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Enhanced thermostability of Rhizopus chinensis lipase by error-prone PCR / 生物工程学报
Chinese Journal of Biotechnology ; (12): 1753-1764, 2013.
Article in Chinese | WPRIM | ID: wpr-242456
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.
Subject(s)
Full text: Available Index: WPRIM (Western Pacific) Main subject: Pichia / Rhizopus / Enzyme Stability / Industrial Microbiology / Protein Engineering / Chemistry / Polymerase Chain Reaction / Cloning, Molecular / Directed Molecular Evolution / Genetics Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2013 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Main subject: Pichia / Rhizopus / Enzyme Stability / Industrial Microbiology / Protein Engineering / Chemistry / Polymerase Chain Reaction / Cloning, Molecular / Directed Molecular Evolution / Genetics Language: Chinese Journal: Chinese Journal of Biotechnology Year: 2013 Type: Article