Aerobic mineralization of chlorobenzoates by a natural polychlorinated biphenyl-degrading mixed bacterial culture

A natural mixed aerobic bacterial culture, designated MIXE1, was found to be capable of degrading several low-chlorinated biphenyls when 4-chlorobiphenyl was used as a co-substrate. MIXE1 was capable of using all the three monochlorobenzoate (CBA) isomers tested as well as 2,5-, 3,4- and 3,5-dichlorobenzoate (dCBA) as the sole carbon and energy source. During MIXE1 growth on these substrates, a nearly stoichiometric amount of chloride was released: 0.5 g/l of each chlorobenzoate was completely mineralized by MIXE1 after 2 or 3 days of culture incubation. Two strains, namely CPE2 and CPE3, were selected from MIXE1: CPE2, referred to the Pseudomonas genus, was found to be capable of totally degrading both 2-CBA and 2,5-dCBA, whereas Alcaligenes strain CPE3 was capable of mineralizing 3-, 4-CBA and 3,4-dCBA. Substrate uptake studies carried out with whole cells of strain CPE2 suggested that 2-CBA was metabolized through catechol, while 2,5-dCBA was degraded via 4-chlorocatechol. 3-CBA, 4-CBA, and 3,4-dCBA appeared to be degraded through 3,4-dihydroxybenzoate by the CPE3 strain. MIXE1, which is capable of degrading several chlorobenzoates, should therefore be able to mineralize a number of low-chlorinated congeners of simple and complex polychlorinated biphenyl mixtures. Correspondence to: F. Fava     

Pusillimonas sp. 5HP degrading 5-hydroxypicolinic acid

A bacterial strain 5HP capable of degrading and utilizing 5-hydroxypicolinic acid as the sole source of carbon and energy was isolated from soil. In addition, the isolate 5HP could also utilize 3-hydroxypyridine and 3-cyanopyridine as well as nicotinic, benzoic and p-hydroxybenzoic acids for growth in the basic salt media. On the basis of 16S rRNA gene sequence analysis, the isolate 5HP was shown to belong to the genus Pusillimonas. Both the bioconversion analysis using resting cells and the enzymatic assay showed that the degradation of 5-hydroxypicolinic acid, 3-hydroxypyridine and nicotinic acid was inducible and proceeded via formation of the same metabolite, 2,5-dihydroxypyridine. The activity of a novel enzyme, 5-hydroxypicolinate 2-monooxygenase, was detected in the cell-free extracts prepared from 5-hydroxypicolinate-grown cells. The enzyme was partially purified and was shown to catalyze the oxidative decarboxylation of 5-hydroxypicolinate to 2,5-dihydroxypyridine. The activity of 5-hydroxypicolinate 2-monooxygenase was dependent on O₂, NADH and FAD.     

Gentisic acid and its 3- and 4-methyl-substituted homologues as intermediates in the bacterial degradation of m-cresol, 3,5-xylenol and 2,5-xylenol

1. Intact cells of a non-fluorescent Pseudomonas grown with m-cresol, 2,5-xylenol, 3,5-xylenol, 3-ethyl-5-methylphenol or 2,3,5-trimethylphenol rapidly oxidized all these phenols to completion. 3-Hydroxybenzoate and 2,5-dihydroxybenzoate (gentisate) were also readily oxidized. 2. 3-Hydroxybenzoic acid and 2,5-dihydroxybenzoic acid were isolated as products of m-cresol oxidation by cells inhibited by alphaalpha'-bipyridyl. Alkyl-substituted 3-hydroxybenzoic acids and alkyl-substituted gentisic acids were formed similarly from 2,5-xylenol, 3,5-xylenol, 3-ethyl-5-methylphenol and 2,3,5-trimethylphenol. 3. When supplemented with NADH, not NADPH, extracts of cells grown with 2,5-xylenol catalysed the oxidation of all five phenols and accumulated the corresponding gentisic acids in the presence of alphaalpha'-bipyridyl. 4. Cells of a fluorescent Pseudomonas grown with m-cresol oxidized m-cresol, 3,5-xylenol and 3-ethyl-5-methylphenol to completion and oxidized 2,5-xylenol and 2,3,5-trimethylphenol partially. The oxidation product of 2,5-xylenol was identified as 3-hydroxy-4-methylbenzoic acid. In the presence of alphaalpha'-bipyridyl, 3-hydroxy-5-methylbenzoic acid and 3-methylgentisic acid were formed from 3,5-xylenol.     

Mathematical modeling of in vitro enzymatic production of 2-Keto-L-gulonic acid using NAD(H) or NADP(H) as cofactors

A 2-Keto-L-gulonic acid (2-KLG) production process using stationary Pantoea citrea cells and a Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase enzyme has been developed which may represent an improved method of vitamin C biosynthesis. Experimental data was collected using the F22Y/A272G 2,5-DKG reductase mutant and NADP(H) as a cofactor. An extensive kinetic analysis was performed and a kinetic rate equation model for this process was developed. A recent protein engineering effort has resulted in several 2,5-DKG reductase mutants exhibiting improved activity with NADH as a cofactor. The use of NAD(H) in the bioreactor may be preferable due to its increased stability and lower cost. The kinetic parameters in the rate equation model have been replaced in order to predict 2-KLG production with NAD(H) as a cofactor. The model was also extended to predict 2-KLG production in the presence of a range of combined cofactor concentrations. This analysis suggests that the use of the F22Y/K232G/R238H/A272G 2,5-DKG reductase mutant with NAD(H) combined with a small amount of NADP(H) could provide a significant cost benefit for in vitro enzymatic 2-KLG production.     

The metabolism of 2-furoic acid by Pseudomonas F2

1. Pseudomonas F2 isolated by enrichment culture on 2-furoic acid and grown with it as carbon source oxidized the compound with a Q(o) (2) of 170mul./mg. dry wt./hr. and the overall consumption of 2.5mumoles of oxygen/mumole of substrate. 2. In the presence of 1mm-sodium arsenite, oxygen uptake was restricted to 0.54mumole/mumole of 2-furoate oxidized, with the formation of 0.86mumole of 2-oxoglutarate/mumole of 2-furoate. 3. Cell suspensions, disrupted in a French pressure cell and centrifuged at 27000g, yielded supernatants capable of catalysing the slow oxidation of 2-furoate (0.17mumole/mg. of protein/hr.). 4. Fractionation of 27000g supernatants at 200000g yielded a soluble enzyme fraction capable of catalysing the oxidation of 2-furoate only in the presence of added 200000g pellet or of Methylene Blue. 5. The 2-furoate-stimulated uptake of oxygen or the anaerobic reduction of Methylene Blue by dialysed 27000g supernatant required the addition of ATP and CoA, and the rate of oxygen uptake was further enhanced by the addition of magnesium chloride and NAD(+). 6. The role of ATP and CoA in the formation of 2-furoyl-CoA was demonstrated by the accumulation of 2-furoylhydroxamic acid in the presence of hydroxylamine. 7. Dialysed 200000g supernatant, treated with Dowex 1, required the addition of ATP, CoA and Methylene Blue before it could oxidize 2-furoate to 2-oxoglutarate, which was trapped in unitary stoicheiometric yield as its phenylhydrazone. Magnesium chloride and NAD(+) were not stimulatory in this system. The oxidation of 2-furoate to 2-oxoglutarate was not inhibited by substrate analogues, metal ion-chelating agents, thiol-active compounds or inhibitors of cytochrome-mediated electron transport. 8. No evidence was obtained for the intervention of 2,5-dioxovalerate as an intermediate in 2-oxoglutarate formation.     

Influence of organic and inorganic growth supplements on the aerobic biodegradation of chlorobenzoic acids

The effect of yeast extract and its less complex substituents on the rate of aerobic dechlorination of 2-chlorobenzoic acid (2-ClBzOH) and 2,5-dichlorobenzoic acid (2,5-Cl₂BzOH) by Pseudomonas sp. CPE2 strain, and of 3-chlorobenzoic acid (3-ClBzOH), 4-chlorobenzoic acid (4-ClBzOH) and 3,4-dichlorobenzoic acid (3,4-Cl₂BzOH) by Alcaligenes sp. CPE3 strain were investigated. Yeast extract at 50 mg/l increased the average dechlorination rate of 200 mg/l of 4-ClBzOH, 2,5-Cl₂BzOH, 3,4-Cl₂BzOH, 3-ClBzOH and 2-ClBzOH by about 75%, 70%, 55%, 7%, and 1%, respectively. However, in the presence of yeast extract the specific dechlorination activity of CPE2 and CPE3 cells (per unit biomass) was always lower than without yeast extract, although it increased significantly during the exponential growth phase. When a mixed vitamin solution or a mixed trace element solution was used instead of yeast extract the rate of 4-ClBzOH dechlorination increased by 30%–35%, whereas the rate of 2,5-Cl₂BzOH and 3,4-Cl₂BzOH dechlorination increased by only 2%–10%. The presence of vitamins or trace elements also resulted in a specific dechlorination activity that was generally higher than that observed for the same cells grown solely on chlorobenzoic acid. The results of this work indicate that yeast extract, a complex mixture of readily oxidizable carbon sources, vitamins, and trace elements, enhances the growth and the dechlorination activity of CPE2 and CPE3 cells, thus resulting in an overall increase in the rate of chlorobenzoic acid utilization and dechlorination.     

Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-D-gluconic acid reductase for use in vitamin C biosynthesis

The strict cofactor specificity of many enzymes can potentially become a liability when these enzymes are to be employed in an artificial metabolic pathway. The preference for NADPH over NADH exhibited by the Corynebacterium 2,5-diketo-D-gluconic acid (2,5-DKG) reductase may not be ideal for use in industrial scale vitamin C biosynthesis. We have previously reported making a number of site-directed mutations at five sites located in the cofactor-binding pocket that interact with the 2'-phosphate group of NADPH. These mutations conferred greater activity with NADH upon the Corynebacterium 2,5-DKG reductase [Banta, S., Swanson, B. A., Wu, S., Jarnagin, A., and Anderson, S. (2002) Protein Eng. 15, 131-140; (1)]. The best of these mutations have now been combined to see if further improvements can be obtained. In addition, several chimeric mutants have been produced that contain the same residues as are found in other members of the aldo-keto reductase superfamily that are naturally able to use NADH as a cofactor. The most active mutants obtained in this work were also combined with a previously reported substrate-binding pocket double mutant, F22Y/A272G. Mutant activity was assayed using activity-stained native polyacrylamide gels. Superior mutants were purified and subjected to a simplified kinetic analysis. The simplified kinetic analysis was extended for the most active mutants in order to obtain the kinetic parameters in the full-ordered bi bi rate equation in the absence of products, with both NADH and NADPH as cofactors. The best mutant 2,5-DKG reductase produced in this work was the F22Y/K232G/R238H/A272G quadruple mutant, which exhibits activity with NADH that is more than 2 orders of magnitude higher than that of the wild-type enzyme, and it retains a high level of activity with NADPH. This new 2,5-DKG reductase may be a valuable new catalyst for use in vitamin C biosynthesis.     

Effect of yeast extract on growth kinetics during aerobic biodegradation of chlorobenzoic acids

The Monod or Andrews kinetic parameters describing the growth of Pseudomonas sp. CPE2 strain on 2,5-dich!orobenzoic acid and 2-chlorobenzoic acid, and Al-caligenes sp. CPE3 strain on 3,4-dichlorobenzoic acid, 4-chlorobenzoic acid, and 3-chlorobenzoic acid were determined from batch and continuous growth experiments conducted in the presence or absence of yeast extract (50 mg/L). Strain CPE2 displayed inhibitory growth kinetics in the absence of yeast extract and a noninhibitory kinetics in the presence of yeast extract. Similar results were obtained for CPE3. The presence of yeast extract also resulted in a significant increase in the affinity of the strains for the chlorobenzoic acids they degraded. (c) 1995 John Wiley & Sons, Inc.     

Mineralization of the herbicide 2,3,6-trichlorobenzoic acid by a co-culture of anaerobic and aerobic bacteria

Abstract Bacteria from an anaerobic enrichment reductively removed chlorine from the ortho- position of 2,3,6-trichlorobenzoic acid (2,3,6-TBA) producing 2,5-dichlorobenzoate (2,5-DBA). The strictly aerobic bacterium Pseudomonas aeruginosa JB2 subsequently used 2,5-DBA as a growth substrate in the presence of oxygen. The anaerobic dechlorinating microbial population was grown with P. aeruginosa JB2 in continuous culture. Inside the liquid culture, a nylon netting, on a stainless-steel support, contained vermiculite particles to provide a strictly anaerobic environment within the aerated culture. Complete mineralization of 2,3,6-TBA depended on the extent of oxygen input into the reactor. Under strictly anaerobic conditions 2,5-DBA and Cl⁻ were produced stoichiometrically through the reductive dechlorination of 2,3,6-TBA. This process of reductive dechlorination was not inhibited by (moderate) aeration resulting in an O₂-concentration of 0.3–0.5 μM in the culture liquid.     

Mineralization of the herbicide 2,3,6-trichlorobenzoic acid by a co-culture of anaerobic and aerobic bacteria

Abstract Bacteria from an anaerobic enrichment reductively removed chlorine from the ortho - position of 2,3,6-trichlorobenzoic acid (2,3,6-TBA) producing 2,5-dichlorobenzoate (2,5-DBA). The strictly aerobic bacterium Pseudomonas aeruginosa JB2 subsequently used 2,5-DBA as a growth substrate in the presence of oxygen. The anaerobic dechlorinating microbial population was grown with P. aeruginosa JB2 in continuous culture. Inside the liquid culture, a nylon netting, on a stainless-steel support, contained vermiculite particles to provide a strictly anaerobic environment within the aerated culture. Complete mineralization of 2,3,6-TBA depended on the extent of oxygen input into the reactor. Under strictly anaerobic conditions 2,5-DBA and Cl⁻ were produced stoichiometrically through the reductive dechlorination of 2,3,6-TBA. This process of reductive dechlorination was not inhibited by (moderate) aeration resulting in an O₂-concentration of 0.3–0.5 μM in the culture liquid.     

Assay of phosphoenolpyruvate carboxykinase in crude yeast extracts.

The applicability of a spectrophotometric assay of phosphoenolpyruvate car?ykinase to crude yeast extracts has been studied. The assay measured oxalacetate production by coupling to the malate dehydrogenase reaction (phosphoenolpyruvate + ADP + bicarbonate → oxalacetate + ATP; oxalacetate + NADH → malate + NAD). Disappearance of NADH depended strictly on the presence of phosphoenolpyruvate, bicarbonate, ADP, and Mn²⁺. Furthermore, the disappearance of NADH was shown to be accompanied by stoichiometric accumulation of malate. Addition of 10 mm quinolinate, which is a known inhibitor of liver phosphoenolpyruvate car?ykinase, completely prevented phosphoenolpyruvate-dependent NADH disappearance. These observations demonstrated that the assay provides a quantitative measure of phosphoenolpyruvate car?ykinase activity in crude extracts. The assay could be applied to crude extracts from yeast cells grown under laboratory conditions but not to extracts from commercially produced baker's yeast, because of an extremely high rate of endogeneous oxidation of NADH in the latter extracts. With the spectrophotometric assay, optimal activity was observed at pH 7.0 with both crude extracts and a 15-fold-purified preparation.     

Degradation and dechlorination of low-chlorinated biphenyls by a three-membered bacterial co-culture

A Pseudomonas sp. strain, designated CPE1, was found to be capable of completely mineralizing 4-chlorobiphenyl via 4-chlorobenzoate and of partially dechlorinating 3,4-dichlorobiphenyl in the presence of biphenyl. A three-membered bacterial consortium, designated ECO3, prepared by combining CPE1 with two chlorobenzoate (CBA)-degrading strains, was capable of extensively degrading and dechlorinating all the monochlorinated biphenyls and several dichlorinated biphenyls in the presence of bipheny. Both CPE1 and ECO3 were capable of co-metabolizing several low-chlorinated biphenyl congeners of Fenclor 42 in the presence of biphenyl; however, only in ECO3 cultures were high degradation rates and chloride release observed. The higher rate of degradation and mineralization of some polychlorinated biphenyls (PCBs) of Fenclor 42 due to the concerted action of ECO3 members both on PCBs and CBAs suggested that the removal of CBAs from the culture medium may favour PCB degradation, and, therefore, that CBAs may be ivollved in the regulation of the degradation process of several chlorinated biphenyl congeners.Correspoeence to: F. Fava     

An improved method for the determination of NADH oxidase in the presence of NADH peroxidase in lactic acid bacteria

The complexity of the coupled NADH oxidase-NADH peroxidase enzyme system in lactic acid bacteria makes it difficult to simultaneously determine the individual levels of both these enzymes spectrophotometrically. This study describes an improved assay to accurately determine low concentrations of NADH oxidase from enzyme suspensions containing NADH oxidase and NADH peroxidase. For the standardisation of the assay, pure NADH oxidase and NADH peroxidase were mixed in various proportions and the percentage recovery was estimated by both the currently available assay as well as by the improved assay reported in this study. The recovery of NADH oxidase using the currently available assay ranged from as low as -200% to as high as +102% as against 90-102% in the improved assay. The recovery percentage of NADH peroxidase ranged from 91% to 112% in both assays. The slopes of NADH oxidation by cell-free extracts of six lactic acid bacteria were also measured by both assays for the estimation of NADH oxidase and NADH peroxidase levels. The improved assay can further distinguish between NADH-H(2)O oxidase and NADH-H(2)O(2) oxidase and was successfully applied to identify the type of NADH oxidase in the lactic acid bacteria tested.     

Pyridine nucleotide specificity of barley nitrate reductase

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH.     

Structural alteration of cofactor specificity in Corynebacterium 2,5-diketo-D-gluconic acid reductase

Corynebacterium 2,5-Diketo-D-gluconic acid reductase (2,5-DKGR) catalyzes the reduction of 2,5-diketo-D-gluconic acid (2,5-DKG) to 2-Keto-L-gulonic acid (2-KLG). 2-KLG is an immediate precursor to L-ascorbic acid (vitamin C), and 2,5-DKGR is, therefore, an important enzyme in a novel industrial method for the production of vitamin C. 2,5-DKGR, as with most other members of the aldo-keto reductase (AKR) superfamily, exhibits a preference for NADPH compared to NADH as a cofactor in the stereo-specific reduction of substrate. The application of 2,5-DKGR in the industrial production of vitamin C would be greatly enhanced if NADH could be efficiently utilized as a cofactor. A mutant form of 2,5-DKGR has previously been identified that exhibits two orders of magnitude higher activity with NADH in comparison to the wild-type enzyme, while retaining a high level of activity with NADPH. We report here an X-ray crystal structure of the holo form of this mutant in complex with NADH cofactor, as well as thermodynamic stability data. By comparing the results to our previously reported X-ray structure of the holo form of wild-type 2,5-DKGR in complex with NADPH, the structural basis of the differential NAD(P)H selectivity of wild-type and mutant 2,5-DKGR enzymes has been identified.     

Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans.

The enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity. 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA. Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.     

Cytotoxicity of extracts from Dolsan leaf mustard Kimchi treated with lactic acid bacteria on lung and gastric cancer cells

The cytotoxicity of extracts from Dolsan leaf mustard Kimchi (DLMK) treated with lactic acid bacteria on A 549 human lung cancer cells and SNU-601 human gastric cancer cells were investigated. Leuconostoc mesenteroides, Leu. Gelidum, and Weissella kimchii previously isolated from properly ripened DLMK were inoculated to DLMK as a starter (1 × 10⁸ CFU/mL). The DLMK was then fractionated by various extracting solvents. The cytotoxicity of MeOH extracts from DLMK on A 549 and SNU-601 cancer cells was found to occur in a dose-dependent manner. Although the cytotoxicity of the MeOH extracts was found to be approximately 20 to 30% at concentrations of 250 μg/mL by MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide) assay, cytotoxicity of chloroform soluble fraction of DLMK treated with W. kimchii showed about 80 to 90%. Consequently, the growth of cancer cells was inhibited significantly in medium containing DLMK extracts. In addition, significant morphological changes such as cell condensation, cell fragmentation, and alterations in the size and shape of the cells were observed in cells grown in medium that contained the DLMK extracts. Taken together, these results suggest that inhibition of the proliferation of cancer cells by apoptosis was induced by DLMK extracts.     

Oxidation of NADH in a Coupled Oxidase-Peroxidase Reaction and its Significance for the Fermentation in Rumen Protozoa of the Genus Isotricha

SYNOPSIS. Cell-free extracts of the anaerobic rumen ciliate Isotricha prostoma possess a strong NADH oxidase activity. Evidence for H₂O₂ as an intermediary product during oxidation of NADH has been obtained. Gatalase activity could not be demonstrated but hydrogen peroxide is removed by a rate limiting NAD peroxidase.
In addition to oxygen several other compounds such as ferricyanide, cytochrome c , menadione and certain dyes may function as electron acceptors during oxidation of NADH. The ferricyanide reductase activity in the Isotricha extracts strongly resembles that of the mitochondrial enzyme from mammalian sources in a number of characteristics.
Partial inhibition of NADH oxidase activity was obtained with the following chelating agents: hydroxylamine, diethyl dithiocarbamate, 2,9-dimethyl-1,10-phenanthroline (DMPH), and 2-thenoyl trifluoroacetone, whereas citrate, tartrate, pyrophosphate, salicylaldoxime, EDTA and 8-hydroxyquinoline had no effect. The peroxidase was blocked completely by 0.42 mM DMPH and this inhibitor was used to block the enzyme in whole cells in experiments on oxygen toxicity. The oxidase was largely insensitive to azide, KCN, and uncouplers. Antimycin A and rotenone caused a partial inhibition of the oxidase when added in very high concentrations. ATP formation occurred during oxidation of NADH, and P/O ratios were 0.1–0.35. Addition of small amounts of oxygen to intact ciliates led to a decrease in the production of hydrogen and butyrate, while the production of acetate was increased and no change in the lactate formation was seen. This shift in fermentation end-products possibly is caused by a competition of oxygen for NADH.     

The bacterial metabolism of 2,4-xylenol

1. Measurements of the rates of oxidation of various compounds by a fluorescent Pseudomonas indicated that metabolism of 2,4-xylenol was initiated by oxidation of the methyl group para to the hydroxyl group. 2. 4-Hydroxy-3-methylbenzoic acid was isolated as the product of oxidation of 2,4-xylenol by cells inhibited with alphaalpha'-bipyridyl. 3. 4-Hydroxyisophthalic acid accumulated at low oxygen concentrations when either 2,4-xylenol or 4-hydroxy-3-methylbenzoic acid was oxidized by cells grown with 2,4-xylenol. 4. When supplemented with NADH, but not with NADPH, cell extracts oxidized 4-hydroxy-3-methylbenzoic acid readily. 2-Hydroxy-5-methylbenzoic acid was not oxidized. 5. Both 4-hydroxyisophthalic acid and p-hydroxybenzoic acid were oxidized to beta-oxoadipic acid by cell extracts supplemented with either NADH or NADPH. 4,5-Dihydroxyisophthalic acid was not oxidized. 6. From measurements of oxygen consumed and carbon dioxide evolved it was concluded that protocatechuic acid is an intermediate in the conversion of 4-hydroxyisophthalic acid into beta-oxoadipic acid.     

Effect of oxygen on the metabolism of Zymomonas mobilis

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was 25–40% lower in the presence of oxygen than under anaerobic conditions, provided the cultures were supplied with a low substrate concentration (20 g glucose/l). However, the molar growth yield of these cultures was not influenced by oxygen. With washed cell suspensions, an oxygen consumption could be initiated by the addition of either glucose, fructose, or ethanol. Cell extracts catalyzed the oxidation of NADH with oxygen at a molar ratio of 2:1. Further experiments showed that this NADH oxidase is located in the cell membrane. The specific oxygen consumption rates of cell suspensions correlated with the intracellular NADH oxidizing activities; both levels decreased with increasing concentrations of the fermentation end-product ethanol. The addition of 5 mM NaCN completely inhibited both the intracellular oxygen reduction and also the oxygen consumption of whole cells. Both catalase and superoxide dismutase were present even in anaerobically grown cells. Aeration seemed to have little effect on the level of catalase, but the superoxide dismutase activity was 5-fold higher in cells grown aerobically. Under aerobic conditions considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide.Dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday