Sharma_2012_Gene_491_264

A highly thermostable mutant lipase was generated and characterized. Mutant enzyme demonstrated 144 fold enhanced thermostability over the wild type enzyme at 60 degrees 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 degrees C, whereas wild type protein structure was completely distorted above 35 degrees 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 A. In contrast to this, Wt enzyme has not shown such H-bonding interaction.