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Although still there are Klebsiella strains which do not harbour plasmids and produce constitutive chromosomal β-lactamases, recently clinical isolates were found in ever increasing numbers carrying mainly TEM-, CARB- and OXA type R-factors. We selected four chromosomal cephalosporinase producing Klebsiella strains to study the p I values of the enzymes and their simultaneous separability from accompanying proteins by chromatofocusing techniques. We compared p I values of the pure and the crude preparations: K. pneumoniae Kl SC 10436: p I pure = 6.4, p I crude = 6.42; K. aerogenes Kl 1082 E: p I pure = 6.5, p I crude = 6.5; K. oxytoca 1082 E: p I pure = 6.42, p I crude = 6.4; K. oxytoca 20: p I pure = 7.62, p I crude = 7.6. Excellent agreement of the p I values among each other, but occasional differences with those obtained by analytical isoelectrofocusing are attributed to methodological diversities and to the presence of satellite enzymes, known to exist in Klebsiella .

N-Acetyl- d -(−)-penicillamine as a stable second-generation biospecific affinity ligand has previously been suggested for purification of Bacillus cereus 569/H β-lactamase I. A complex spacer arm is coupled with the matrix by using epichlorohydrin and phloroglucinol doubly activated with divinyl sulphone in the meta position. Coupling of d -(−)-penicillamine ligand resulted in an active affigel. However, we found that two affinity ligands in close proximity prevents simultaneous binding of two penicillinase molecules, therefore one ligand is superfluous. Our results show that: (1) shortening the spacer arm by direct activation of the matrix with divinyl sulphone is satisfactory to produce the affinity material with N-acetyl- d -(−)-penicillamine; (2) incorporation of 15 μmol of N-acetyl- d -(−)-penicillamine per ml of wet Sepharose 4B satisfies the maximum binding capacity requirements of the affigel (about half of the originally incorporated amount of ligand); (3) our simplified affinity adsorbent is generally applicable for large-scale purification of penicillinases to homogeneity from various bacterial sources by the convenient batch method without prior concentration of these enzymes; (4) reacetylation for four/five times can regenerate the original binding capacity of the affigel.