title: Characterization and evolution of a metagenome-derived lipase towards enhanced enzyme activity and thermostability.
abstract: In the present investigation, we used directed evolution approach to engineer a lipase from metagenomic origin. A variant S311C, was generated, characterized in detail and compared with wild type. Wild type and variant lipases were overexpressed and purified to homogeneity. The temperature optima of the purified lipases (Variant and wild type) were almost same, and found to be 45 and 50 °C, respectively. The variant protein was highly thermostable (54 times) as compared with the wild type at 60 °C. The variant displayed very high kinetic efficiency over the wild type protein. Analysis of the homology models of wild type and variant lipase showed that the substitution is on the surface of the protein. This substitution, along with hydrophobic residues in near vicinity may be involved in formation of strong hydrophobic channel leading to active site. This study identifies the role of hydrophobic interactions in protein stability along with enhancement of enzyme activity.
authors.affiliation: Department of Biotechnology, Sector 14, Panjab University, Chandigarh 160014, India.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Kumar
authors.forename: Monika
authors.initials: M
authors.lastname: Sharma
authors.forename: Ranvir
authors.initials: R
authors.lastname: Singh
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D020134:Catalytic Domain; D003001:Cloning, Molecular; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D054892:Metagenome; D008958:Models, Molecular; D012988:Soil Microbiology; D040681:Structural Homology, Protein
chemical_list: D001426:Bacterial Proteins; D008049:Lipase