beta-lactamase from Streptomyces cacaoi. Purification and properties.
A beta-lactamase was purified to an apparently homogeneous state from Streptomyces cacaoi. The molecular weight calculated from the mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis was 34,000. pI was 4.7 and the optimal pH was 6.5. The optimum temperature was found to be between 40 degrees C and 45 degrees C, but the enzyme lost activity above 50 degrees C. N-Bromosuccinimide was the strongest inhibitor among the reagents tested, followed by iodine. p-Chloromercuribenzoate showed a weak inhibitory effect. Diisopropylfluorophosphate and sodium chloride did not show any inhibitory effect on the enzyme. The beta-lactamase catalyzed the hydrolysis of methicillin and cloxacillin at two-thirds to one-third the rate of benzylpenicillin. On the other hand, the enzyme hydrolyzed cephalosporins and 7-methoxycephalosporin only slowly. With benzylpenicillin as a substrate, the Km increased sharply with decreasing pH and the pK alpha estimated from the Km versus pH curve was 6.5 to 7.0. In contrast, with cloxacillin as a substrate, the Km showed a minimum at pH 7.5. The Vmax changed with pH in a bell-shaped curve in the case of benzylpenicillin, but the Vmax for cloxacillin changed only within a small range. In addition, the ratio of the hydrolysis rate of benzylpenicillin and cloxacillin at 30 degrees C and 20 degrees C (V30 degrees/V20 degrees) was found to be 1.23 and 1.55, respectively. These results indicate that the S. cacaoi beta-lactamase behaves differently toward benzylpenicillin and cloxacillin, although both are penicillins. S. cacaoi seems to release beta-lactamase into the culture medium soon after its biosynthesis without retaining it in the membrane and the soluble fraction. The possible relationships between beta-lactamases from Streptomyces and those from pathogenic bacteria are discussed.
D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D001618:beta-Lactamases / Q000302:isolation & purification / Q000378:metabolism*
D001618:beta-Lactamases