title: Point mutation Gln121-Arg increased temperature optima of Bacillus lipase (1.4 subfamily) by fifteen degrees.
abstract: Small molecular weight Bacillus lipases are industrially attractive because of its alkaline optimum pH, broad substrate specificity and production in high yield by overexpression both in Escherichia coli and Bacillus subtilis. Its major limitation of being mesophilic in nature is constantly targeted by laboratory evolution studies. Herein metagenomically isolated Bacillus LipJ was randomly evolved by error prone PCR and library of variants were screened for enhanced thermostability. Point mutant Gln121Arg was extensively characterized and it showed dramatic shift of Temp. opt to 50°C compared to 37°C for parent enzyme. Thermostability studies at 45°C and 50°C determined six fold increase in half life for point variant Gln121Arg compared to LipJ. Circular dichroism (CD) and tryptophan fluorescence study established enhanced thermostability of Gln121Arg. Specific activity of point variant Gln121Arg was comparable to wild type with increased substrate affinity (Km reduced). Reduced kcat for variant Gln121Arg infer that kinetic and catalytic efficiency of mutant was compromised. Structural implications by homolog modelling predicted Gln121 to be placed within longest loop of the structure at surface. Localization of loop due to additional polar interactions by Arg121 to protein core defines molecular basis of enhanced thermostability of random point variant Gln121Arg.
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh 160014, India. Electronic address: shelly.sahni@rediffmail.com.
authors.forename: Shelly
authors.initials: S
authors.lastname: Goomber
authors.affiliation: Department of Microbiology and Cell Biology, Indian Institute Of Science, Bangalore, Karnatka 560012, 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: National Institute of Malaria Research, Dwarka, New Delhi, Delhi 110077, India.
authors.forename: Neelima
authors.initials: N
authors.lastname: Mishra
authors.affiliation: Department of Biotechnology, Panjab University, Chandigarh 160014, India.
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
mesh_terms: D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D007700:Kinetics; D008049:Lipase / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D008049:Lipase
keywords: Directed evolution; Error prone PCR; Homolog modelling; Lipase; Thermostability