Optimization of site-directed mutagenesis. 2. Application of random-centroid optimization to one-site mutation of Bacillus stearothermophilus neutral protease to improve thermostability

摘要

The random-centroid optimization program was modified for application to site-directed mutagenesis of proteins and renamed random-centroid optimization for genetics (RCG). RCG was used to select both the site in the protein sequence for mutation and the amino acid for substitution. The substituting amino acid was selected on the basis of a hydrophobicity scale. Other indices of amino acids, namely propensities of alpha-helix, beta-strand, and beta-turn as well as bulkiness, were also included in the RCG program. For validating the RCG approach, single-site mutations were conducted in the 16 amino acid residues of the active-site helix (G139-Y154) of Bacillus stearothermophilus neutral protease. After 13 mutations, it was found that the substitution V143E substantially improved the thremostability of the recombinant enzyme. The half-survival temperature of mutant V143E was higher by 6.5 degrees C than the 68.3 degrees C of the wild-type enzyme. Lower hydrophobicity and bulkiness at the N-terminal side of the active region of enzyme were important for maintaining high thermostability as determined from mapping, based on the different amino acid scales. [References: 27]