Acid-Base Control for Biocatalysis in Organic Media: New Solid-State Proton/Cation Buffers and an Indicator

• Published in: Chemistry : a European journal 2000-06, Vol.6 (11), p.1923-1929
• Main Authors: Harper, Neil, Dolman, Mark, Moore, Barry D., Halling, Peter J.
• Format: Article
• Language: English
• Subjects: buffers; enzyme catalysis; protonations; solid-state chemistry; solvent effects
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/1521-3765(20000602)6:11<1923::AID-CHEM1923>3.0.CO;2-T

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 zwitterion and its sodium salt, are able to set and maintain the ionisation state of an enzyme by the exchange of H+ and Na+ ions. Surprisingly, equilibrium is established between the different solid components quickly enough to provide a practical means of controlling acid‐base conditions during biocatalysed reactions. We developed an organosoluble chromoionophore indicator to screen the behaviour of possible buffer pairs and quantify their relative H+/Na+ exchange potential. The transesterification activity of an immobilised 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 maximum water activities accessible without formation of hydrates or solutions of the buffers are reported here. The indicator was also used to monitor, for the first time in situ, changes in the acid‐base conditions of an enzyme‐catalysed transesterification reaction in toluene. We found that even very minor amounts of an acidic by‐product of hydrolysis were leading to protonation of the enzyme, resulting in rapid loss of activity. Addition of solid‐state buffer was able to prevent this process, shortening reaction times and improving yields. Solid‐state buffers offer a general and inexpensive way of precisely controlling acid‐base conditions in organic solvents and thus also have potential applications outside of biocatalysis. Solid acid‐base buffers can be made from zwitterions containing an amino and sulfonic acid group and their sodium salts suspended in organic solvents. These crystalline buffers were shown to be able to control the ionisation state, and thereby the enzymatic activity, of subtilisin Carlsberg suspended in toluene [Eq. (1)].