Exploiting hysteresis for high activity enzymes in organic media

Abstract

Common methods for removing water from protein preparations such as freeze drying and air drying are shown here to be much more detrimental to enzyme activity than simple rapid dehydration. In a comparison of drying methods for cross-linked enzyme crystals of subtilisin Carlsberg, the highest rates of reaction in organic media were obtained with crystals dried by washing with anhydrous polar solvent. Crystals dried over molecular sieves in air or solvent showed significantly lower activity. In all cases, full activity could be recovered in aqueous buffer. When different solvents were used to dry the enzyme crystals, the catalytic rate was found to vary significantly - longer chain alcohols gave the highest rates while smaller “water-like” solvents such as methanol gave much lower rates. The study was extended to immobilised forms of subtilisin Carlsberg and α-chymotrypsin. When these preparations were dried by solvent washing, high catalytic rates similar to those of crystals prepared by the same procedure were obtained. Catalytic efficiency in polar solvents was 1000 fold greater than that of freeze-dried powders. As with the crystals, subsequent drying over molecular sieves resulted in a substantial loss in enzymatic activity. The activities of solvent washed immobilised enzymes and of cross-linked crystals were found to vary significantly as a function of system water content.