Solid-state proton/sodium buffers: “chemical pH stats” for biocatalysts in organic solvents

Abstract

The useful application of enzymes in organic synthesis requires reliable and straightforward methods for maximising efficiency and selectivity. Here we describe how to control the protonation state of enzymes using a new class of solid-state buffers, applied for the first time in polar organic solvents. Remarkably these insoluble buffers are able to rapidly exchange H⁺ and Na⁺ ions with both the protein and reaction mixture as demonstrated here using propanol rinsed enzyme preparations (PREPs) of subtilisin Carlsberg and chymotrypsin. The buffers tested were generally mixtures of a zwitterionic biological buffer and its Na⁺ salt with each buffer pair setting a characteristic fixed ratio of H⁺ activity to Na⁺ activity (a_H+/a_Na+) within the system. Dependent upon the solid buffer pair selected a wide range of different enzymatic activities could be observed. The variation in rate showed a fairly good but not exact correlation with the aqueous pK_a of the buffers, indicating that crystal lattice energies have less affect on acid–base strength than might be expected. The solid-state buffers were able to prevent detrimental changes to enzyme activity caused by the presence or build up of acids or bases in the organic reaction mixture (often undetected). They could also be used advantageously to tune the enzyme protonation state in solvent if a previous aqueous preparation step needs to be carried out at a pH not optimal for catalysis. Such buffering systems are expected to find wide-spread use as ‘chemical pH stats’ for reactions in non-aqueous media.