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

Abstract

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 byproduct of hydrolysis were leading to protonation of the enzyme, resulting in rapid loss of activity. Addition of solidstate 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.

Synthesis of propyl gallate by mycelium-bound tannase from Aspergillus niger in organic solvent

“…In solvents of low dielectric constant, opposite charges interact more strongly, as there is less shielding between them. Therefore, counterions are more closely associated with their opposite charges and their presence must be taken into consideration in determining the protonation state of an enzyme (Harper et al 2000). Researchers have reported that solid-state acid-base buffers added in organic media could control enzyme activity (Dolman et al 1997;Zacharis et al 1997).…”

Enzymic Conversions in Organic and Other Low‐Water Media

“…Again, to cover the range of acid-base conditions that might be appropriate for different enzymic syntheses, a series of different buffer pairs is required. A number have been identified [32][33][34], but the known range probably needs extending.…”

Organic-phase biological buffers for biochemical and biological research in organic media

“…These organo-soluble buffers, although limited in number, effectively erase the enzyme's pH memory and control its protonation state [10]. Solid-state acid-base buffers have been also proposed for use in organic media [10][11][12][13][14]. These solid buffers consist of a zwitterionic compound (Good's buffers among the others) and its sodium salt.…”

“…These solid buffers consist of a zwitterionic compound (Good's buffers among the others) and its sodium salt. Although these buffers were able to control the ionization state of the catalytic triad of subtilisin in organic solvents [13], they failed with papain [10].…”