Pressure dependence of subtilisin crystallization kinetics

The effect of pressures from atmospheric to 100 MPa on crystallization of Purafect subtilisin was investigated. The overall crystallization rate was lower at high pressures and short exposures to high pressure followed by atmospheric crystallization did not increase the overall crystallization rate. Subtilisin solubility increased with pressure from 1.1 mg/ml at 0.1 MPa (atmospheric pressure) to 2.9 mg/ml at 68 MPa giving a calculated volume change for crystallization as 30 +/- 7 cm(3)/mol. Linear crystal growth rate dropped with increasing pressure in the range 0.1-13.6 MPa. The dependence of growth rate on supersaturation at 0.1 MPa changed from an exponent of 0.18 at high supersaturation to 2.8 at lower supersaturation indicating a possible change in the rate limiting step. At high pressures, single values of the exponent sufficed but the value increased to 4.2 at 6.8 MPa and 6.3 at 13.6 MPa. Crystal size distributions (CSD) were obtained after crystallization with agitation. Pressure decreased the crystallization rate in these experiments also. Nucleation rate expressions were obtained by simultaneously fitting a population balance model to the residual soluble protein concentration and final crystal size distribution data. (C) 2000 Elsevier Science B.V. All rights reserved.