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Engineering of a metagenome derived lipase toward thermal tolerance: effect of asparagine to lysine mutation on the protein surface
Authors:Sharma Pushpender Kumar  Kumar Rajender  Kumar Rakesh  Mohammad Owais  Singh Ranvir  Kaur Jagdeep
Institution:
  • a Department of Biotechnology, Sector 14, Panjab University, Chandigarh 160014, India
  • b Indian Institute of Science Education and Research (IISER) Mohali, S.A.S. Nagar, Sector 81, Mohali-140306, Punjab, India
  • c Computer Centre, National Institute of Pharmaceutical Education and Research (NIPER), Sector-67, S.A.S. Nagar 160062, Punjab, India
  • d Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India
  • e 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 HZ1single bondOE2 Glu284) with a distance 2.44 Å. In contrast to this, Wt enzyme has not shown such H-bonding interaction.
    Keywords:CD  Circular dichroism  IPTG  Isopropyl-beta-thio-galactopyranoside  PMSF  Phenylmethylsulphonyl fluoride  DEPC  Diethyl pyrocarbonate  pNP  Para-nitrophenyl  SDS  Sodium dodecyl sulfate  PDB  Protein database  NCBI  National Centre for Biotechnology Information  BLAST  Basic Local Alignment Search Tools  SAVES  Structural Analysis and Verification Server  ORF  Open reading frame  OD  Optical density  3D  Three dimensional  Asn (N)  Asparagine  Lys (K)  Lysine
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