Computer-Aided Optimization of Site-Directed Mutagenesis of Bacillus stearothermphilus Neutral Protease for Improving Thermostability

摘要

A graphical slution using Random-Centroid Optimization (RCO) was successful in finding the global optimum in multimodal functions. The RCO program was modified to apply to site-directed mutagenesis (RCG). This RCG program was used to mutate one site at-a-time in the active-site helix region (G139-Y154) of B. stearothermophilus neutral protease to improve thermostability. Mutant V143E increased the halfsurvival temperature (T_(50)) by 6.3 deg C (T_(50) of the wild-type enzyme = 68.3 deg C)) after 13 mutations. Meanwhile by the proline mutant (I140P) increased the T_(50) by 7.5 deg C. The increase in stability in both mutations was explained by molecular rigidification. Mutant I140P also increased the yield of themolysin-mediated synthesis of Z-L-Asp-Met-OMe by a factor of 5. The RCG program written for mutating up to 3 sites may be useful in elucidating the structure-function relationships of proteins.