title: Combinatorial reshaping of a lipase structure for thermostability: additive role of surface stabilizing single point mutations.
abstract: Thermostable lipases are of high priority for industrial applications. In the present study, targeted improvement of the thermostability of a lipase from metagenomic origin was examined by using a combinatorial protein engineering approach exploring additive effects of single amino acid substitutions. A variant (LipR5) was generated after combination of two thermostabilizing mutations (R214C & N355K). Thermostability of the variant enzyme was analyzed by half-life measurement and circular dichroism (CD). To assess whether catalytic properties were affected by mutation, the optimal reaction conditions were determined. The protein LipR5, displayed optimum activity at 50°C and pH 8.0. It showed two fold enhancement in thermostability (at 60°C) as compared to LipR3 (R214C) and nearly 168 fold enhancement as compared to parent enzyme (LipR1). Circular dichroism and fluorescence study suggest that the protein structure had become more rigid and stable to denaturation. Study of 3D model suggested that Lys355 was involved in formation of a Hydrogen bond with OE1 of Glu284. Lys355 was also making salt bridge with OE2 of Glu284.
authors.affiliation: Department of Biotechnology, Sector 14, Panjab University, Chandigarh 160014, India.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Kumar
authors.affiliation: National Centre for Human Genome Studies and Research, Panjab University, Chandigarh 160014, India.
authors.forename: Ranvir
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Biotechnology, Sector 14, Panjab University, Chandigarh 160014, India. Electronic address: jagsekhon@yahoo.com.
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D054892:Metagenome; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D008049:Lipase
keywords: Circular dichroism; Lipase; Mutagenesis; Salt bridge; Thermostability