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A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.

Both a penicillinase and a cephalosporinase are present in a strain of Citrobacter freundii (GN7391) resistant to beta-lactam antibiotics. The penicillinase was identical to the type Ia penicillinases (Type III by Richmond classification), mediated by Rms212 and R-TEM. A cephalosporinase, typical of enterobacteriaceae chromosomal beta-lactamase (Type I by Richmond classification), was purified from the strain. It gave a single protein band on polyacrylamide gel electrophoresis and immunoelectrophoresis; the pI was 8.6 and its molecular weight was approximately 38 000. Cysteine was not found among its amino acids. The specific activity was 388 units (mg protein)-1 for the hydrolysis of cephaloridine, and the optimal pH was 8.0. Rabbit antiserum obtained against the purified enzyme showed cross-reaction with cephalosporinases produced by strains of Enterobacter cloacae in a neutralization test.

BETA-Lactamases (EC. 3.5.2.6) from strains of Gram-negative bacteria have been studied using analytical isoelectric focusing. This permits a visual comparison of the patterns of beta-lactamase bands produced by enzymes from different organisms. Purification of crude intracellular preparations is unnecessary and the technique is sufficiently sensitive to demonstrate beta-lactamase in mutants previously reported to lack the enzyme. R that have not been distinguished from one another biochemically or immunologically can be differentiated by isoelectric focusing. Conversely, the enzymes specified by the R factors RTEM, R1 and RGN14, with identical isoelectric focusing patterns have the same biochemical properties. Chromosomal and R-factor-mediated beta-lactamases from single strains have been separated and their identities confirmed by immunoisoelectric focusing. R factor-mediated enzymes gave identical isoelectric focusing patterns irrespective of the host strain. Isoelectric focusing can therefore be used to observe the transfer of beta-lactamases carried by R factors.