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 (Western Pacific)
| ID: wpr-242456
Responsible library:
WPRO
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.
Full text:
Available
Database:
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
Document type:
Article