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- Title
In Vitro Evolution of a Polyhydroxybutyrate Synthase by Intragenic Suppression-Type Mutagenesis1.
- Authors
Taguchi, Seiichi; Nakamura, Hirofumi; Hiraishi, Tomohiro; Yamato, Ichiro; Doi, Yoshiharu
- Abstract
In vitro evolution was applied to obtain highly active mutants of Ralstonia eutropha polyester synthase (PhbCRe), which is a key enzyme catalyzing the formation of polyhy-droxybutyrate (PHB) from (f?)-3-hydroxybutyryl-CoA (3HB-CoA). To search for beneficial mutations for activity improvement of this enzyme, we have conducted multi-step mutations, including activity loss and intragenic suppression-type activity reversion. Among 259 revertants, triple mutant E11S12 was obtained as the most active one via PCR-medi-ated secondary mutagenesis from mutant E11 with a single mutation (Ser to Pro at position 80), which exhibited reduced activity (as low as 27%of the wild-type level) but higher thermostability compared to the wild-type enzyme. Mutant E11S12 exhibited up to 79% of the wild-type enzyme activity. Mutation separation of E11S12 revealed that the replacement of Phe by Ser at position 420 (F420S), located in a highly conserved a⊘p hydrolase fold region, of the E11S12 mutant contributes to the improvement of the enzyme activity. A purified sample of the genetically engineered mutant, termed E11S12-1, with the F420S mutation alone was found to exhibit a 2.4-fold increase in specific activity toward 3HB-CoA, compared to the wild-type.
- Subjects
POLYHYDROXYBUTYRATE; POLYHYDROXYALKANOATES; GENETIC mutation; GENETICS; BIOLOGY
- Publication
Journal of Biochemistry, 2002, Vol 131, Issue 6, p801
- ISSN
0021-924X
- Publication type
Article
- DOI
10.1093/oxfordjournals.jbchem.a003168