Immobilization of a cephalosporin acetylesterase by containment within an ultrafiltration device

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.