Reengineering of subtilisin Carlsberg for oxidative resistance

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Mild bleaching conditions by in situ production of hydrogen peroxide or peroxycarboxylic acid is attractive for pulp, textile, and cosmetics industries. The enzymatic generation of chemical oxidants is often limited by enzyme stability. The subtilisin Carlsberg variant T58A/L216W/M221 is a promiscuous protease that was improved in producing peroxycarboxylic acids. In the current article, we identified two amino acid positions (Trp216 and Met221) that are important for the oxidative resistance of subtilisin Carlsberg T58A/L216W/M221. Site-saturation mutagenesis at positions Trp216 and Met221, which are located close to the active site, resulted in variants M4 (T58/W216M/M221) and M6 (T58A/W216L/M221C). Variants M4 (T58/W216M/M221) and M6 (T58A/W216L/M221C) have a 2.6-fold (M4) and 1.5-fold (M6) increased oxidative resistance and 1.4-fold increased kcat values for peroxycarboxylic acid formation, compared with wild-type subtilisin Carlsberg.

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