Preparation and Characterization of Immobilized β-Lactamase for Destruction of Penicillin in Milk

Abstract

β-Lactamase I (Bacillus cereus) was covalently bound to cyanogen bromide-activated, crosslinked agarose. An initial 5.00 mg of soluble β-lactamase were used in the immobilization reaction for each preparation, and average coupling yield was 80.5%. Of the enzyme immobilized on the matrix, an average 53.4% remained active. To minimize diffusional effects on immobilized enzyme activity, reaction mixtures were rotated at 250 rpm throughout the study. The shape of the pH activity curve of the immobilized enzyme was identical to that of the soluble enzyme; both exhibited optimum pH around 7.0. In general, only 2-fold differences in Michaelis constant and maximum volume were observed between native and immobilized enzyme when penicillin G was used as the substrate. However, the Michaelis constant of the immobilized enzyme increased up to 22-fold that of the native enzyme when cephaloridine was used as the substrate. The immobilized enzyme exhibited enhanced stability in the acidic pH region in contrast to the native enzyme, which had superior stability in the alkaline pH region. The heat stability of the immobilized enzyme was about twice that of native enzyme after heat treatment at 60°C for 30 min. Approximately a 10% increase of storage stability on immobilization of β-lactamase was observed when stored at room temperature (23 ± 1°C) for up to 6 d in the absence of antimicrobial agents. Little loss of activity (<2%) was noted after repeated use of the immobilized enzyme up to seven times each in 10.0 ml of skim milk containing .5 U/ml penicillin G. At an immobilized enzyme concentration of 2.33 U/ml of milk, it totally destroyed .5 U penicillin G/ml in 10.0 ml whole milk and in 10.0 ml skimmilk at 4°C, when rotated at 250 rpm, for 9 and 4 min, respectively. © 1987, American Dairy Science Association. All rights reserved.