Pressure effect on subtilisin crystallization and solubility

High pressures (135 and 200 MPa) arrested the crystallization process in unseeded batch crystallizations of subtilisin conducted at 25 degrees C over a period of seven days. Crystallization resumed at the normal atmospheric pressure rate once batches were depressurized and the active soluble enzyme concentration decayed to the same value as seen in atmospheric crystallizations. Solubility experiments conducted at 25 degrees C showed an exponential increase in apparent solubility with pressure. The solubility increased approximately five-fold at 200 MPa. The partial molar volume change of crystallization for this increase in solubility with pressure is + 21 +/- 1 ml/mol. Solubility changes with pressure are largely responsible for the arrest in crystallization rate at high pressure. Experiments on pH-pressure and ionic strength-pressure interactions showed that the change in apparent solubility is not due to pressure-induced changes in pH and ionic strength. For comparative purposes, solubility data as a function of pressure were collected on lysozyme. A linear dependence of lysozyme solubility with pressure was observed, with a 40% increase in apparent solubility after incubation for seven days at 210 MPa and 25 degrees C. (C) 1999 Elsevier Science B.V. All rights reserved.