Institution: | a Department of Biotechnology, Sector 14, Panjab University, Chandigarh 160014, Indiab Indian Institute of Science Education and Research (IISER) Mohali, S.A.S. Nagar, Sector 81, Mohali-140306, Punjab, Indiac Computer Centre, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar 160062, Punjab, Indiad Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, Indiae National Centre for Human Genome Studies and Research (NCHGSR) Panjab University Chandigarh, 160014, India |
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 (kcat/Km) 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. |