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Molecular level investigations and mathematical modeling of immobilized alpha-chymotrypsin preparation, utilization and deactivation

Citation

Clark, Douglas S. (1984) Molecular level investigations and mathematical modeling of immobilized alpha-chymotrypsin preparation, utilization and deactivation. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-11082005-133605

Abstract

Radiation-mediated grafting of polyacrolein onto polymethyl methacrylate microspheres activated the particles for chymotrypsin immobilization. Treatment of porous polystyrene/magnetite particles with polyacrolein produced very small enzyme loading enhancement and significantly increased substrate diffusional resistance. These results nonetheless demonstrate the feasibility of obtaining grafted materials and of utilizing this approach to improve the capacity of microspheres for protein immobilization. Enzyme immobilization on a more conventional support material, CNBr-activated Sepharose 4B, was studied extensively. Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy. When EPR spectra were recorded in the presence of indole, direct evidence for the existence of two distinct forms of active alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B was obtained. The indole EPR spectra of five different spin-labeled immobilized enzyme formulations were all resolved into the same two spectral components. Both subpopulation spectra were approximately identified experimentally, and the subpopulation exhibiting greatly restricted spin-label motion was shown also to be relatively inaccessible to solvent. Using overall specific activity data and subpopulation fractions from EPR spectral analysis, the specific activity of the more constricted immobilized enzyme active form was shown to be approximately fifteen times smaller than that of the other class of immobilized enzyme molecules with an indole EPR spectrum similar to that of chymotrypsin in solution. Variations in overall specific activity of formulations with different loadings and different supports result entirely from changes in the proportions of the same two subpopulations of immobilized enzyme molecules. Deactivation in aliphatic alcohols of alpha-chymotrypsin and alpha-chymotrypsin-CNBr Sepharose 4B conjugates was also studied by several characterization methods. Active site titration measurements, which were used to determine the amount of catalytically active enzyme, revealed appreciable differences between the deactivation kinetics of free and immobilized chymotrypsin. In all cases for the immobilized enzyme, the kinetics of active enzyme disappearance differed significantly from first-order. Interestingly, the estimated intrinsic activity of immobilized chymotrypsin remaining active after different exposure times to 50% n-propanol solution increased somewhat as a result of exposure to alcohol. These findings were complemented by direct information, provided by EPR spectroscopy, on the effects of alcohols on the active site configuration of spin-labeled chymotrypsin. EPR spectra of the free enzyme illustrated the appearance in different alcohol solutions of different enzyme forms with different active site structures. EPR experiments also showed that denaturation of immobilized chymotrypsin was accompanied by unfolding of the active site that followed similar multistep kinetics as the loss of active enzyme. Further insight into the deactivation in 50% n-propanol of immobilized alpha-chymotrypsin was provided by analyses that focused on the behavior of the two distinct active forms of immobilized enzyme, designated here A and B, identified previously. Raw data provided by EPR spectroscopy clearly show that the relative quantities of active chymotrypsin-A and active chymotrypsin-B change as a result of exposure to alcohol, with the relative quantity of the B form increasing with time. These and additional results provide evidence that the distribution of A and B forms is a function of active enzyme loading but independent of the means used to obtain the loading. Different kinetic models in conjunction with experimental observations consistently indicate that the activity of enzyme form B, by far the more active enzyme form, does not change significantly during the initial 60 min. of catalyst deactivation but then decreases appreciably. Finally, theoretical analyses of enzyme immobilization, more general in scope than the alpha-chymotrypsin work, were also performed. Enzymes are often immobilized on the internal surfaces of porous solid supports by immersing enzyme-free particles in a well-mixed solution of enzyme. The ensuing impregnation process involves coupled transient mass transfer and surface attachment of enzyme. Mathematical models were employed to explore the influences of process parameters on the amount of enzyme loaded and the distribution of immobilized enzyme within the support particles. Nonuniform loading of the support occurs under some conditions, particularly when diffusion of unattached enzyme through the support is restricted by the large size of the enzyme relative to the accessible cross-sectional area of the support's pores. This is significant since the distribution of enzyme within the support particle influences the overall activity and stability of the immobilized enzyme catalyst. The models developed here may also be used to describe removal of reversibly immobilized enzyme during washing or utilization of the immobilized enzyme catalyst.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemical Engineering
Degree Grantor:California Institute of Technology
Major Option:Chemical Engineering
Thesis Availability:Restricted to Caltech community only
Thesis Committee:
  • Bailey, James E. (chair)
Defense Date:7 December 1983
Record Number:CaltechETD:etd-11082005-133605
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-11082005-133605
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:4455
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:09 Nov 2005
Last Modified:03 Dec 2014 00:06

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