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The penicillinase from an Escherichia coli strain harboring an R factor R(GN823) was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S(20,w) of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type Ib penicillinase, and the original type I penicillinase was renamed type Ia-penicillinase.

On the assumption that the penicillinase determinants on a group of R factors conferring ampicillin resistance have a phylogenetically close relationship to the penicillinase gene of the Klebsiella group, the penicillinases from four strains of K. pneumoniae, GN69, GN1103R(-), GN422, and GN118, were purified 230- to 1,000-fold and compared with the known two R-factor-mediated penicillinases. By gel filtration on Sephadex G-75, the molecular weights were estimated to be 17,400, 18,100, 20,000 and 18,300, respectively, which are slightly lower than those of the R-factor penicillinases. The isoelectric points of the Klebsiella penicillinases were not in agreement with those of the R-factor penicillinases. All the enzymes showed a pH optimum between 6.3 to 7.2 and a temperature optimum of 45 C, and those properties, together with behavior towards inhibitors, were about the same as those in the R-factor penicillinases. The substrate specificity and the Michaelis constants of the Klebsiella penicillinases for penicillins and cephaloridine were broadly similar to those of the R-factor penicillinases, however, some variations were found even among the four penicillinases of K. pneumoniae. The reactivities of the four penicillinases of K. pneumoniae with the antiserum against one R-factor penicillinase were tested, and three of the four Klebsiella penicillinases were found to be indistinguishable immunologically from both R-factor penicillinases. The remaining Klebsiella penicillinase, from GN1103R(-), showed an immunological partial homology with the R-factor penicillinases.