Biochemical Characterization of Type A and Type B -Lactamase from Enterobacter cloacae

Abstract

Different types of chromosomally coded β-lactamases arefound in Enterobacter cloacae. E. cloacae M6300 produces β-lactamase type A, which has an isoelectric point of 8.8, whereas E. cloacae 908 R produces β-lactamase type B, which has an isoelectric point of 7.9. Bothenzymes were purified to homogeneity by a procedure that included affinity chromatography on amino phenylboronic acid-modified Sepharose. The two enzymes were closely related as shown bysodium dodecyl sulfate-polyacrylamide gel electrophoresis, kinetic constants with several substrates, amino acid composition, NH₂-terminal amino acid sequence, and reaction with antisera. In addition to having different isoelectric points, the two enzymes migrated to slightly different positions on polyacrylamide gels and differed significantly in rate of catalysis for cephalothin, imipenem, and the penem Sch 34343. One of three antisera seemed to recognize an epitope that differs in the two enzymes. The diversity of cephalosporinases found in E. cloacae with respect to the evolution of novel β-lactamases was considered.