Preparation and properties of penicillin amidase immobilized in polyelectrolyte complexes

Abstract

(1) Immobilization of penicillin amidase (acylamide amidohydrolase, EC 3.5.1.4) from Escherichia coli was carried out on negatively charged particles of the water-soluble polyelectrolyte complex formed by poly(4-vinyl- N-ethylpyridinium bromide) and poly(methacrylic acid) in the ratio 1 : 3. The enzyme was covalently cross-linked to the polycation which was previously modified by cyanuric chloride. (2) Kinetic parameters of benzylpenicillin hydrolysis, catalyzed by immobilized penicillin amidase, were determined and it was shown that with a given method of immobilization, the catalytic efficiency of enzyme action changes slightly. The reaction proceeds in homogeneous aqueous solution without any diffusional difficulties. (3) Deformation of the pH optimum of catalytic activity, an increase of k cat in alkaline media and a sharp increase of the inhibition constant from 2 · 10 −5 to 1 · 10 −3 M caused by the product of the reaction (phenylacetic acid) show a remarkable effect of the negatively charged shell of the polyelectrolyte complex on kinetic parameters of the reaction. (4) Immobilization of the enzyme in polyelectrolyte complex particles leads to the appearance of new properties of the biocatalyst; immobilized penicillin amidase can be reversibly converted to the insoluble state with a slight change in pH and ionic strength of the solution. Transition of the enzyme into the insoluble state results in interruption of the reaction. The conditions for the phase separation of immobilized enzyme in solutions of salts are determined by the composition of polyelectrolyte complex and the nature of the low molecular weight electrolytes. Dissolution of the precipitate leads to quantitative recovery of the initial catalytic activity. (5) The self-regulating enzymatic system was simulated. Control of the activity in the system takes place according to the following scheme: accumulation of product → change in ionic strength of the solution → alteration of microenvironment of the enzyme → decrease in catalytic activity.