Cometabolic biodegradation of 4-chlorophenol by sequencing batch reactors at different temperatures

The simultaneous removal of 4-chlorophenol (4-CP) and phenol in lab-scale sequencing batch reactors at different temperatures has been studied. Phenol feed concentration was fixed at 525 mg/L and 4-CP concentration was increased from 105 to 2100 mg/L at a constant hydraulic residence time (HRT) of 10.5 d. Complete phenol and 4-CP biodegradation was achieved during the aerobic stage working with 4-CP concentrations up to 1470 mg/L in the feed. Both 4-CP and phenol specific initial removal rates were strongly affected by 4-CP feed concentration and temperature. Only at the highest temperature tested (35 °C) it was possible to increase the maximum assimilative 4-CP concentration by the biological sludge up to 2100 mg/L, and a significant reduction of the ecotoxicity of the effluents was observed. 4-chlorocatechol (4-CC) was identified as the major intermediate in the aerobic cometabolic 4-CP degradation, being the ecotoxicity of that species substantially lower than that of 4-CP.     

Biodegradation of 2-Chlorophenol by Rhodopseudomonas palustris

A phototrophic bacterial culture that assimilates 2-chlorophenol (2-CP) was enriched from effluents of paper and wood industry. The isolated bacterium was identified as Rhodopseudomonas palustris based on its morphological characteristics, biochemical reactions, and fatty acid methyl ester gas chromatography (FAME-GC) analysis. The bacterium R. palustris being photoheterotrophic in nutrition was also able to switch between phototropic, aerobic, and anaerobic modes of metabolism, as evidenced by modulation of the bacterial photopigments. The switching of anaerobic to aerobic metabolism was evidenced by the presence of catechol, a product of aerobic oxidation of 2-CP in the spent medium and the disappearance of photopigment-specific absorbance in the organism. R. palustris degraded about 97% of the supplemented 2-CP from the culture medium in 40 days along with production of an exo-polysaccharide, which probably provides protection from substrate toxicity and enhances its bioavailability. Thin-layer chromatography (TLC) and gas chromoatography–mass spectrometry (GC-MS) analysis of the R. palustris spent medium extracts indicated the presence of phenol. The GC-MS data also indicated that phenol is metabolized through an ortho-cleavage pathway. Further analysis of the R. palustris cell-free extracts for enzymes showed the presence of a chlorophenol dehalogenase (CD) and a chlorophenol nicotinamide adenine dinucleotide phosphate (NADPH)-oxidoreductase (CNOR) with respective specific activities of 0.114 and 0.26 μmol/min/mg, suggesting an initial reductive dechlorination step during 2-CP catabolism. The study thus highlights the important roles of phototrophic bacteria in the decontamination of environmental pollutants from the polluted sites.     

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

Biodegradation of the mixtures of 4-chlorophenol and phenol by Comamonas testosteroni CPW301

A 4-chlorophenol (4-CP)-degrading bacterium, strain CPW301, was isolated from soil and identified as Comamonas testosteroni. This strain dechlorinated and degraded 4-CP via a meta-cleavage pathway. CPW301 could also utilize phenol as a carbon and energy source without the accumulation of any metabolites via the same meta-cleavage pathway. When phenol was added as a additional substrate, CPW301 could degrade 4-CP and phenol simultaneously. The addition of phenol greatly accelerated the degradation of 4-CP due to the increased cell mass. The simultaneous degradation of the 4-CP and phenol is useful not only for enhanced cell growth but also for the bioremediation of both compounds, which are normally present in hazardous waste sites as a mixture.     

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant: Isolation and Characterization 1

Nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisum sativum L.) seedlings and was purified 65-fold. The purified enzyme exhibits one predominant protein band by polyacrylamide gel electrophoresis, which corresponds to the dehydrogenase activity as measured by the nitro blue tetrazolium technique. The reaction is readily reversible, the pH optima for the forward (nicotinamide adenine dinucleotide phosphate reduction) and reverse reactions being 8.4 and 6.0, respectively. The enzyme has different cofactor and inhibitor characteristics in the two directions. Manganese ions can be used as a cofactor for the reaction in each direction but magnesium ions only act as a cofactor in the forward reaction. Zinc ions, and to a lesser extent calcium ions, inhibit the enzyme at low concentrations when magnesium but not manganese is the metal activator. It is suggested that there is a fundamental difference between magnesium and manganese in the activation of the enzyme. The enzyme shows normal kinetics and the Michaelis contant for each substrate was determined. The inhibition by nucleotides, nucleosides, reaction products, and related compounds was studied. The enzyme shows a linear response to the mole fraction of reduced nicotinamide adenine dinucleotide phosphate when total nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate plus reduced nicotinamide adenine dinucleotide phosphate) is kept constant. Isocitrate in the presence of divalent metal ions will protect the enzyme from inactivation by p-chloromercuribenzoate. Protection is also afforded by manganese ions alone but not by magnesium ions alone There is a concerted inhibition of the enzyme by oxalacetate and glyoxylate.     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Cometabolic bioregeneration of activated carbons loaded with 2-chlorophenol

Thermally and chemically activated carbons were used to investigate the extent of cometabolic bioregeneration in laboratory scale activated sludge reactors. Bioregeneration was determined and quantified by measuring the substrate and chloride concentrations, oxygen uptake rates, and deterioration in adsorption capacities. Activated carbons loaded with 2-chlorophenol could be partially bioregenerated in the presence of phenol as the growth substrate. The occurrence of exoenzymatic bioregeneration was also possible during cometabolic bioregeneration of thermally activated carbons. However, cometabolic bioregeneration of chemically activated carbons was much superior compared with thermally activated carbons. In cometabolic bioregeneration of activated carbons loaded with 2-chlorophenol, biodegradation, rather than desorption, was the rate-limiting step. Environmental Scanning Electron Microscopy analyses showed that groups of cocci-shaped phenol-oxidizers were attached to the outer surface or internal cavities of activated carbon as a fingerprint of bioregeneration.     

Sequential anaerobic degradation of 2,4-dichlorophenol in freshwater sediments.

2,4-Dichlorophenol (2,4-DCP) was anaerobically degraded in freshwater lake sediments. From observed intermediates in incubated sediment samples and from enrichment cultures, the following sequence of transformations was postulated. 2,4-DCP is dechlorinated to 4-chlorophenol (4-CP), 4-CP is dechlorinated to phenol, phenol is carboxylated to benzoate, and benzoate is degraded via acetate to methane and CO2; at least five different organisms are involved sequentially. The rate-limiting step was the transformation of 4-CP to phenol. Sediment-free enrichment cultures were obtained which catalyzed only the dechlorination of 2,4-DCP, the carboxylation of phenol, and the degradation of benzoate, respectively. Whereas the dechlorination of 2,4-DCP was not inhibited by H2, the dechlorination of 4-CP, and the transformation of phenol and benzoate were. Low concentrations of 4-CP inhibited phenol and benzoate degradation. Transformation rates and maximum concentrations allowing degradation were determined in both freshly collected sediments and in adapted samples: at 31 degrees C, which was the optimal temperature for the dechlorination, the average adaptation time for 2,4-DCP, 4-CP, phenol, and benzoate transformations were 7, 37, 11 and 2 days, respectively. The maximal observed transformation rates for these compounds in acclimated sediments were 300, 78, 2, 130, and 2,080 micromol/liter(-1)/day(-1), respectively. The highest concentrations which still allowed the transformation of the compound in acclimated sediments were 3.1 m/M 2,4-DCP, 3.1 mM 4-CP, 13 mM phenol, and greater than 52 mM benzoate. The corresponding values were lower for sediments which had not been adapted for the transformation steps.     

Impact of iron-complex (Fe(III)-NTA) on photoinduced degradation of 4-chlorophenol in aqueous solution.

In this research work, the photochemical impact of Fe(III)-nitrilotriacetic acid complex (FeNTA) on the fate of an organic pollutant (4-chlorophenol (4-CP)) was investigated in natural waters. The quantum yields of the photodecomposition of the FeNTA complex and of Fe(II) formation, by an intra-molecular photoredox process (the first stage of the reaction) are high. This photoredox reaction represents the first step of the process leading to 4-CP disappearance. Whereas oxygen does not affect FeNTA photodegradation, 4-CP depletion requires the presence of oxygen. The radical species HO and CO3*- responsible of the degradation were identified by ESR spectroscopy under irradiation. Two different wavelength-dependent mechanisms of 4-chlorophenol degradation are proposed. It clearly appears that under solar irradiation, iron organic complexes like FeNTA can play a significant role on the fate of the organic compounds present in natural waters.     

Glutamate dehydrogenase from Mycoplasma laidlawii

Mycoplasma laidlawii possesses a single glutamate dehydrogenase (GDH) with dual coenzyme specificity [specificity for nicotinamide adenine dinucleotide (H) and nicotinamide adenine dinucleotide phosphate (H)]. A purification procedure is reported which results in an enzyme preparation with a specific activity of 79.5 units/mg and which displays only one significant protein band after gel electrophoresis. This one band was determined, by activity staining, to have all of the GDH nucleotide specificities. The molecular weight of the enzyme is 250,000 +/- 10%, and it has a subunit size of about 48,000. The enzyme exhibits measurable activity with aspartate and pyruvate but is inactive with eight other possible substrates. Purine nucleotides do not affect the activity. The K(m) for reduced nicotinamide adenine dinucleotide was 1.8 x 10(-4)m. The optimal substrate concentrations and pH optimum for each of the respective GDH activities are also reported.     

Characterization of 4-hydroxyphenylacetate 3-hydroxylase (HpaB) of Escherichia coli as a reduced flavin adenine dinucleotide-utilizing monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH(2))-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH(2) in vitro, HpaB was able to use FADH(2) and O(2) for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH(2) for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH(2)-utilizing monooxygenase. FADH(2) generated by Fre was rapidly oxidized by O(2) to form H(2)O(2) in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH(2) and transitorily protected it from rapid autoxidation by O(2). When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O(2) for FADH(2) utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH(2) produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH(2) was autoxidized by O(2), causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH(2)-utilizing monooxygenase that uses FADH(2) as a substrate rather than as a cofactor.     

Enzymatic oxidation of NADP+ to its 4-oxo derivative is a side-reaction displayed only by the adrenodoxin reductase type of ferredoxin-NADP+ reductases

We have previously shown that Mycobacterium tuberculosis FprA, an NADPH-ferredoxin reductase homologous to mammalian adrenodoxin reductase, promotes the oxidation of NADP(+) to its 4-oxo derivative 3-carboxamide-4-pyridone adenine dinucleotide phosphate [Bossi RT, Aliverti A, Raimondi D, Fischer F, Zanetti G, Ferrari D, Tahallah N, Maier CS, Heck AJ, Rizzi M et al. (2002) Biochemistry41, 8807-8818]. Here, we provide a detailed study of this unusual enzyme reaction, showing that it occurs at a very slow rate (0.14 h(-1)), requires the participation of the enzyme-bound FAD, and is regiospecific in affecting only the C4 of the NADP nicotinamide ring. By protein engineering, we excluded the involvement in catalysis of residues Glu214 and His57, previously suggested to be implicated on the basis of their localization in the three-dimensional structure of the enzyme. Our results substantiate a catalytic mechanism for 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation in which the initial and rate-determining step is the nucleophilic attack of the nicotinamide moiety by an active site water molecule. Whereas plant-type ferredoxin reductases were unable to oxidize NADP(+), the mammalian adrenodoxin reductase also catalyzed this unusual reaction. Thus, the 3-carboxamide-4-pyridone adenine dinucleotide phosphate formation reaction seems to be a peculiar feature of the mitochondrial type of ferredoxin reductases, possibly reflecting conserved properties of their active sites. Furthermore, we showed that 3-carboxamide-4-pyridone adenine dinucleotide phosphate is good ligand and a competitive inhibitor of various dehydrogenases, making this nucleotide analog a useful tool for the characterization of the cosubstrate-binding site of NADPH-dependent enzymes.     

Purification and properties of alpha-ketoglutarate reductase from Micrococcus aerogenes

Micrococcus aerogenes grown in media containing glutamate has high levels of glutamate dehydrogenase and alpha-ketoglutarate reductase. The latter enzyme catalyzes the reversible reduction of alpha-ketoglutarate to alpha-hydroxyglutarate in the presence of reduced nicotinamide adenine dinucleotide (NADH). The enzyme has a high specificity for both substrates in either direction and displays Michaelis-Menten kinetics at moderate substrate concentrations. K(m) values of 0.12 to 0.17 mm alpha-ketoglutarate and 0.3 mm NADH for the forward reaction were calculated from data obtained at low substrate concentrations. At high concentrations, this reaction was inhibited by both substrates. The reverse reaction, which proceeded at 0.1 to 0.2 times the rate of the forward reactions, was inhibited by one of the products, alpha-ketoglutarate. K(m) values for the substrates of this reaction were 10 mm for alpha-hydroxyglutarate and 1 mm for nicotinamide adenine dinucleotide. alpha-Ketoglutarate reductase has a molecular weight of 7.5 x 10(4) to 8.2 x 10(4) and is composed of identical polypeptide chains with a molecular weight of 3.6 x 10(4) to 3.8 x 10(4).     

Chlorophenol degradation coupled to sulfate reduction.

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Chlorophenol degradation coupled to sulfate reduction

We studied chlorophenol degradation under sulfate-reducing conditions with an estuarine sediment inoculum. These cultures degraded 0.1 mM 2-, 3-, and 4-chlorophenol and 2,4-dichlorophenol within 120 to 220 days, but after refeeding with chlorophenols degradation took place in 40 days or less. Further refeeding greatly enhanced the rate of degradation. Sulfate consumption by the cultures corresponded to the stoichiometric values expected for complete oxidation of the chlorophenol to CO2. Formation of sulfide from sulfate was confirmed with a radiotracer technique. No methane was formed, verifying that sulfate reduction was the electron sink. Addition of molybdate, a specific inhibitor of sulfate reduction, inhibited chlorophenol degradation completely. These results indicate that the chlorophenols were mineralized under sulfidogenic conditions and that substrate oxidation was coupled to sulfate reduction. In acclimated cultures the three monochlorophenol isomers and 2,4-dichlorophenol were degraded at rates of 8 to 37 mumol liter-1 day-1. The relative rates of degradation were 4-chlorophenol greater than 3-chlorophenol greater than 2-chlorophenol, 2,4-dichlorophenol. Sulfidogenic cultures initiated with biomass from an anaerobic bioreactor used in treatment of pulp-bleaching effluents dechlorinated 2,4-dichlorophenol to 4-chlorophenol, which persisted, whereas 2,6-dichlorophenol was sequentially dechlorinated first to 2-chlorophenol and then to phenol.     

Optimization of trichloroethylene oxidation by methanotrophs and the use of a colorimetric assay to detect soluble methane monooxygenase activity

Methylosinus trichosporium OB3b biosynthesizes a broad specificity soluble methane monooxygenase that rapidly oxidizes trichloroethylene (TCE). The selective expression of the soluble methane monooxygenase was followed in vivo by a rapid colorimetric assay. Naphthalene was oxidized by purified soluble methane monooxygenase or by cells grown in copper-deficient media to a mixture of 1-naphthol and 2-naphthol. The naphthols were detected by reaction with tetrazotized o-dianisidine to form purple diazo dyes with large molar absorptivities. The rate of color formation with the rapid assay correlated with the velocity of TCE oxidation that was determined by gas chromatography. Both assays were used to optimize conditions for TCE oxidation by M. trichosporium OB3b and to test several methanotrophic bacteria for the ability to oxidize TCE and naphthalene.Abbreviations A₆₀₀ absorbance due to cell density measured at 600 nm - HPLC high pressure liquid chromatography - NADH reduced nicotinamide adenine dinucleotide - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - sMMO soluble methane monooxygenase - TCE trichloroethylene     

Changes in fatty acid composition of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> KB2 during co-metabolic degradation of monochlorophenols

The changes in the cellular fatty acid composition of Stenotrophomonas maltophilia KB2 during co-metabolic degradation of monochlorophenols in the presence of phenol as well as its adaptive mechanisms to these compounds were studied. It was found that bacteria were capable of degrading 4-chlorophenol (4-CP) completely in the presence of phenol, while 2-chlorophenol (2-CP) and 3-chlorophenol (3-CP) they degraded partially. The analysis of the fatty acid profiles indicated that adaptive mechanisms of bacteria depended on earlier exposure to phenol, which isomer they degraded, and on incubation time. In bacteria unexposed to phenol the permeability and structure of their membranes could be modified through the increase of hydroxylated and cyclopropane fatty acids, and straight-chain and hydroxylated fatty acids under 2-CP, 3-CP and 4-CP exposure, respectively. In the exposed cells, regardless of the isomer they degraded, the most important changes were connected with the increase of the contribution of branched fatty acid on day 4 and the content of hydroxylated fatty acids on day 7. The changes, particularly in the proportion of branched fatty acids, could be a good indicator for assessing the progress of the degradation of monochlorophenols by S. maltophilia KB2. In comparison, in phenol-degrading cells the increase of cyclopropane and straight-chain fatty acid content was established. These findings indicated the degradative potential of the tested strain towards the co-metabolic degradation of persistent chlorophenols, and extended the current knowledge about the adaptive mechanisms of these bacteria to such chemicals.     

The Development of a Phytoremediation Technique for the Detoxification of Soils Contaminated with Phenolic Compounds Using Horseradish Peroxidase (Armoracia rusticana): Preliminary Results

The proposed phytoremediation technique is based on the successful exploitation and optimization of oxidative coupling, mediated by horseradish peroxidase. Susceptibility to degradation of a selection of phenolic compounds, in solution, by horseradish peroxidase appears to be structurally related and was found to be of the order 2,4-dichlorophenol (2,4-DCP) > 4-chlorophenol (4-CP) > 2-chlorophenol (2-CP). Only 1.89% of 2,4-DCP, at an initial concentration of 5 mM, remained unchanged at the end of the experiment. Reaction rates between purified horseradish peroxidase and 2,4-DCP were found to be extremely rapid with 74% of the substrate removed from solution during the first 30 s. Inhibition of the reaction by the heavy metals Cd, Zn, Ni, and Pb at concentrations of 100 mg/l is of concern because these metals are often present in contaminated soils. H₂O₂ has a dominant role in optimizing peroxidase activity in crude horseradish extracts. Fluctuations in temperature and pH, normally experienced in soils, did not appear to have a detrimental impact on peroxidase activity. However, the functioning of the enzyme is seriously affected at a pH ≤ 3. All reactions in this study were carried out in solution.     

Modified carbon paste electrodes for flow injection amperometric determination of isocitrate dehydrogenase activity in serum

A carbon paste electrode modified with the adsorbed products of the electrochemical oxidation of adenosine triphosphate is described. The electrode was applied to the amperometric electrocatalytic detection of the reduced form of both nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate. The catalytic oxidation current shows a linear dependence on the concentration of the reduced form of nicotinamide adenine dinucleotide up to 1x10(-4)M, with a detection limit of 5x10(-9)M. Modified carbon paste electrodes were coated with an electrogenerated film of nonconducting poly(o-phenylenediamine) to obtain a stable amperometric response for at least 150h. In addition to static measurements, determination of both reduced cofactors was carried out in a flow injection analysis system with a thin-layer amperometric detection cell. The electrocatalytic monitoring of reduced nicotinamide adenine dinucleotide phosphate was applied to flow injection measurement of isocitrate dehydrogenase activity in serum. The results were in good agreement with those for the standard spectrophotometric test kit. The proposed method consumed less time and reagents and provided better precision than the standard method.     

Effects of diethylstilbestrol, 2,2'-dithiodipyridine, and chloral hydrate on the esterase activity of sheep liver cytoplasmic aldehyde dehydrogenase

The binding of diethylstilbestrol (DES) to aldehyde dehydrogenase (ALDH) has a very similar effect on the dehydrogenase activity of the enzyme as has modification of the enzyme by 2,2'-dithiodipyridine [Kitson, T.M. (1982) Biochem. J. 207, 81-89]. The latter modification may occur at the site of the esterase activity of the enzyme [Kitson, T.M. (1985) Biochem. J. 228, 765-767]. This suggests that DES might be a competitive inhibitor of the esterase reaction. However, in the absence of oxidized nicotinamide adenine dinucleotide (NAD+) or reduced nicotinamide adenine dinucleotide (NADH), and at low concentrations of substrate (4-nitrophenyl acetate, PNPA), DES is a potent partial noncompetitive inhibitor. It is concluded therefore that DES binds at a site different from the esterase active site and that the enzyme-DES complex retains some ability to act as an esterase. High concentrations of PNPA appear to displace DES from its binding site. In the presence of NAD+, DES is a weaker inhibitor, and in the presence of NADH, DES has very little effect. Esterase activity is enhanced by NADH when PNPA concentrations are high but is inhibited when they are low. The rate of reaction of ALDH with 2,2'-dithiodipyridine is only slightly reduced by DES, suggesting that the site at which thiol modifiers react and the DES binding site are different. When ALDH is modified by 2,2'-dithiodipyridine, it has reduced esterase activity, which declines further as the modified enzyme loses its 2-thiopyridyl label. In the presence of NAD+, chloral hydrate is a simple competitive inhibitor of the esterase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)