Open AccessPreparation and Properties of a Cephalosporin Acetylesterase Adsorbed onto Bentonite
Author(s) -
Bernard J. Abbott,
D. S. Fukuda
Publication year - 1975
Publication title -
antimicrobial agents and chemotherapy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.07
H-Index - 259
eISSN - 1070-6283
pISSN - 0066-4804
DOI - 10.1128/aac.8.3.282
Subject(s) - bentonite , chemistry , bacillus subtilis , enzyme kinetics , adsorption , kinetics , nuclear chemistry , immobilized enzyme , cephalosporin c , substrate (aquarium) , glutaraldehyde , stereochemistry , chromatography , enzyme , cephalosporin , organic chemistry , biochemistry , active site , chemical engineering , bacteria , genetics , physics , oceanography , quantum mechanics , engineering , biology , geology , antibiotics
A cephalosporin acetylesterase produced byBacillus 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 andKm = 3.2 × 10−3 M, respectively. Similar kinetics were observed with 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin), but theKm value measured with EI (3.7 × 10−3 M) was less than one-half that measured with this substrate and ES . The reduction inKm 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 . TheKi 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.