Date of Award
Doctor of Philosophy
Chemical and Biological Engineering
C. E. Glatz
The aim of this work was to study the effect of high pressure on kinetics of subtilisin crystallization and to use high pressure as a means to better understand the mechanism of crystallization;It is known that the effect of hydrostatic pressure on conformation varies with the protein being studied. To determine the effect of high pressure on the conformation of subtilisin, activity assays were used in addition to FTIR spectroscopy. Pressures up to 100 MPa were found to cause no irreversible conformational changes in subtilisin;To study the effect of high pressure on subtilisin crystallization, a factorial experiment was designed with level of pressure (0.1, 33, 68, 100 MPa) and duration of pressurization (2, 24 hours) as two factors. Unlike lysozyme, no overall enhancement of yield was observed by short exposures to high pressures followed by crystallization at atmospheric pressure. Subtilisin solubility increased with pressure from 1.1 mg/ml at 0.1 MPa to 2.9 mg/ml at 68 MPa giving a calculated volume change for crystallization of 30 +/- 7 cm 3/mol;Crystal growth rate for subtilisin was observed to decrease with increasing pressure in the range 0.1--13.6 MPa. The dependence of growth rate on supersaturation changed with pressure indicating a possible change in the rate-limiting step at high pressures. A possible change in the rate-limiting step was also observed at lower supersaturations at 0.1 MPa;The nucleation rate for subtilisin decreased by a factor of about 60 with an increase in pressure from 0.1 to 34 MPa. The dependence of nucleation rate on supersaturation was of the order of 1.6 for all the pressures. The activation volume for subtilisin nucleation was calculated as +330 cm 3/mol;Crystal size distributions (CSD) were obtained for crystallization with agitation. Agitation prevented aggregation and led to secondary nucleation. Pressure decreased the rate of crystallization for crystallization with agitation also. Nucleation and growth rate expressions were obtained by fitting a population balance model to residual soluble protein concentration and CSD data.
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Ruta Yeshwant Waghmare
Waghmare, Ruta Yeshwant, "High pressure crystallization of Purafect® subtilisin " (1999). Retrospective Theses and Dissertations. 12620.