The catalatic activity of chloroperoxidase (CPO) was demonstrated to exhibit saturation kinetics under steady-state conditions, which were not observed with catalase under comparable conditions. Results were obtained using reaction mixtures of CPO and H2O2 at pH 6.2, rapid spectral scan and single wavelength measurements, and transient- and steady-state reaction conditions. The observed rectangular hyperbolae (measurement of rates of disappearance of H2O2 and appearance of O2) could be fit quantitatively to [equation: see text] where v is rate of O2 evolution, [CPO]0 is total enzyme concentration, B1 = (9 +/- 1) x 10(2) s-1, and B2 = (3.3 +/- 0.4) x 10(-3) M. The results indicated formation of a complex of compound I (CPO-I) and H2O2, which dissociated to native CPO, O2, and H2O with a rate constant of (9 +/- 1) x 10(2) s-1. The determination of the peroxidatic activity of CPO was performed using demethylation of N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) under steady-state conditions. Attempts to determine Michaelis-Menten constants for the substrates TMPD and H2O2 gave rise to apparently anomalous data. Our data showed that the modified ping-pong mechanism established for horseradish peroxidase is applicable to the peroxidatic reaction catalyzed by chloroperoxidase. Both peroxidatic and catalatic reactions occurred in the reaction system containing H2O2, a reducing substrate, and CPO. A combined reaction mechanism was proposed for CPO-catalyzed reactions in which the modified ping-pong mechanism was applicable for the peroxidatic reactions and the formation of a CPO-I-H2O2 complex occurred for the catalatic reaction.
Resistance to piperacillin in several isolates of Citrobacter freundii and Enterobacter cloacae was investigated and confirmed to occur at a frequency of 10(-7) to 10(-6). Development of resistance to piperacillin was significantly suppressed by tazobactam but not by clavulanic acid. To elucidate the mechanism by which resistance suppression occurs, the effect of piperacillin plus tazobactam on the induction of AmpC beta-lactamase was analyzed by monitoring the beta-galactosidase activity of an inducible ampC-lacZ gene fusion in Escherichia coli. The combination exerted no inhibitory effect on AmpC beta-lactamase induction. Tazobactam also had no effect on the accumulation of a key intermediate in the AmpC beta-lactamase induction pathway, 1,6-anhydromurotripeptide, in an ampD mutant strain of E. coli. However, the addition of tazobactam to liquid cultures of E. cloacae 40001 in the presence of piperacillin at four times the MIC caused a delay in the recovery of the culture to piperacillin-induced stress. At 16 times the MIC, a complete suppression of regrowth occurred. Analysis of culture viability on piperacillin plates showed that the culture recovery was due to growth by moderately resistant mutants preexisting in the cell population, which at 16 times the MIC became susceptible to the combination. Evidence from the kinetics of inhibition of the E. cloacae 40001 AmpC beta-lactamase by clavulanic acid, sulbactam, and tazobactam and from the effects of these drugs on the frequency of resistance to piperacillin suggests that the suppressive effect of tazobactam on the appearance of resistance is primarily mediated by the beta-lactamase inhibitory activity.
Chloroperoxidase (CPO) purified from Caldariomyces fumago CMI 89362 was covalently bound to aminopropyl-glass by using a modification of an established method. Acid-washed glass was derivatized by using aminopropyltriethoxysilane, and the enzyme was ionically bound at low ionic strength. Further treatment with glutaraldehyde covalently linked the enzyme to the glass beads in an active form. No elution of bound activity from glass beads could be detected with a variety of washings. The loading of enzyme protein to the glass beads was highest, 100 mg of CPO per g of glass, at high reaction ratios of CPO to glass, but the specific activity of the immobilized enzyme was highest, 36% of theoretical, at low enzyme-to-carrier ratios. No differences in the properties of the soluble and immobilized enzymes could be detected by a number of criteria: their pH-activity and pH-stability profiles were similar, as were their thermal stabilities. After five uses, the immobilized enzyme retained full activity between pH 6.0 and 6.7.
A rapid and sensitive colorimetric assay was developed for the estimation of chloroperoxidase activity. N,N,N',N'-Tetramethyl-p-phenylenediamine was chosen from four potential chromogenic substrates because the blue product resulting from chloroperoxidase conversion gave the highest molar absorption. This product exhibited two absorbance maxima, at 563 and 610 nm. Activity was monitored at 563 nm, and the product absorbance was stable for at least 1 h at 10 degrees C after treatment with an equal volume of a mixture (40:1) of methanol and phosphoric acid (85% w/v), pH 2. The linear range of the assay with respect to enzyme amount was determined. The assay was developed using soluble chloroperoxidase but worked well with the enzyme immobilized on glass beads.
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