Influence of Internal Diffusional Limitations on the Performance of an Immobilized Enzyme in Organic Medium

University dissertation from Department of Biotechnology, Lund University

Abstract: The influence of mass-transfer limitations on the performance of immobilized a-chymotrypsin operating in acetonitrile medium has been investigated. The enzyme was immobilized by deposition on various porous carrier materials such as celite, polyamides of different particle sizes, controlled-pore glasses, polypropylene or polyacrylamides. When carrying out kinetically controlled peptide synthesis reactions catalyzed by these preparations, mass-transfer effects have been observed. It has been shown that internal diffusional limitations are the mechanism responsible such mass-transfer effects. A correlation between observed activity towards a given reaction at a given enzyme loading and the morphological characteristics of the support material has been developed, taking into account the effects of internal diffusion. The factors that affect substrate diffusion and enzyme kinetics are discussed, because both processes must happen at the same rate in simultaneous reaction and diffusion. The extent of internal diffusional limitations can be controlled by changing enzyme loading, support material, or the intrinsic velocity of the reaction carried out. The intrinsic kinetics of the studied reactions have been studied in the absence of mass-transfer effects by immobilizing the enzyme on nonporous glass beads. Internal diffusional limitations influence the performance of the immobilized enzyme in many ways: Strong diffusional limitations reduce the specific activity of the enzyme preparations. The initial rate per amount of preparation can even decrease with increased enzyme loadings in the case of very low porosity supports, due to reduced available space for substrate diffusion. Internal diffusional limitations can be relaxed by the presence of insoluble hydrochlorinated nucleophile, presumably by promoting the desorption of part of the enzyme from "overloaded" supports. Internal diffusional limitations also decrease the apparent specificity of the chemical transformations. Acyl donor specificity and enantioselectivity in the conversion of a racemic acyl donor are much more affected than nucleophile specificity. The extent of the effect on nucleophile specificity depends on the size of the used nucleophile. Strong internal diffusional limitations still increase the apparent stability, both storage and operational, of the enzyme preparations. A numeric model has been developed to describe the effects of internal diffusional on initial activity and nucleophile specificity. The model accurately describes experimental observations such as plateau-shaped initial rate profiles, the stronger influence on initial rate than on nucleophile specificity and reduction of initial rate with increased enzyme loading with low porosity supports. Besides showing that internal diffusional limitations influence all aspects of enzyme performance, conceptual and numerical explanations to the observed effects have also been provided.

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