pH Control of the Catalytic Activity of Cross-Linked Enzyme Crystals in Organic Solvents

Abstract

Lyophilized subtilisin Carlsberg exhibits “pH memory” in organic solvents, i.e., its catalytic activity in such media is profoundly affected by the pH of the aqueous solution from which the enzyme was lyophilized. In contrast, cross-linked crystalline subtilisin displays no appreciable pH memory. This disparate behavior is rationalized in terms of the different protonation states of the two enzyme preparations in organic solvents. Since cross-linked crystals (CLCs) of enzymes in organic solvents offer performance and mechanistic advantages over their lyophilized counterparts, the opportunities for pH maximizing their catalytic activity in such media are explored. It is found that subtilisin CLCs in pentanone can be activated more than 100-fold by adding a “buffer” consisting of a mixture of a suitable acid and its sodium salt (conjugate base). The maximal activation is achieved (i) when the pKa value of the buffer acid in water lies in the appropriate range, (ii) at an optimal acid/base composition of the buffer mixture, and (iii) when the buffer mixture is present in a sufficient concentration.