE |
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