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.
Copyright © 2011 Elsevier B.V. All rights reserved.