The Effects of Organic Solvents on Wild-Type and Mutant Subtilisin-Catalyzed Hydrolyses

The kinetic parameters, kcat and KM, for the hydrolysis of N-α-tosyl-L-arginine methyl ester (1, TAME) by the wild-type subtilisins Carlsberg and BPN′ as well as the BPN′ mutants Glyl66Ser, GLyl66Asn, and Met222Phe, were determined in the presence of 5 and 15% (v/v) of a selection of water-soluble organic solvents. The goals were to compare and evaluate the solvent effects with a view to expanding their use in organic synthetic applications of the WT and mutant subtilisins. The results showed that subtilisin BPN′ and its mutants were much less affected by organic solvents than subtilisin Carlsberg. The BPN′ mutant Met222Phe demonstrated the greatest resistance to cosolvent inactivation, making it a particularly attractive mutant for peptide synthesis. Dimethyl sulfoxide, acetone, and branched alcohols were found to be the most benign solvents, whereas dioxane, THF, and N-methyl-2-pyrrolidinone seriously reduced catalytic activities, even at low concentrations. The results parallel the solvent-effect data available for other proteinases, including α-chymotrypsin.