Immobilization of enzymes in porous supports: Effects of support–enzyme solution contacting

Abstract

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.

Immobilization of enzymes in porous solids under restricted diffusion conditions

“…The overall rate is often largest when the active site is located near the external surface rather than uniformly distributed (Wang and Varma, 1980). This conclusion has been demonstrated for immobilized enzyme catalysts as reported by Borchert and Buchholz (1979) and Dennis et al (1984). However, from a deactivation standpoint, the work by Corbett and Luss ( 1 974) has shown that uniformly loaded catalyst may be more stable than nonuniformly loaded catalyst.…”

“…Recently it was suggested by Dennis et al (1984) that restricted diffusion may cause nonuniform distribution of enzymes in porous supports. On the basis of this, a mathematical model has been formulated by Clark et al ( 1 985).…”

A novel method of determination of the internal enzyme distribution within porous solid supports and the deactivation rate constant

“…Clearly, it is not unity because of the main reaction. However, its initial location Xo can be found by matching equation (19) at zero time to the quasi-steady-state solution for Ab given in equation (13). We find (19) and (22) yields…”

Affinity adsorption

“…[41) Similarly, immobilized antibodies have been found to have reduced affinity and capacity (on a molar basis) for binding compared with soluble antibodies.l l l .4 s . 46 ) Reduced enzymatic activity has been attributed to phenomena such as immobilizing bond-induced conformational changes,[41.4 7 ) increased immobilized enzyme density, [48) and support/substrate ionic repulsion. [49) Immobilized antibodies have also been shown to have reduced capacity (on a per mole basis) at higher antibody concentrations and higher activation levels.…”