Preparation and properties of a cephalosporin acetylesterase adsorbed onto bentonite.
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 x 10(-3) M and K(m) = 3.2 x 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 x 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 x 10(-2) M for acetate and 3.6 x 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.
D000115:Acetylesterase / Q000032:analysis* / Q000302:isolation & purification; D000327:Adsorption; D001412:Bacillus subtilis / Q000201:enzymology; D001546:Bentonite; D002511:Cephalosporins; D055598:Chemical Phenomena; D002621:Chemistry; D004355:Drug Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
D002511:Cephalosporins; D001546:Bentonite; D000115:Acetylesterase