Effects of one amino acid substitutions at the C-terminal region of thermostable L2 lipase from Bacillus sp. L2

The substitutions of the amino acid residue at the predetermined critical point of the Cterminal of L2 lipase may increase its thermostability and lipase activity. N and C-terminal regions in most proteins are often disordered and flexible. However, some protein function was closely related to flexibility as well as play a role in the enzyme reaction. The critical point of the stability of L2 lipase structure was predicted at position 385 (wild type residue Serine) of the L2 sequence based on I-Mutant2.0 software. The effects of substitution of the amino acids at the critical point with Glutamic acid, Isoleucine, and Valine were analyzed with Molecular Dynamics (MD) simulation by using Yet Another Scientific Artificial Reality Application (YASARA) software and it showed that the best predicted mutant L2 lipases had lower RMSD value as compared to L2 lipase. It indicated that the three mutants had higher compactness in the structure consequently enhancing the stability. From RMSF analysis, mutations had reduced the flexibility of the enzyme. The best predicted mutants (S385E, S385I, and S385V) were produced in the experimental lab by site-directed mutagenesis. The mutant L2 lipases (60.4 kDa) were purified to homogeneity by a single chromatography step before proceeding with characterization. There were high lipase activities produced by purified mutant L2 lipases at a temperature range of 60-85 °C with the optimum temperature of 80 ºC, 75 °C and 70 °C for S385E, S385V, and S385I lipases respectively. The optimum temperature for recombinant L2 lipase was at 70 °C. Mutant L2 lipases (S385E and S385V) had higher optimum temperature compared to recombinant L2 lipase. The optimum pH for mutant L2 lipases (S385E and S385V) was found to be at pH 8 and for S385I was at pH 9, whereas the optimum pH for recombinant L2 lipase was at pH 9. S385I lipase was more thermostable as compared to recombinant L2 lipase and other mutants at temperature 60 °C within 16 hours preincubation. The stability of S385V lipase in varies organic solvents was higher as compared to recombinant L2 lipase. S385V lipase had relative activities higher than 100% which 111% in DMSO, 105% in acetone, 123% in diethyl ether and 124% in nhexane. Tm values for S385V and S385E lipases were at 85.96 °C and 84.85 °C and the values were higher as compared to recombinant L2 lipase which is only 66.73 °C. This showed the higher thermal stability of S385E and S38V lipases as compared to recombinant L2 lipase. Thus, the substitutions at the predetermined critical point of the Cterminal (Ser385) changed the functionality of the protein structure towards the activity, stability, and flexibility of L2 lipase. The critical point mutation towards the structure of L2 lipase provided a very advantageous strategy for the improvement of enzyme with better function to adapt with harsh environment.

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