Comparative evaluation of stability of benzylpenicillin in acid media of natural solutions and in culture fluid]

Inactivation of benzylpenicillin in real media i.e. fermentation broths and their filtrates was studied in comparison with the published data on inactivation of commercial benzylpenicillin in aqueous solutions as dependent on the medium pH and temperature. The lowest constant of benzylpenicillin inactivation was shown to be in the fermentation broths.     

Integrated L-phenylalanine separation in an E. coli fed-batch process: from laboratory to pilot scale

Pilot-scale reactive-extraction technology for fully integrated L-phenylalanine (L-Phe) separation in Escherichia coli fed-batch fermentations was investigated in order to prevent an inhibition of microbial L-Phe production by-product accumulation. An optimal reactive-extraction system, consisting of an organic kerosene phase with the cation-selective carrier DEHPA (di-2-ethylhexyl phosphonic acid) and an aqueous stripping phase including sulphuric acid, was found particularly efficient. Using this system with two membrane contactors, mass-transfer coefficients of up to 288 x 10(-7) cm s(-1) for the aqueous/organic and 77 x 10(-7) cm s(-1) for the organic/stripping phase were derived from experimental data using a simple modelling approach. Concentration factors higher than 4 were achieved in the stripping phase as compared to the aqueous donor phase. Reactive extraction enabled a 98% cation portion of L-Phe in the stripping phase, leading to final product purity higher than 99% after L-Phe precipitation. A doubling of L-Phe/glucose yield was observed when kerosene/DEHPA was added to the fermentation solution in the bioreactor to experimentally simulate a fully integrated L-Phe separation process.     

Does Bioavailability Limit Biodegradation? A Comparison of Hydrocarbon Biodegradation and Desorption Rates in Aged Soils

In order to determine whether bioavailability limits the biodegradability of petroleum hydrocarbons in aged soils, both the biodegradation and abiotic desorption rates of PAHs and n-alkanes were measured at various time points in six different aged soils undergoing slurry bioremediation treatment. Alkane biodegradation rates were always much greater than the respective desorption rates, indicating that these saturated hydrocarbons apparently do not need to be dissolved into the aqueous phase prior to metabolism by soil microorganisms. The biodegradation of PAHs was generally not mass-transfer rate limited during the initial phase, while it often became so at the end of the treatment period when biodegradation rates equaled abiotic desorption rates. However, in all cases where PAH biodegradation was not observed or PAH removal temporarily stalled, bioavailability limitations were not deemed responsible for this recalcitrance since these PAHs desorbed rapidly from the soil into the aqueous phase. Consequently, aged PAHs that are often thought to be recalcitrant due to bioavailability limitations may not be so and therefore may pose a greater risk to environmental receptors than previously thought.     

Acyloxymethyl as a drug protecting group. Part 7: Tertiary sulfonamidomethyl ester prodrugs of benzylpenicillin: chemical hydrolysis and anti-bacterial activity

Tertiary sulfonamidomethyl esters of benzylpenicillin (4) were synthesised and evaluated as a new class of potential prodrugs for beta-lactam antibiotics. Their hydrolysis in aqueous buffers was studied by HPLC and reveal a U-shaped pH rate profile with a pH-independent process extending from ca. pH 2 to ca. pH 10. This pathway is characterised by kinetic data that are consistent with a unimolecular mechanism involving rate-limiting iminium ion formation and penicillinoate expulsion. Benzylpenicillin and the corresponding sulfonamide are the ultimate products detected and isolated, indicating that beta-lactam ring opening is much slower than ester hydrolysis. As expected from the high reactivity, benzylpenicillin esters (4) displayed similar in vitro antibacterial activity to benzylpenicillin itself. Compared to the benzylpenicillin derivatives, sulfonamidomethyl esters of benzoic, clofibric and valproic acids display a much higher stability, giving rise to a Br?nsted beta1g value of -0.96 and suggesting that tertiary sulfonamidomethyl esters may be useful prodrugs for carboxylic acid drugs with pKa > 4.     

Determination of lipid loss during aqueous and phase partition fixation using formalin and glutaraldehyde

Phase partition fixation permits fixation of tissue in a nonaqueous environment, thus eliminating osmotic effects. It was shown in an earlier investigation that retention of protein in liver blocks can be improved by phase partition fixation. By using radioisotopic labeling techniques, the effects of phase partition fixation on lipid retention during fixation, dehydration, and clearing have been determined and compared with those of standard aqueous fixation techniques. In this article we show that retention of total lipid in liver blocks following phase partition fixation using formalin was comparable to or better than that with aqueous formalin fixation and processing. Fixation with glutaraldehyde using phase partition fixation resulted in somewhat greater loss of total lipid than that observed for aqueous buffered glutaraldehyde-fixed blocks.     

Influence of soil-water ratio on the performance of slurry phase bioreactor treating herbicide contaminated soil

The influence of soil-water ratio was studied on the performance of the slurry phase bioreactor operated in sequencing batch mode (anoxic-aerobic-anoxic microenvironments) during the bioremediation of soil contaminated with pendimethalin. The performance of the reactors was evaluated at different soil-water ratios (1:5-1:25; at soil loading rate (60 kg of soil/cum-day to 12 kg of soil/cum-day)) keeping the loading rate of pendimethalin constant (133.2 g/kg of soil-day) in six reactors and variable (66.6 g/kg of soil-day to 166.6 g/kg of soil-day) in other four reactors. At 1:20 soil-water ratio, the slurry phase system showed enhanced degradation of substrate (629 microg pendimethalin/g soil). The removal efficiency of pendimethalin in the reactors was dependent on the mass-transfer rates of the substrate from the soil to the aqueous phase. Soil-water ratio and substrate loading rates showed significant influence on the substrate portioning, substrate degradation efficiency and substrate desorption rate.     

Influence of hydrodynamic conditions on naphthalene dissolution and subsequent biodegradation

The influence of hydrodynamic conditions on the dissolution rate of crystalline naphthalene as a model polycyclic aromatic hydrocarbon (PAH) was studied in stirred batch reactors with varying impeller speeds. Mass transfer from naphthalene melts of different surface areas to the aqueous phase was measured and results were modeled according to the film theory. Results were generalized using dimensionless numbers (Reynolds, Schmidt, and Sherwood). In combined mass transfer and biodegradation experiments, the effect of hydrodynamic conditions on the degradation rate of naphthalene by Pseudomonas 8909N was studied. Experimental results were mathematically described using mass-transfer and microbiological models. The experiments allowed determination of mass-transfer and microbiological parameters separately in a single run. The biomass formation rate under mass transfer limited conditions, which is related to the naphthalene biodegradation rate, was correlated to the dimensionless Reynolds number, indicating increased bioavailability at increased mixing in the reactor liquid. The methodology presented in which mass transfer processes are quantified under sterile conditions followed by a biodegradation experiment can also be adapted to more complex and realistic systems, such as particulate, suspended PAH solids or soils with intrapartically sorbed contaminants when the appropriate mass-transfer equations are incorporated.     

Sensitivity to antibiotics of pneumococci isolated from patients with chronic nonspecific pneumonia and bronchitis

Four hundred and twenty two pneumococcal strains isolated from 300 patients with chronic nonspecific pneumonia and bronchitis were studied with respect to their sensitivity to 18 antibiotics within a period from 1982 to 1985. It was shown with the method of serial dilutions on solid media that 91.7, 87.8, 85 and 81 per cent of the isolates were sensitive to benzylpenicillin, ampicillin, lincomycin and cefuroxime, respectively. A significant percentage of the pneumococcal strains had decreased sensitivity to benzylpenicillin (MIC close to the therapeutic concentration). On this basis it was recommended to use lower concentrations of benzylpenicillin (less than 0.25 units/ml) in assay of sensitivity in clinical strains of Pneumococcus.     

The inhibition of mucopeptide synthesis by benzylpenicillin in relation to irreversible fixation of the antibiotic by staphylococci

1. Benzylpenicillin is irreversibly fixed to staphyloccoci by a reaction that obeys second-order kinetics, whereas the progress of inhibition of mucopeptide synthesis obeys first-order kinetics after a short lag during which the antibiotic has no effect. 2. When the micro-organisms are saturated with benzylpenicillin they can still make mucopeptide in solutions containing chloramphenicol at a normal rate after a lag period. 3. About 90% of the benzylpenicillin stays fixed to the cells after mucopeptide synthesis has reached its maximum and constant rate. 4. During the phase when mucopeptide synthesis by cells saturated with benzylpenicillin is accelerating, a small number of additional sites that fix benzylpenicillin is revealed. The number of these sites reaches a maximum and constant value at about the same time as mucopeptide biosynthesis reaches a maximum and constant rate. 5. Staphylococci saturated with benzylpenicillin are exceedingly sensitive to fresh additions of the antibiotic. 6. The degree of inhibition of mucopeptide synthesis caused by these small amounts of antibiotic agrees with the degree of substitution by benzylpenicillin of the newly revealed or `sensitive' sites. 7. Since these sensitive sites are revealed during incubation of the bacteria with chloramphenicol it is unlikely that they are due to newly formed protein. 8. On the basis of these results, a hypothesis for the inhibition by penicillin of the cross-linking reaction in the terminal stages of mucopeptide synthesis is suggested.     

Concentration of ultrafiltered benzylpenicillin broths by reverse osmosis

Concentration of benzylpenicillin filtered broths purified by ultrafiltration and fermented broths clarified by ultrafiltration was carried out by reverse osmosis. This study was done using a reverse osmosis laboratory/pilot installation from Paterson Candy International Ltd. whose module has a tubular configuration and a membrane with a molecular weight cut-off of 100 Dalton (ZF99) made of a polyamide film with a rejection of 99% of NaCl. It was concluded that reverse osmosis is an adequate technique to concentrate benzylpenicillin ultrafiltered broths, leading to low losses of benzylpenicillin in the permeate and high benzylpenicillin recovery for high volumetric concentration factors.     

The effect of temperature on the kinetics of a reverse micellar protein extraction process

Summary The kinetics of transfer of chymotrypsin from an aqueous into a reverse micellar phase and vice versa has been examined for a two-phase system with planar interface equipped with vibrating disks for separate mixing of both phases. The temperature dependence of the mass-transfer coefficients gives two values for the forward transfer (30 and 90 kJ/mol) depending on the temperature range. Only one value is obtained for the backward transfer (60kJ/mol).     

The effect of benzylpenicillin on the activity of antioxidative enzymes in the erythrocytes and on methemoglobin formation in children

Activity of erythrocyte antioxidative enzymes and the content of methemoglobin were studied in 36 healthy children under preventive treatment with benzylpenicillin and in 65 healthy children of the control group. It was shown that there was relationship between the changes in the activity of superoxidodismutase and catalase and the antibiotic dose and duration of the use. After benzylpenicillin intramuscular administration for 3-4 days (the total dose of 80,000-3000000 units) the catalase activity decreased to 65.6 per cent while the activity of superoxidodismutase did not change. When the antibiotic was used for 1-2.5 weeks (the total dose of 3000000-6000,000 units) the activity of catalase and superoxidodismutase decreased to 53.6 and 82 per cent respectively. Beginning from the 3rd week of the antibiotic use the catalase activity increased to 71.6 per cent while the superoxidodismutase activity did not change and remained at the level of 84.9 per cent. The content of methemoglobin in the children treated with benzylpenicillin was about 3 times higher than that in the controls. The correlation between the activity of the antioxidative enzymes and the content of erythrocyte methemoglobin was inverse: the lower was the enzyme activity, the higher was the content of methemoglobin and vice versa. It was concluded that benzylpenicillin impaired definite balance between single electron reduction of oxygen and antioxidative protection resulting in the antibiotic adverse action i. e. increased methemoglobin formation. The effects of benzylpenicillin should be considered when it is used in combination with oxidants in treatment of children.     

Cryoenzymology of Bacillus cereus beta-lactamase II

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental studies to set hygienic standards for benzylpenicillin in the air of a work area

The character and level of benzylpenicillin toxic effect was studied in accordance with the methodic instruction for performance of the experiments on toxicological estimation and hygienic norms of antibiotics in the air of working areas in production premises. It was shown that the antibiotic was of low toxicity under conditions of a single exposure, did not cumulate and had no local irritating and resorbing effect. It was shown to be an allergen. Prolonged inhalation of benzylpenicillin in a concentration of 14 mg/m3 resulted in reversible changes in the function of the kidneys and liver of the animals, their sensitization and development of pronounced dysbacteriosis of the mouth and intestine mucosa. In a concentration of 1 mg/m3 benzylpenicillin had no general toxic and sensitizing effect but induced reversible changes in the normal autoflora. This allowed the authors to consider the above concentration to be maximum with respect to the antibacterial effect. On the basis of the experiments the maximum permissible norm (MPN) of the aerosol of benzylpenicillin disintegration was recommended at the level of 0.1 mg/m3.     

Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus beta-lactamase II

Rapid-scanning stopped-flow spectroscopy (425-700 nm) has been used to study spectral changes in cobalt(II)-substituted Bacillus cereus beta-lactamase II during the binding and hydrolysis of benzylpenicillin. The experiments were carried out in aqueous solution over a temperature range of 3-20 degrees C. Three metallointermediates have been characterized by their visible absorption spectra. Two of them have visible absorption spectra identical with the intermediates ES1 and ES2 previously observed at subzero temperatures in a mixed aqueous/organic solvent [Bicknell, R., & Waley, S.G. (1985) Biochemistry 24, 6876-6887]. In addition, the branched kinetic pathway observed with the zinc(II) and cobalt(II) beta-lactamase II at subzero temperatures has been shown to occur with the cobalt(II)-substituted enzyme in aqueous solution at above-zero temperatures; thus, at pH 6.0 and 3 degrees C, the rate and equilibrium constants are readily determined for the reaction scheme: (Formula: see text). A third transient intermediate (called ES*) was found to precede ES1 in the pre-steady-state time period. The identity of the intermediates formed in aqueous solution with those previously observed in the cryostudy confirms that the mechanism is not changed either by the presence of an organic cosolvent or by subzero temperatures. Further characterization of ES1 and the steady-state intermediate ES2 at subzero temperatures, where their lifetime may be extended for up to several hours, has involved circular and magnetic circular dichroic studies. The magnetic circular dichroic spectra identify changes in the coordination sphere of the active-site metal during catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of sulfalene and furosemide on benzylpenicillin kinetics in rats with aseptic inflammation

The effect of sulfalen and furosemide on benzylpenicillin kinetics in blood serum, intact tissues and aseptic inflammation foci was studied on rats. It was shown that under the action of sulfalen and furosemide protein binding of benzylpenicillin lowered by 30 per cent. The changes in the antibiotic kinetics after combined use with sulfalen and furosemide were of the same type: markedly increased concentrations in blood serum and tissues and retarded elimination.     

Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.     

Immunomodulating effect of levamisole in antibiotic therapy

The use of ampicillin, benzylpenicillin, gentamicin, erythromycin, cefamezine or cephaloridine in combination with levamisole aimed at increasing their efficacy and correcting the adverse reactions was studied. Immunological activity of the antibiotics was comparatively characterized by using standard methods. It was shown that the character of the antibiotic effect on the basic factors of nonspecific resistance and immunity was heterogeneous. Schemes for using the antibiotics in combination with levamisole for correcting the detected disorders in the immunity status of the experimental animals were developed.     

Dibenzothiophene Biodegradation by a Pseudomonas sp. in Model Solutions

The presence of a fatty acid and an n-alkane may affect the biodegradation rate of aromatic sulphur compounds such as dibenzothiophene (DBT). A fatty acid (hexadecanoic acid) may form micellar structures favouring DBT bioavailability. n-Alkanes, such as n-dodecane or n-hexadecane, form a film around the aromatic sulphur molecule as a consequence of solvation, thus increasing DBT bioavailability. The mass-transfer rate from the solid to the aqueous phase controls the DBT biodegradation rate when DBT is the only carbon source. Diffusional coassimilation and microbial hydrophobic effects are rate-limiting steps in DBT biodegradation in the presence of aliphatic compounds. Diffusion depends on the DBT concentration in n-alkane, while cometabolism is associated with different n-alkane biodegradation rates. Through the definition of biodesulphurization selectivity and biodesulphurization efficiency, our investigations have shown that a selective aerobic biodesulphurization process is possible by using an unselective biocatalyst, such as a Pseudomonas sp.     

Destabilization of emulsions during extraction of antibiotics from natural solutions]

Various means for changing the aggregative stability of emulsions in the system of benzylpenicillin fermentation broth filtrate and butyl acetate were tested. It was shown that the use of desemulgators was more efficient than the use of some means for removing the desemulgating admixtures. Moreover, addition of surface active substances after the preliminary thermocoagulation resulted in maximum decreasing of the emulsion aggregative stability.