SOLVENT EFFECTS ON THE CATALYTIC ACTIVITY OF SUBTILISIN SUSPENDED IN ORGANIC-SOLVENTS

We studied a model transesterification reaction catalyzed by subtilisi n Carlsberg suspended in toluene, n-hexane, diisopropyl ether, and mix tures of these solvents. To account for solvent effects due to differe nces in water partitioning between the enzyme and the bulk solvents, w e measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obt ained bell-shaped curves with maxima at the same enzyme hydration in a ll the solvents. However, the activity maxima were different in all th e media, being the lowest in toluene. Differences in the partitioning of substrates and product between the bulk solvent phase and the enzym e active site were accou nted for but could not explain the lower cata lytic activity observed in toluene. The fact that toluene is very simi lar to one of the substrates suggested the possibility of competitive inhibition by this solvent. We derived a model allowing for difference s in solvation of the substrates, by using thermodynamic activities in stead of concentrations, as well as for competitive inhibition by tolu ene. The model fit the experimental data well, confirming that toluene had a direct adverse effect on the catalytic activity of the enzyme. (C) 1996 John Wiley & Sons, Inc.