title: Thirty-degree shift in optimum temperature of a thermophilic lipase by a single-point mutation: effect of serine to threonine mutation on structural flexibility.
abstract: In order to understand the molecular basis of cold adaptation, we have used directed evolution to transform a thermophilic lipase LipR1 into its psychrophilic counterpart. A single round of random mutagenesis followed by screening for improved variants yielded a mutant with single-point mutation LipR1M1 (S130T), with optimum activity at 20 °C. Its activity at 50 °C is only 20% as compared to wild type (100%). It showed catalytic rate constant (kcat) 3 times higher and a catalytic efficiency (kcat/Km) 4 times that of wild type. Circular dichroism and fluorescence studies also supported our observation of mutant structural flexibility. Structure analysis using homology models showed that Threonine 130 is exposed to solvent and has lost H-bond interaction with neighboring amino acid, thereby increasing flexibility of this lipase structure.
authors.affiliation: Department of Biotechnology, Sector 25, Panjab University, Chandigarh, 160014, India.
authors.forename: Monika
authors.initials: M
authors.lastname: Sharma
authors.affiliation: Department of Biotechnology, Sector 25, Panjab University, Chandigarh, 160014, India.|Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Kumar
authors.affiliation: Department Cum 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 25, Panjab University, Chandigarh, 160014, India. jagsekhon@yahoo.com.
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
mesh_terms: D019020:Directed Molecular Evolution; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D008049:Lipase / Q000737:chemistry* / Q000235:genetics*; D017354:Point Mutation
chemical_list: D008049:Lipase
keywords: Circular dichroism; Cold adaptation; Directed evolution; Lipase