Exploiting hydration hysteresis for high activity of cross-linked subtilisin crystals in acetonitrile

The catalytic behavior of cross-linked subtilisin crystals in acetonitrile exhibits pronounced hysteresis. The transesterification activity of the crystals in anhydrous solvent varies as much as 80-foId, depending on the hydration history of the enzyme, In a comparison of drying methods, the highest rates of reaction were obtained with crystals dried by washing with organic 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. Evidence for hysteresis in hydration was obtained using a H-2-NMR method. Crystals rinsed with anhydrous solvent were found to retain approximately 70 waters per enzyme molecule more than those dried over molecular sieves, When different solvents were used to dry the enzyme crystals, the catalytic rate in anhydrous acetonitrile was found to vary significantly-methanol-washed CLEC giving the lowest rates. The transesterification activity of the solvent-washed enzyme was found to be profoundly effected by the concentration of water in the system. Optimum conditions for eater production were obtained in anhydrous acetonitrile. Under these conditions, despite a lower initial rate, absence of the hydrolysis byproduct which inhibits the enzyme led to better synthetic yields.