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 described the kinetic constants that characterize the reaction: Km = 2.8 .times. 10-3 M, Ki acetate = 5 .times. 10-2 M, and Ki deacetyl-7-ACA = 3.6 .times. 10-2 M. These constants were used to predict the time course of the reaction using the following equation for dual competitive product inhibition. .**GRAPHIC**. where St = mg/ml 7-ACA, At = mg/ml acetate, Dt = 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 4-24 mg/ml 7-ACA. The specific activity after the 20th use was the same as the activity prior to the 1st use, indicating little enzyme inactivation occurred.