Towards more active biocatalysts in organic media: Increasing the activityof salt-activated enzymes

The activation of freeze-dried subtilisin Carlsberg (SC) in hexane has been systematically studied and partially optimized with respect to the freezin g method, the addition of inorganic salts and lyoprotectants, the initial c oncentration and final weight percent of additives, and the amount of water added to the organic solvent. Activity and water content were found to cor relate directly with the kosmotropicity of the activating salt (kosmotropic salts bind water molecules strongly relative to the strength of water-wate r interactions in bulk solution). Combinations of kosmotropic salts with kn own lyoprotectants such as poly(ethylene glycol) (PEG) and sugars did not y ield an appreciably more active catalyst. However, the combination of the k osmotropic sodium acetate with the strongly buffering sodium carbonate acti vated the enzyme more than the individual additives alone. Enzyme activity was enhanced further by the addition of small amounts of water to the organ ic solvent. Under optimal conditions, enzyme activity in hexane was improve d over 27,000-fold relative to the salt-free enzyme, reaching a catalytic e fficiency that was within one order of magnitude of k(cat)/K-m for hydrolys is of the same substrate in aqueous buffer. Further activation to attain ev en higher catalytic efficiencies may be possible with additional optimizati on. (C) 2001 John Wiley & Sons, Inc.