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Engineering of class I lactate-polymerizing polyhydroxyalkanoate synthases from Ralstonia eutropha that synthesize lactate-based polyester with a block nature.
MedLine Citation:
PMID:  22801709     Owner:  NLM     Status:  Publisher    
Class I polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (PhaC(Re)) was engineered so as to acquire an unusual lactate (LA)-polymerizing activity. To achieve this, the site-directed saturation mutagenesis of PhaC(Re) was conducted at position 510, which corresponds to position 481 in the initially discovered class II LA-polymerizing PHA synthase (PhaC1(Ps)STQK), a mutation in which (Gln481Lys) was shown to be essential to its LA-polymerizing activity (Taguchi et al., Proc Natl Acad Sci USA 105(45):17323-17327, 2008). The LA-polymerizing activity of the PhaC(Re)A510X mutants was evaluated based on the incorporation of LA units into the P[3-hydroxybutyrate(3HB)] backbone in vivo using recombinant Escherichia coli LS5218. Among 19 PhaC(Re)(A510X) mutants, 15 synthesized P (LA-co-3HB), indicating that the 510 residue plays a critical role in LA polymerization. The polymer synthesized by PhaC(Re)A510S was fractionated using gel permeation chromatography in order to remove the low molecular weight fractions. The (13)C and (1)H NMR analyses of the high molecular weight fraction revealed that the polymer was a P(7 mol% LA-co-3HB) copolymer with a weight-averaged molecular weight of 3.2 × 10(5) Da. Interestingly, the polymer contained an unexpectedly high ratio of an LA-LA*-LA triad sequence, suggesting that the polymer synthesized by PhaC(Re) mutant may not be a random copolymer, but presumably had a block sequence.
Anna Ochi; Ken'ichiro Matsumoto; Takashi Ooba; Kohei Sakai; Takeharu Tsuge; Seiichi Taguchi
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-7-17
Journal Detail:
Title:  Applied microbiology and biotechnology     Volume:  -     ISSN:  1432-0614     ISO Abbreviation:  -     Publication Date:  2012 Jul 
Date Detail:
Created Date:  2012-7-17     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  8406612     Medline TA:  Appl Microbiol Biotechnol     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Division of Biotechnology and Molecular Chemistry, Graduate School of Engineering, Hokkaido University, N13-W8, Kita-ku, Sapporo, 060-8628, Japan.
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