Purification and properties of two extracellular β-lactamases from Bacillus cereus 569/H

Abstract

1. When Bacillus cereus 569/H was grown in a casamino acid (casein-hydrolysate) medium containing zinc sulphate rapid production of extracellular beta-lactamase II preceded that of beta-lactamase I. 2. beta-Lactamase I was separated from beta-lactamase II by fractional precipitation with ammonium sulphate. 3. beta-Lactamase I was purified by a process involving chromatography on Celite and DEAE-cellulose and beta-lactamase II by chromatography on DEAE-cellulose after denaturation of beta-lactamase I by heat. Both enzymes were obtained in crystalline form. 4. beta-Lactamase II prepared in this way appeared to have a higher molecular weight than beta-lactamase I and required Zn(2+) as a cofactor for both cephalosporinase and penicillinase activities.

The composition of β-lactamase I and β-lactamase II from Bacillus cereus 569/H

“…Table 1. Amino acid and amino sugar compo8ition off,-lactamase I and P-lactama8e II The crystalline enzyme preparations analysed were (a) those used for the determination of molecular weights (Kuwabara, 1970;Lloyd & Peacocke, 1970) and (b) similar preparations from different culture supernatants. Hydrolysis and analysis in amino acid analysers were carried out as described in the Materials and Methods section.…”

“…The molecular weight calculated by Lloyd & Peacocke (1970) from sedimentation-equilibrium studies for P-lactamase II (35600) is higher than that for ,B-lactamase I (27 800) and that calculated Table 2. Composition of the carbohydrate moiety of /3-lactamase II Sample A was a preparation of fi-lactamase that had been precipitated by (NH4)2SO4 immediately before crystallization (Kuwabara, 1970). Sample B was material from the same batch after two crystallizations.…”

“…However, the nature of the association of carbohydrate with protein and whether the carbohydrate is essential for the characteristic enzymic activity of ,B-lactamase II remain to be determined. The carbohydrate and protein were not dissociated by chromatography on DEAE-cellulose under the conditions used, but the loss of f-lactamase II activity when ,B-lactamase I is purified by adsorption on powdered glass or Celite (Sabath & Abraham, 1965;Kuwabara, 1970) raises the question whether a protein-carbohydrate complex is dissociated by this procedure. The finding that ,B-lactamase II appears in the culture fluid before f-lactamase I is of interest in relation to Eylar's (1965) suggestion that the carbohydrate moiety of glycoproteins promotes the transport of these proteins from cells and needs play no functional role in their biological activity.…”

“…Enzyme preparations. These were the crystalline preparations of ,8-lactamase I (stage 8) and ,-lactamase II (stage 7) described by Kuwabara (1970). Buffers B and BZ are also as described by Kuwabara (1970).…”

Protein and carbohydrate moieties of a preparation of β-lactamase II

“…both produced by Bacillus cereus 569H the latter enzyme differs from the former in requiring Zn2+ as a cofactor (Sabath & Abraham, 1966), in showing a wider range of activity, and in being less readily inactivated by heat (Crompton, Jago, Crawford, Newton & Abraham, 1962; Kuwabara & Abraham, 1967). Crystalline fi-lactamase II prepared by Kuwabara (1970) had a higher molecular weight than crystalline ,-lactamase I (Lloyd & Peacocke, 1970) and unlike the latter, which gave only amino acids on acid hydrolysis, yielded substantial quantities of neutral sugars and amino sugars in addition to amino acids (Kuwabara, Adams & Abraham, 1970). However, the nature of the association of carbohydrate with protein in this preparation of ,-lactamase II and the extent to which the carbohydrate moiety was responsible for its characteristic fl-lactamase activity remained unknown.…”