High pressure crystallization of Purafect® subtilisin

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Waghmare, Ruta
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C. E. Glatz
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Chemical and Biological Engineering

The function of the Department of Chemical and Biological Engineering has been to prepare students for the study and application of chemistry in industry. This focus has included preparation for employment in various industries as well as the development, design, and operation of equipment and processes within industry.Through the CBE Department, Iowa State University is nationally recognized for its initiatives in bioinformatics, biomaterials, bioproducts, metabolic/tissue engineering, multiphase computational fluid dynamics, advanced polymeric materials and nanostructured materials.

The Department of Chemical Engineering was founded in 1913 under the Department of Physics and Illuminating Engineering. From 1915 to 1931 it was jointly administered by the Divisions of Industrial Science and Engineering, and from 1931 onward it has been under the Division/College of Engineering. In 1928 it merged with Mining Engineering, and from 1973–1979 it merged with Nuclear Engineering. It became Chemical and Biological Engineering in 2005.

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1913 - present

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  • Department of Chemical Engineering (1913–1928)
  • Department of Chemical and Mining Engineering (1928–1957)
  • Department of Chemical Engineering (1957–1973, 1979–2005)
    • Department of Chemical and Biological Engineering (2005–present)

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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.

Fri Jan 01 00:00:00 UTC 1999