Salt‐activation of nonhydrolase enzymes for use in organic solvents

Abstract

Enzymatic reactions are important for the synthesis of chiral molecules. One factor limiting synthetic applications of enzymes is the poor aqueous solubility of numerous substrates. To overcome this limitation, enzymes can be used directly in organic solvents; however, in nonaqueous media enzymes usually exhibit only a fraction of their aqueous-level activity. Salt-activation, a technique previously demonstrated to substantially increase the transesterification activity of hydrolytic enzymes in organic solvents, was applied to horse liver alcohol dehydrogenase, soybean peroxidase, galactose oxidase, and xanthine oxidase, which are oxidoreductase and oxygenase enzymes. Assays of the lyophilized enzyme preparations demonstrated that the presence of salt protected enzymes from irreversible inactivation. In organic solvents, there were significant increases in activity for the salt-activated enzymes compared to nonsalt-activated controls for every enzyme tested. The increased enzymatic activity in organic solvents was shown to result from a combination of protection against inactivation during the freeze-drying process and other as-yet undetermined factors.

Protein-coated Microcrystals for use in Organic Solvents: Application to Oxidoreductases

“…The transesterification activity of subtilisin Carlsberg-PCMC in acetonitrile was also three orders of magnitude higher when compared to enzyme powders as received (Kreiner et al 2001, Kreiner & Parker 2004. In contrast, the effect of salt activation on oxidoreductases was moderate (4À50 fold) compared to the control that contained no salt (Morgan & Clark 2004), and as such in a similar range as obtained with oxidoreductases in the PCMC form. Co-lyophilisation of peroxidases with common lyoprotectants and other compounds that presumably bind to the hydrophobic pocket of the enzyme active site could also increase the catalytic activity.…”

“…The method by Morgan & Clark (2004) was adapted as follows: SBP (2À10 mg) as received or in PCMC form, was suspended in 2959 ll dry TBME, containing indole (10 mM). The reaction was started by addition of 41 ll tert-butylhydrosperoxide (t-BuOOH) (from a 0.73 M stock solution in TBME, freshly made before running the reactions).…”

Reflections on Charge State Distributions, Protein Structure, and the Mystical Mechanism of Electrospray Ionization

“…This further supports retention of protein structure as being important for successful catalysis. In addition, experiments have shown that the addition of salts or crown ethers to solution prior to lyophilization greatly enhances catalytic activity [14,15]. There are multiple potential explanations for this observation.…”

Horseradish and soybean peroxidases: comparable tools for alternative niches?

“…The major problems of 278 substrate solubility and unwanted side reactions promoted by water are also overcome 279 during organic solvent based synthesis. Additionally, in some anhydrous solvents 280 peroxidase (HRP and SBP) activity was actually increased [75], with additional 281 methods, such as salt activation [76] and excipient aided lyophilisation [77] also 282 resulting in increased peroxidase activity. However, in some low water solvents, 283 peroxidases can lose their confirmational structure [78]; although recent advances in 284 peroxidase encapsulation in amphiphilic matrices [79], the use of reverse micelles 285 [80] and oil emulsions [81] allow for peroxidase activity in an extended range of 286 anhydrous solvents.…”