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Preparation and Properties of a Cephalosporin Acetylesterase Adsorbed onto Bentonite

By Bernard J. Abbott and D. S. Fukuda


A cephalosporin acetylesterase produced by Bacillus subtilis was immobilized by adsorption onto bentonite. The immobilized enzyme (E(I)) and the soluble enzyme (E(S)) exhibited Michaelis-Menton kinetics with 7-aminocephalosporanic acid (7-ACA): K(m) = 2.8 × 10(−3) M and K(m) = 3.2 × 10(−3) M, respectively. Similar kinetics were observed with 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin), but the K(m) value measured with E(I) (3.7 × 10(−3) M) was less than one-half that measured with this substrate and E(S). The reduction in K(m) value was correlated with the ability of bentonite to adsorb cephalothin. The reaction products, acetate and deacetyl-7-ACA, were weak competitive inhibitors of E(S) and E(I). The K(i) values for E(I) were 5.0 × 10(−2) M for acetate and 3.6 × 10(−2) M for deacetyl-7-ACA. Similar values were measured with E(S) and these substrates. E(I) 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 E(I) with glutaraldehyde or bis-dimethyladipimidate, or by adding Al(OH)(3) to the suspension. With the latter addition, E(I) was stabilized so that it could be reused nine times before one-half of the initial activity was lost

Topics: Chemistry; Mechanisms of Action and Resistance
Year: 1975
DOI identifier: 10.1128/aac.8.3.282
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Provided by: PubMed Central
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