Enhanced thermostability of Rhizopus chinensis lipase by error-prone PCR.
- Author:
Rui WANG
;
Xiaowei YU
;
Yan XU
- Publication Type:Journal Article
- MeSH:
Cloning, Molecular;
Directed Molecular Evolution;
methods;
Enzyme Stability;
genetics;
Hot Temperature;
Industrial Microbiology;
Lipase;
chemistry;
genetics;
Mutation;
Pichia;
genetics;
metabolism;
Polymerase Chain Reaction;
methods;
Protein Engineering;
methods;
Rhizopus;
enzymology
- From:
Chinese Journal of Biotechnology
2013;29(12):1753-1764
- CountryChina
- Language:Chinese
-
Abstract:
Directed evolution was conducted to improve the thermostability of lipase from Rhizopus chinensis CCTCC M201021. Mutations were introduced by two rounds of error-prone PCR and mutant lipase was selected by fast-blue RR top agar screening. Two positive variants were selected in the first-round and four in the second-round screening process. Ep2-4 was proved as the most thermostable lipase and its DNA sequencing revealed three amino acid substitutions: A129S, P168L and V329A. Compared with the parent, its half-life at 60 degrees C was 5.4- times longer and T50 was 7.8 degrees higher. Purified lipase of Ep2-4 was characterized and the result shows that its thermostability improved without compromising enzyme activity. According to the mimicked protein structure, mutation A129S formed a hydrogen bond with Gln133 and improved the thermostability by increasing the hydrophilicity and polarity of protein; mutation P168L by forming a hydrophobic bond with the nearby Leu164.
