Bacillus cereus substrates

Massing U, Eibl H (1994) Substrates for phospholipase C and sphingomyelinase from Bacillus cereus. In Woolley P, Petersen SB (eds) Lipases. Their structure, biochemistry and application. Cambridge University Press, Cambridge, p 225... 

The suggestion has been made that certain peptides which have been studied ns inducers of /3-lactamase in Bacillus cereus and in S. aureus may serve as substrates for the enzyme [A. K. Saz and D. L. Lowery, BBRC 15, 525 (1964) 19, 102 (1965)1. However, there is no evidence that such peptides are in fact hydrolyzed by /3-lactamase. 

More recent staining procedures largely use fluorescent dyes to characterize the physiological and biochemical states of cells. Fluorescein Diacetate (FDA), a non-polar substance which crosses the membrane and is hydrolyzed by intracellular esterases in viable cells to produce fluorescein, exhibits yellow-green fluorescence when excited at 490 nm. Damaged or non-viable cells in general are unable to hydrolyze FDA or to retain fluorescein within the cell [172,173]. In combination with Ethidium Homodimer or Propidium Iodide, a similar esterase substrate, calcein acetoxy methyl ester (CAM) has been found to be reliable for viability assessment of protozoans, but not on Candida yeast, neither on bacteria such as Bacillus cereus and Escherichia coli [174]. 

A considerable amount of data has been reported on the substrate preference of the phospholipase C present in the organism Bacillus cereus. Interestingly, three phospholipases C have been isolated and purified, the first of which has high specificity for phosphatidylcholine, the second for phospha-tidylinositol, and the third for sphingomyelin (often termed sphingomyelinase). Similar substrate requirements have been noted in the phospholipase C isolated from other bacteria. 

The mechanism shown in Scheme 3 leaves open the question of whether the intermediate Gl,6-diP dissociates from the enzyme during catalysis. Substrate reorientation within the active site occurs at a faster rate than dissociation with PGM from rabbit muscle and from rat.201 This means that the substrate reorients by flipping within the confines of the active site, rather than by dissociating and rebinding. In contrast, dissociation is more rapid with the PGM from Bacillus cereus and Micrococcus lysodekticus, and as a result, the latter enzymes have an absolute requirement for Gl,6-diP as a cofactor to rephosphorylate the enzyme. 

Valero, M., Leontidis, S., Fernandez, P.S., Martinez, A., and Salmeron, M.C. 2000. Growth of Bacillus cereus in natural and acidified carrot substrates over the temperature range 5-30°C. Food Microbiology 17 605-612. 

Fernandez A, Cofiaudo J, Cunha EM, Ocio MJ, Martinez A (2002) Empirical model building based on Weibull distribution to describe the joint effect oh pH and temperature on the thermal resistance of Bacillus cereus in vegetale substrate. Int J Food Microbiol 77 147-153 Forney LJ, Zhou X, Brown CJ (2004) Molecular microbial ecology land of the one-eyed king. Curr Opin Microbiol 7 210-220... 

Chitosanases are useful enzymes to hydrolyze chitosan, thus producing dichitooligosaccharides, trichitooligosaccharides, and tetrachitooligosac-charides (Kurakake et al., 2000). A chitosanase with an MW of 45 kDa from Bacillus cereus SI was purified and characterized (Kurakake et al., 2000). Optimum pH for reaction incubation was about 6 at an optimum temperature of about 60 °C. This chitosanase was stable in basic solutions. Purified chitosanase has been used for many substrates, such as... 

Figure 8 L-Canavanine (9) is a slow substrate and de facto inhibitor of ADI and an irreversible inactivator of the ADI from Bacillus cereus. (a) Canavanine, in its neutral form, favors the amino tautomer, (b) Proposed mechanism of slow turnover of canavanine by ADI and irreversible inactivation of B. cereus ADI. Filled circles represent 0.

Figure 16 Schematic drawing of the active site of Bacillus cereus phospholipase C complexed with a phosphonate substrate analog inhibitor (reproduced by permission of Springer from Hergenrother and...

Many mathematical models of situations like the foregoing have been published see (, 18-25). Analyses of these models suggests that pure but not simple competition should often result in coexistence of competitors, even in systems in which spatial homogeneity of the environment is imposed and for which all external influences are time-invariant. Experimental data of Yoon et al. (22) on competition of Bacillus cereus and Candida tropicalis for the substitutable resources glucose and fructose show that coexistence in a chemostat is indeed possible here. Experimental testing of model predictions in situations of elementary but not simple competition is quite important, because the models used are necessarily those for multiple substrate limitation of growth, and all such models have low credibility, in my opinion. 

Continuous enzymatic production of xylitol with simultaneous coenzyme regeneration in a charged membrane reactor was studied (210). An NADH dependent xylose reductase from C. tenuis catalyzed the reduction of xylose. This was coupled to enzymatic oxidation of glucose by glucose dehydrogenase from Bacillus cereus to make achievable an up to 10,000-fold regeneration of NADH per cycle of discontinuous conversion. Under suitoble conditions, 300 g/L of substrate could be converted in yields above 96% in one single batch reaction. 

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 exploring the structural features of the active site, derivatives of penicillins which are substrates or competitive inhibitors of all )3-lac tamases should be useful for afiSnity labeling and active-site analysis. The method of preparation of penicillin isocyanates and the inactivation of an E. coli -lactamase, /8-lactamase75, and a -lactamase from Bacillus cereus are described herein. 

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-... 

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