Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device

Abstract

A cephalosporin acetylesterase produced by Bacillus subtilis catalyzes the deacetylation of 7‐aminocephalosporanic acid (7‐ACA). Previous reports from our laboratory described the kinetic constants that characterize the reaction: K_m = 2.8 × 10⁻³M, K_ia acetate = 5 × 10⁻²M, and K_id deacetyl‐7‐ACA = 3.6 × 10⁻²M. These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition. where S_t = mg/ml 7‐ACA, A_t = mg/ml acetate, D_t = mg/ml deacetyl‐7‐ACA. The predicted time course closely matched the time course measured experimentally. The equation also was solved without the inhibition terms and the solution indicated that product inhibition caused about a 30% increase in the time required for complete (>97%) hydrolysis of a 24 mg/ml 7‐ACA solution. The esterase was immobilized by containment within an ultrafiltration device. With this technique the enzyme was reused 20 times over an 11 day span to deacetylate 7‐ACA solutions containing 4 to 24 mg/ml 7‐ACA. The specific activity after the 20th use was the same as the activity prior to the first use, indicating little enzyme inactivation occurred.