Bacillus cereus penicillinase

B. The reagent contains a mixture of SchenLabs (SchenLabs Pharmaceuticals Inc., New York) purified Bacillus cereus penicillinase (10,000 units/ml), 1% starch, 0.1 iV iodine and M sodium phosphate buffer, pH 7, in the ratio 5 50 1 1. The rate of decolorization of the spray by the penicillin substrate is found to be dependent on the penicillinase cone, and, with this mixture, development is complete at room temperature (ca. 22°) in 10-15 min after hydrolysis of the j8-lactam ring with alkali or penicillinase. The resulting penicilloic acid rapidly consumes nine equivalents of iodine. Under suitable conditions it is found that both penicillins and the related products cepha-... 

It should be appreciated, however, that the enzymes of a functionally similar type may not infrequently be cell-bound in one species of bacterium and in another they may be extracellular—e.g.9 penicillinase and alkaline phosphatase of E. coli are cell-bound, whereas those of Bacillus cereus are extracellular (6). Even in the same strain of bacterium, an enzyme may be partly cell-bound and partially extracellular penicillinase of Bacillus licheniformis is an example (60). Consequently, the above classification is not so distinctive and may change, depending on the circumstances of cells or by their mutation. 

Derivatives of cellulose continue to be used as supports for new affinity chromatography media and for immobilization of enzymes. Trypsin has been immobilized on 4-aminobenzyl-cellulose. A penicillinase from Bacillus cereus has been immobilized by glutaraldehyde-mediated reaction with aminoethyl-cellulose. Tannin has been activated with cyanogen bromide and coupled with... 

Penicillin G and V Very useful procedures for the assay of jS-lactams, such as penicillin G and V, have been designed using j6-lactamases, such as penicillinase type 1 from Bacillus cereus (EC 3.5.2.6). The useful linear range is 0.005-200 mmol 1 Several industrial applications have been developed using both discrete samples and continuous monitoring on pilot-plant and production-scale fermentors. Alternatively, the penicillin amidase (EC 3.5.1.11) has been used which shows better specificity in fermentation broths. The sensitivity is, however, lower although sufficient for many purposes. In both cases, the enzyme columns are very stable and can be used for several months or for thousands of samples. 

Fig. 1. Time course of penicillinase induction in Bacillus cereus 569 after induction by 1 unit of benzylpenicillin/ml. Data from Pollock [12]. Cultures diluted 1 2 in fresh medium at 2 hours and every i hour thereafter. X = growth of induced culture = growth of control culture = total penicillinase in induced culture (units/ml original culture) = total penicillinase in control culture (units/ml original culture) O = specific activity of induced culture (penicillinase units/mg cells) A = specific activity of control culture.

Fig. 2. Comparison of the fixation of penicillin to cells of Bacillus cereus 569 and the rate of penicillinase synthesis reached by the cells during subsequent growth. Data from Pollock and Perret [13]. = bound penicillin O = rate of penicillinase synthesis.

Fig. 3. Induction of penicillinase by penicillin (6 /xg/ml) after growth of Bacillus cereus 569 cells in different media. Data from Csanyi et al. [15]. O = in standard casein hydrolysate medium A = in standard medium-1-0.1 M phosphate =in standard medium - - 0.3 M phosphate.

Fig. 4. A. Changes in penicillinase specific activity of Bacillus cereus 569 cells on re-growth after washing. Data from Csanyi [29]. A = control culture (untreated) = culture of density 0.25 mg dry weight/ml O = culture of density 0.125 mg dry weight/ml = culture of density 0.075 mg dry, weight/ml. All cultures were resuspended at a density of 0.1 mg dry weight/ml. B. Penicillinase specific activity 60 minutes after the treatment described in A [Csanyi, 29], related to the density of the original culture.

Fig. 5. Time course of penicillinase induction in Bacillus cereus 569 with different inducer concentrations [Csanyi, 29]. A = 10 M benzylpenicillin B = 10 M benzyl-penicillin C = 10" M benzylpenicillin.

Cephalosporin C, which contains a dihydrothiazine-j8-lactam ring system, shows at least 10% of the activity of cephalosporin N against Staph, aureus and induces the formation of the enzyme penicillinase by Staph, aureus and Bacillus cereus. This may be attributed to the fact that the stereochemistry of a major part of the cephalosporin C nucleus is similar to that of 6-aminopenicillanic acid. The precise factors which determine the ability of a substance to act as an inducer of penicillinase are clearly different from those which make the substance a good substrate of the enzyme. The /9-lactam ring in cephalosporin C is insensitive to penicillinase. However, the affinity of a substance for a penicillinase varies with the source of the enzyme. Cephalosporin C acts as a competitive inhibitor of the action of penicillinase from Bacillus cereus, but not from Staph, aureus, on benzylpenicillin. In contrast, the V-phenylacetyl derivative of 7-aminocephalosporanic acid is a powerful inhibitor of the staphylococcal enzyme12... 

In this way Staphylococcus aureus becomes resistant to penicillin in the clinic. Penicillin-resistant strains isolated from patients secrete the enzyme p-lactamase ( penicillinase ). This enzyme hydrolyses the drug to penicil-loic acid, which is biologically inert (see Section 12.i). Penicillinase-producing staphylococci are inherently quite sensitive to penicillin. Hence small inocula can be inhibited by low concentrations of the antibiotic. It is, in effect, a race between the speed with which penicillin can kill the bacteria and the speed with which they can produce enough of the enzyme to destroy the penicillin (Knox, 1962). Actually penicillin can be made to induce some strains of Staph, aureus to produce penicillinase. No permanently resistant population of this bacterium has arisen in this way, and the organisms return fairly rapidly to the uninduced susceptible state when the penicillin is withdrawn. Much of the detail of penicillinase-induction was first worked out in Bacillus cereus (Pollock and Ferret, 1951). 

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