APPLICATION OF HIGH HYDROSTATIC-PRESSURE FOR INCREASING ACTIVITY AND STABILITY OF ENZYMES

Elevated hydrostatic pressure has been used to increase catalytic acti vity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activatio n volume (Delta V-not equal), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyze d by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar ). Due to a strong temperature dependence of Delta V-not equal, the ac celeration effect of high pressure becomes more pronounced at high tem peratures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activit y at normal conditions (20 degrees C, 1 atm). At pressures of higher t han 3.6 kbar, the enzymatic activity is decreased due to a pressure-in duced denaturation. Elevated hydrostatic pressure is also efficient fo r increasing stability of CT against thermal denaturation. For example , at 55 degrees C, CT is almost instantaneously inactivated at atmosph eric pressure, whereas under a pressure of 1.8 kbar CT retains its ani lide-hydrolyzing activity during several dozen minutes. Additional sta bilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabi lized CT can be decelerated in a supplementary manner by addition of 4 0% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both e xtreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activit y of CT in the presence of 40% (v/v) glycerol is severalfold higher th an in aqueous buffer. The results of this study are discussed in terms of the hypotheses which explain the action of external and medium eff ects on protein structure, such as preferential hydration and osmotic pressure. (C) 1996 John Wiley & Sons, Inc.