Dramatic enhancement of enzymatic activity in organic solvents by lyoprotectants

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Abstract
When seven different hydrolytic enzymes (four proteases and three lipases) were lyophilized from aqueous solution containing a ligand, N‐Ac‐L‐Phe‐NH2, their catalytic activity in anhydrous solvents was far greater (one to two orders of magnitude) than that of the enzymes lyophilized without the ligand. This ligand‐induced activation was expressed regardless of whether the substrate employed in organic solvents structurally resembled the ligand. Furthermore, nonligand lyoprotectants [sorbitol, other sugars, and poly(ethylene glycol)] also dramaticaliy enhanced enzymatic activity in anhydrous solvents when present in enzyme aqueous solution prior to lyophilization. The effects of the ligand and of the lyoprotectants were nonadditive, suggesting the same mechanism of action. Excipient activated and nonactivated enzymes exhibited identical activities in water. Also, addition of the excipients directly to suspensions of nonactivated enzymes in organic solvents had no appreciable effect on catalytic activity. These observations indicate that the mechanism of the excipient‐induced activation is based on the ability of the excipients to alleviate reversible denaturation of enzymes upon lyophilization. Activity enhancement induced by the excipients is displayed even after their removal by washing enzymes with anhydrous solvents. Subtilisin Carlsberg, lyophilized with sorbitol, was found to be a much more efficient practical catalyst than its “regular” counterpart. © 1993 John Wiley & Sons, Inc.