title: [Enhanced thermostability of Rhizopus chinensis lipase by error-prone PCR].
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.
authors.forename: Rui
authors.initials: R
authors.lastname: Wang
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Yu
authors.forename: Yan
authors.initials: Y
authors.lastname: Xu
mesh_terms: D003001:Cloning, Molecular; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics*; D006358:Hot Temperature; D007218:Industrial Microbiology; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D015202:Protein Engineering / Q000379:methods; D012233:Rhizopus / Q000201:enzymology*
chemical_list: D008049:Lipase