Preparation and Properties of a Cephalosporin Acetylesterase Adsorbed onto Bentonite

Author:

Abbott Bernard J.1,Fukuda D. S.1

Affiliation:

1. The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206

Abstract

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.

Publisher

American Society for Microbiology

Subject

Infectious Diseases,Pharmacology (medical),Pharmacology

Reference22 articles.

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3. Semipermeable aqueous microcapsules. I. Preparation and properties;Chang T. M. S.;Can. J. Physiol. Pharmacol.,1966

4. Chibata I. T. Tosa T. Sato T. Mori and Y. Matsuo. 1972. Preparation and industrial application of immobilized aminoacylases p. 383-389. In G. Terui (ed.) Fermentation technology today. Society of Fermentation Technology Osaka Japan.

5. Bifunctional reagents. Cross-linking of pancreatic ribonuclease with a diimido ester;Hartman F. C.;J. Am. Chem. Soc.,1966

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