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Preparation and Properties of a Cephalosporin Acetylesterase Adsorbed onto Bentonite
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| Zeitschriftentitel: | Antimicrobial Agents and Chemotherapy |
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| Personen und Körperschaften: | , |
| In: | Antimicrobial Agents and Chemotherapy, 8, 1975, 3, S. 282-288 |
| Format: | E-Article |
| Sprache: | Englisch |
| veröffentlicht: |
American Society for Microbiology
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| Schlagwörter: |
| Zusammenfassung: | <jats:p> A cephalosporin acetylesterase produced by <jats:italic>Bacillus subtilis</jats:italic> was immobilized by adsorption onto bentonite. The immobilized enzyme (E <jats:sub>I</jats:sub> ) and the soluble enzyme (E <jats:sub>S</jats:sub> ) exhibited Michaelis-Menton kinetics with 7-aminocephalosporanic acid (7-ACA): <jats:italic> K <jats:sub>m</jats:sub> </jats:italic> = 2.8 × 10 <jats:sup>−3</jats:sup> M and <jats:italic> K <jats:sub>m</jats:sub> </jats:italic> = 3.2 × 10 <jats:sup>−3</jats:sup> M, respectively. Similar kinetics were observed with 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin), but the <jats:italic> K <jats:sub>m</jats:sub> </jats:italic> value measured with E <jats:sub>I</jats:sub> (3.7 × 10 <jats:sup>−3</jats:sup> M) was less than one-half that measured with this substrate and E <jats:sub>S</jats:sub> . The reduction in <jats:italic> K <jats:sub>m</jats:sub> </jats:italic> 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 <jats:sub>S</jats:sub> and E <jats:sub>I</jats:sub> . The <jats:italic> K <jats:sub>i</jats:sub> </jats:italic> values for E <jats:sub>I</jats:sub> were 5.0 × 10 <jats:sup>−2</jats:sup> M for acetate and 3.6 × 10 <jats:sup>−2</jats:sup> M for deacetyl-7-ACA. Similar values were measured with E <jats:sub>S</jats:sub> and these substrates. E <jats:sub>I</jats:sub> 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 <jats:sub>I</jats:sub> with glutaraldehyde or bis-dimethyladipimidate, or by adding Al(OH) <jats:sub>3</jats:sub> to the suspension. With the latter addition, E <jats:sub>I</jats:sub> was stabilized so that it could be reused nine times before one-half of the initial activity was lost. </jats:p> |
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| Umfang: | 282-288 |
| ISSN: |
0066-4804
1098-6596 |
| DOI: | 10.1128/aac.8.3.282 |