Acid-base control for biocatalysis in organic media: New solid-state proton/cation buffers and an indicator

Although great care is generally taken to buffer aqueous enzyme reactions, active control of acid-base conditions for biocatalysis in low-water media is rarely considered. Here we describe a new class of solid-state acid-base buffers suitable for use in organic media. The buffers, composed of a zwit terion and its sodium salt, are able to set and maintain the ionisation sta te of an enzyme by the exchange of H+ and Na+ ions. Surprisingly, equilibri um is established between the different solid components quickly enough to provide a practical means of controlling acid-base conditions during biocat alysed reactions. We developed an organosoluble chromoionophore indicator t o screen the behaviour of possible buffer pairs and quantify their relative H+/Na+ exchange potential. The transesterification activity of an immobili sed protease, subtilisin Carlsberg, was measured in toluene in the presence of a range of buffers. The large observed difference in rates showed good correlation with that expected from the measured exchange potentials. The m aximum water activities accessible without formation of hydrates or solutio ns of the buffers are reported here. The indicator was also used to used to monitor, for the first time in situ, changes in the acid-base conditions o f an enzyme-catalysed transesterification reaction in toluene. We found tha t even very minor amounts of an acidic byproduct of hydrolysis. were leadin g to protonation of the enzyme, resulting in rapid loss of activity. Additi on of solid-state buffer was able to prevent this process,shortening reacti on times and improving yields. Solid-state buffers offer a general and inex pensive way of precisely controlling acid-base conditions in organic solven ts and thus also have potential applications outside of biocatalysis.