title: Engineering of a metagenome derived lipase toward thermal tolerance: effect of asparagine to lysine mutation on the protein surface.
abstract: A highly thermostable mutant lipase was generated and characterized. Mutant enzyme demonstrated 144 fold enhanced thermostability over the wild type enzyme at 60°C. Interestingly, the overall catalytic efficiency (k(cat/)K(m)) of mutant was also enhanced (~20 folds). Circular dichroism spectroscopy, studied as function of temperature, demonstrated that the mutant lipase retained its secondary structure up to 70-80°C, whereas wild type protein structure was completely distorted above 35°C. Additionally, the intrinsic tryptophan fluorescence (a probe for the tertiary structure) also displayed difference in the conformation of two enzymes during temperature dependent unfolding. Furthermore, mutation N355K resulted in extensive H-bonding (Lys355 HZ1OE2 Glu284) with a distance 2.44 Å. In contrast to this, Wt enzyme has not shown such H-bonding interaction.
authors: Department of Biotechnology, Sector 14, Panjab University, Chandigarh 160014, India.
authors: Pushpender Kumar
authors: PK
authors: Sharma
authors: Rajender
authors: R
authors: Kumar
authors: Rakesh
authors: R
authors: Kumar
authors: Owais
authors: O
authors: Mohammad
authors: Ranvir
authors: R
authors: Singh
authors: Jagdeep
authors: J
authors: Kaur
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine / Q000235:genetics; D002942:Circular Dichroism; D004795:Enzyme Stability; D008049:Lipase / Q000235:genetics*; D008239:Lysine / Q000235:genetics; D054892:Metagenome; D011487:Protein Conformation; D017510:Protein Folding; D013696:Temperature
chemical_list: D001216:Asparagine; D008049:Lipase; D008239:Lysine