Preparation and Properties of a Cephalosporin Acetylesterase Adsorbed onto Bentonite

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A cephalosporin acetylesterase produced by Bacillus subtilis was immobilized by adsorption onto bentonite. The immobilized enzyme (EI) and the soluble enzyme (ES) exhibited Michaelis-Menton kinetics with 7-aminocephalosporanic acid (7-ACA): Km = 2.8 × 10−3 M and Km = 3.2 × 10−3 M, respectively. Similar kinetics were observed with 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin), but the Km value measured with EI (3.7 × 10−3 M) was less than one-half that measured with this substrate and ES. The reduction in Km value was correlated with the ability of bentonite to adsorb cephalothin. The reaction products, acetate and deacetyl-7-ACA, were weak competitive inhibitors of ES and EI. The Ki values for EI were 5.0 × 10−2 M for acetate and 3.6 × 10−2 M for deacetyl-7-ACA. Similar values were measured with ES and these substrates. EI retained about 80% of its initial activity after 3 weeks of storage in solution at 25 C. However, the enzyme dissociated from the bentonite particles during the deacetylation reaction. This dissociation was minimized by cross-linking EI with glutaraldehyde or bis-dimethyladipimidate, or by adding Al(OH)3 to the suspension. With the latter addition, EI was stabilized so that it could be reused nine times before one-half of the initial activity was lost.

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