A carboxylic acid functionalized dendritic polybenzyl ether has been synthesized and used with its sodium salt to generate a novel acid/base buffer soluble in nonpolar organic solvents. The effect of different ratios of the two buffer forms on the catalytic activity of subtilisin Carlsberg and chymotrypsin was investigated in toluene. It was found that reproducible transesterification rates were obtained at each molar ratio consistent with a buffering effect. As the molar ratio of the sodium salt to acid was increased there was a corresponding increase in the catalytic activity of both enzymes although their profiles were not identical. This is consistent with a requirement for deprotonation of a residue at active site of the enzyme as observed in aqueous solution. The ability to alter and precisely control the ionization state of biocatalysts in nonpolar solvents may find useful applications for both fundamental studies and in syntheses where reactants or products have acid/base properties.