Enhancement of catalytic activity of chemically modified subtilisin Carlsberg in benzene by adjustment of lyophilization conditions

Alteration of the lyophilization conditions significantly enhanced not only the activity of insoluble native subtilisin Carlsberg but also that of solubilized enzyme in benzene. The catalytic activity (k(cat)/K-m) for the transesterification reaction of N-acetyl-L-phenylalanine ethyl ester with l-propanol in benzene was enhanced more than 1000 times by the solubilization (100-fold enhancement) and the adjustment of lyophilization conditions (10-fold enhancement). Subtilisin Carlsberg was modified with polyethylene glycol (PEG-Sub) so that it could be solubilized in organic solvents. The effects of pH of the aqueous enzyme solution, of salts, or of a substrate analog in the aqueous solution before the lyophilization process, on the catalytic activity of native subtilisin and PEG-Sub were investigated. The activities of both the native subtilisin and PEG-Sub depended on the pH. The activities of both PEG-Sub and the native subtilisin were enhanced by the presence of salts. The presence of a substrate analog, N-Ac-L-Phe, also enhanced the activity of the native subtilisin and PEG-Sub. These results demonstrated that the organic solvent-solubilized subtilisin was affected by the lyophilization conditions.