Studies on Browning Reactions between Sugars and Amino Compounds

N-d-Glucosyl-p-aminobenzoic acid has been found to form melanoidins in methanol solution acidified with hydrogen chloride at 25°. From the reaction mixture 5-hydroxymethyl-furfural (HMF) has been isolated and identified as its 2,4-dinitrophenylhydrazone. So, comparisons between the browning reaction of HMF or furfural with aromatic amine and that of the corresponding n-glycosides have been made under the same condition. From the results obtained, it has been shown that, under the described condition, furfural is almost inactive for browning, while on the contrary, HMF is active and plays an important role in the browning reaction.     

Methanogenesis from furfural by defined mixed cultures

Methanogenesis from furfural by defined mixed cultures was studied. Under sulfate-reducing conditions, a Desulfovibrio strain was used as the furfural-degrading species producing acetic acid. This sulfate-reducing bacterium (SRB) Desulfovibrio strain B is an incomplete oxidizer, unable to carry out the terminal oxidation of organic substrates, leaving acetic acid as the end product. Introduction of acetate-utilizing methanogenic archaeon Methanosarcina barkeri 227 converted acetic acid to methane. This well-defined mixed consortium used furfural as its sole source of carbon and converted it to methane and CO₂. In the mixed culture, when a methanogen inhibitor was used in the culture medium, furfural was converted to acetic acid by the Desulfovibrio strain B, but acetic acid did not undergo further metabolism. On the other hand, when the growth of Desulfovibrio in the consortium was suppressed with a specific SRB inhibitor, namely molybdate, furfural was not degraded. Thus, the metabolic activities of both Desulfovibrio strain B and M. barkeri 227 were essential for the complete degradation of furfural. Received: 15 August 2001 / Accepted: 20 September 2001     

Effects of Oxygen Concentration and Leghemoglobin on Organic Acid Degradation by Isolated Soybean Nodule Bacteriods

Bacteroids retaining high acetylene reduction activity (nitrogenase activity) were prepared anaerobically from soybean nodules. Addition of succinate (or of both leghemoglobin and succinate) to the acetylene reduction assay system greatly increased the activity of the isolated bacteroids.

When various organic acids were incubated with the bacteroids at 2% oxygen concentration, an optimum condition for bacteroid acetylene reduction, the organic acid degradation by bacteroids was very slow, and both lactate and acetate were accumulated in the incubation system, suggesting the operation of fermentative pathway in bacteroids under such low oxygen conditions.

With 20% oxygen, the added organic acids were degraded rapidly by bacteroids without addition of leghemoglobin to the incubation system.

With leghemoglobin in the incubation system, the organic acid degradation by bacteroids was accelerated extensively even at 2% oxygen, and the formation of lactate and acetate were negligible. No significant difference in the organic acid degradation rate was observed between the 2% and 20% oxygen concentrations when the leghemoglobin was present in the incubation system. Addition of acetylene to the assay system slightly inhibited the organic acid degradation.

This data suggests that bacteroids are unable to oxidize organic acid in low oxygen concentration and that the leghemoglobin allows the rapid organic acid dagradation by bacteroids even in such low oxygen concentrations.     

Bacterial metabolism of 4-chloro-2-methylphenoxyacetate. Formation of glyoxylate by side-chain cleavage

Crude extracts of Pseudomonas sp. grown on 4-chloro-2-methylphenoxyacetate as sole source of carbon were shown to oxidize 4-chloro-2-methylphenoxyacetate to 5-chloro-o-cresol and glyoxylate. A labelled 2,4-dinitrophenylhydrazone was isolated from an incubation mixture in which 4-chloro-2-methylphenoxy[carboxy-¹⁴C]acetate was used. The hydrazone was shown to behave identically on thin-layer chromatograms with the authentic 2,4-dinitrophenylhydrazone of glyoxylate. Radioactivity assay showed that 82% of the side chain of 4-chloro-2-methylphenoxyacetate was recovered as glyoxylate.     

Simultaneous degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 2-(2-methyl-4-chlorophenoxy)propionic acid by mixed bacterial cultures

The simultaneous degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) was achieved by two mixed cultures in the absence of any additional carbon or energy substrates. Mecoprop was not completely degraded by either of the two cultures, nor did addition of 2,4-D affect the degradation of mecoprop. The cultures completely degraded 2,4-D, and the degradation was uninfluenced by the addition of mecoprop. Nearly complete dechlorination of the mixture of two herbicides was achieved by both cultures, on the basis of the total amount of the two herbicides degraded. During the course of the reaction, however, the expected values of chloride were not met. Cell growth continued after the degradation of the parent substrates ceased. Although the mecoprop degradation did not continue to completion, spectral and growth data indicated that the metabolites which had accumulated during the reaction were degraded upon further incubation.     

Formation of 1-Alkyl-5-(hydroxymethyl)pyrrole-2-aldehydes in the Browning Reaction between Hexoses and Alkylamines

d-Glucose and butyl-, ethyl-, or methyl-amine were reacted at 100°C or 70°C in an aqueous or methanol solution neutralized with acetic acid to obtain browning reaction products, and the formation of N-substituted 5-(hydroxymethyl)pyrrole-2-aldehydes in the reaction was investigated.

At first, the reaction products were treated with 2,4-dinitrophenylhydrazine reagent and the resulting 2,4-dinitrophenylhydrazones (2,4-DNPs) were fractionated by column chromatography to isolate crystalline 2,4-DNPs. When the products obtained from d-glucose and butylamine were treated with the reagent consisting of 2,4-dinitrophenylhydrazine, sulfuric acid, water and either ethanol or methanol, 2,4-DNP of l-butyl-5-(ethoxymethyl or meth-oxymethyl)pyrrole-2-aldehyde (I or II) was isolated. In this formation ethanol or methanol was considered to be directly involved. On the other hand, 2,4-DNP of 5-hydroxymethyl derivative (III) was not detected, even when the reagent containing water alone as a solvent was used.

Secondly, in order to isolate the free pyrrolealdehyde, the above browning reaction products were extracted with ethyl acetate and the extract was chromatographed on a silica gel column. From the main carbonyl fraction was isolated 1-butyl-, ethyl-, or methyl-5-(hydroxymethyl)-pyrrole-2-aldehyde (III, IV, or V), which was characterized by elementary analyses and ultraviolet, infrared, and nuclear magnetic resonance spectra.     

Studies on the Changes of the Milk Casein by Various Treatments

The volatile carbonyl compounds generated in casein solution heated at 140°C for 60 minutes were isolated as 2,4-dinitrophenylhydrazone derivatives. The mixture of derivatives were fractionated into 8 derivatives by column chromatography and preparative thin layer chromatography. Compounds identified were ethanal, propanai, 2-methylpropanal, 3-methylbutanai and 2-methylbutanal. Evidence was also presented for the occurrence of methanal, acetone and crotonal.

These compounds seemed to have some relation with heat induced flavor compounds in dairy products.     

Enzymatic reaction of beta-N-methylaminoalanine with L-amino acid oxidase

The reaction of beta-N-methylaminoalanine (BMAA) with L-amino acid oxidase (L-AAO) in the presence of catalase yields ammonia and beta-N-methylaminopyruvate, which was trapped as its 2,4-dinitrophenylhydrazone, as products. Incubation of BMAA with L-AAO in the presence of semicarbazide led to the formation of a semicarbazone, indicating intermediate iminium ion formation; when potassium cyanide (5 mM) was added, semicarbazone formation was blocked. The formation of beta-N-methylaminopyruvate was decreased by omission of catalase and was reduced in the presence of hydrogen peroxide (100 mM). These results indicate that BMAA is converted by L-AAO to the corresponding alpha-imino acid, which undergoes hydrolysis to beta-N-methylaminopyruvate. The alpha-keto acid is readily oxidized to N-methylglycine by hydrogen peroxide.     

Identification of S-carboxymethyl-thiopyruvate as the product of oxidative deamination of S-carboxymethyl-cysteine

Summary Bromopyruvic acid and thioglycolic acid react to form carboxymethylthiopyruvic acid, which was then isolated as the 2,4-dinitrophenylhydrazone. Chromatographic and spectral properties of the compound have been investigated. Using this as a reference standard, it has been possible to demonstrate that carboxymethylthiopyruvate is the main product of the oxidative deamination of carboxymethylcysteine, D-isomer, catalyzed by D-aspartate oxidase. It has been demonstrated moreover that carboxymethylcysteine, L-isomer, may be a substrate for snake venom L-aminoacid oxidase.CMC S-carboxymethyl-cysteine - CMTP S-carboxymethyl-thiopyruvic acid - 2,4-DNPH 2,4-dinitrophelylhydrazine - DNPH 2,4-dinitrophenylhydrazone - d-aspartate oxidase d-aspartate: O₂ oxidoreductase, deaminating, E.C. 1.4.3.1 - l-aminoacid oxidase l-aminoacid: O₂ oxidoreductase, deaminating, E.C. 1.4.3.2 - d-aminoacid oxidase d-aminoacid: O₂ oxidoreductase, deaminating, E.C. 1.4.3.3 - l-glutamic dehydrogenase l-glutamate: NAD oxidoreductase, deaminating, E.C. 1.4.1.2.     

Fate of the herbicides mecoprop, dichlorprop, and 2,4-D in aerobic and anaerobic sewage sludge as determined by laboratory batch studies and enantiomer-specific analysis

Aerobic degradation experiments with the racemic mixtures of mecoprop and dichlorprop revealed that activated sludge collected from the aeration tank of a municipal waste water treatment plant degraded both enantiomers of mecoprop and dichlorprop within 7 days, albeit in an enantioselective manner; the (S) enantiomers were preferentially degraded. Mecoprop, dichlorprop, and 2,4-D were completely metabolized under aerobic conditions, as shown by the 86–98% elimination of dissolved organic carbon. Under anaerobic conditions, the concentration of 2,4-D decreased exponentially with a first-order reaction rate constant of 0.24 per day and without a lag-phase. After an incubation time of 17 days, 2,4-D was completely removed. 2,4-Dichlorophenol was the main metabolite of anaerobic 2,4-D degradation; only traces of 4-chlorophenol were detected. In contrast, the chiral phenoxypropionic acid herbicides mecoprop and dichlorprop persisted under anaerobic conditions during 49 days of incubation.     

Dehalogenation and Deamination of l-2-Amino-4-chloro-4-pentenoic Acid by Proteus mirabilis

Eighty-one strains of bacteria were tested for their ability to catalyze the release of chloride ion from Dl-2-amino-4-chloro-4-pentenoic acid. A dehalogenating enzyme was obtained from the cells of Proteus mirabilis IFO 3849, which can use the l-isomer. The enzyme was constitutively produced. The conversion of l-2-amino-4-chloro-4-pentenoic acid to 2-keto-4-pentenoic acid, ammonia, and chloride ion was demonstrated. The reaction product, 2-keto-4-pentenoic acid, was isolated as its 2,4-dinitrophenylhydrazone and identified by catalytic hydrogenolysis of the hydrazone to the corresponding amino acid, norvaline.     

Biodegradation of 2,4,6-Tribromophenol by Ochrobactrum sp. Strain TB01

A bacterium that utilizes 2,4,6-tribromophenol (2,4,6-TBP) as sole carbon and energy source was isolated from soil contaminated with brominated pollutants. This bacterium, designated strain TB01, was identified as an Ochrobactrum species. The organism degraded 100 μM of 2,4,6-TBP within 36 h in a growing culture. In addition, it released 3 mol of bromine ions from 1 mol of 2,4,6-TBP during the complete degradation of 2,4,6-TBP in a resting cell assay. Moreover, cells grown on 2,4,6-TBP degraded 2,6-dibromophenol (2,6-DBP), 4-bromophenol (4-BP), 2,4,6-trichlorophenol (2,4,6-TCP) and phenol. Metabolic intermediates were detected in the reaction mixture of an in vitro assay for 2,4,6-TBP, and they were identified as 2,4-DBP and 2-BP. NADH was required for the debromination of 2,4,6-TBP. These results suggest that 2,4,6-TBP is converted to phenol through sequential reductive debromination reactions via 2,4-DBP and 2-BP by this strain.     

Biodegradation of mixtures of chlorophenoxyalkanoic acid herbicides by Alcaligenes denitrificans

An Alcaligenes denitrificans strain able to degrade (R)-2-(2-methyl-4-chlorophenoxy)propionic acid [(R)-MCPP, mecoprop] was assessed for its ability to utilise a range of chlorophenoxyalkanoic acid herbicides in single, binary, tertiary and quaternary combinations in batch culture. Degradation rates were rapid with single growth substrates; complete degradation occurred within 29 h for 2,4-dichlorophenoxyacetic acid (2,4-D), 43 h for 4-chloro-2-methylphenoxyacetic acid (MCPA) and 50 h for (R)-MCPP, respectively. After 20 h, the degradation of (RS)-2-(2,4-dichlorophenoxy)propionic acid [(RS)-2,4-DP] had ceased, with only the (R)-enantiomer being degraded. In binary combination, 2,4-D and MCPP degraded within 55 h. Degradation rates decreased when herbicides were added in tertiary and quaternary combinations. Thus, at the whole cell level, catalysis of closely related herbicides is likely to be facilitated by diverse enzymatic activity in A. denitrificans. Journal of Industrial Microbiology & Biotechnology (2000) 25, 255–259. Received 16 April 2000/ Accepted in revised form 07 August 2000     

A new test for chorismate mutase activity.

Chorismate mutase (EC 5.4.99.5) catalyzes the conversion of chorismic acid to prephenic acid. A continuous test of the enzymatic activity is based on the decrease of the absorption of the substrate chorismate at 274 nm (1). In a sensitive but discontinuous test, prephenic acid, the product of the enzymatic reaction is converted to phenylpyruvic acid. The absorbance of its enolic form is determined in alkaline solution at 320 nm (2,3). Another discontinuous test makes use of the absorption of the phenylpyruvate enol-borate complex (4,5) at 300 nm. The continuous test cannot be used when aromatic compounds are to be tested as modifiers of the enzymatic activity. Similarly, the tests based on the absorption of the enolic form of phenylpyruvic acid cannot be used when compounds which show a high absorbance in the 300 to 320 nm wavelength region are tested. This paper describes a test for chrismate mutase based on the determination of the 2,4-dinitrophenylhydrazone of the α-keto acid, phenylpyruvic acid. In alkaline solution the 2,4-dinitrophenylhydrazone of phenylpyruvic acid shows an absorption maximum at 440 nm, thus allowing one to test compounds like 3-hydroxybenzoic acid and 5-hydroxyisophthalic acid as potential inhibitors of the enzymatic reaction.     

Influence of environmental factors on 2,4-dichlorophenoxyacetic acid degradation by Pseudomonas cepacia isolated from peat

A Pseudomonas cepacia, designated strain BRI6001, was isolated from peat by enrichment culture using 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. BRI6001 grew at up to 13 mM 2,4-D, and degraded 1 mM 2,4-D at an average starting population density as low as 1.5 cells/ml. Degradation was optimal at acidic pH, but could also be inhibited at low pH, associated with chloride release from the substrate, and the limited buffering capacity of the growth medium. The only metabolite detected during growth on 2,4-D was 2,4-dichlorophenol (2,4-DCP), and degradation of the aromatic nucleus was by intradiol cleavage. Growth lag times prior to the on-set of degradation, and the total time required for degradation, were linearly related to the starting population density and the initial 2,4-D concentration. BRI6001, grown on 2,4-D, oxidized a variety of structurally similar chlorinated aromatic compounds accompanied by stoichiometric chloride release.     

Biodegradation kinetics of 2,4-D by bacterial strains isolated from soil

The aim of the study was to characterize the 2,4-dichlorophenoxyacetic acid (2,4-D) degradative potential of three bacterial strains identified by MIDI-FAME profiling as Burkholderia cepacia (DS-1), Pseudomonas sp. (DS-2) and Sphingomonas paucimobilis (DS-3) isolated from soil with herbicide treatment history. All strains were capable of using herbicide as the only source of carbon and energy when grown in mineral salt medium (MSM) containing 2,4-D (50 mg/l). Over a 10 day incubation period, 69%, 73% and 54% of the initial dose of 2,4-D were degraded by strains DS-1, DS-2 and DS-3, respectively. Analysis of 2,4-dichlorophenol (2,4-DCP) concentration, the main metabolite of 2,4-D degradation, revealed that strains DS-1 and DS-2 may also have the potential to metabolize this compound. The percentage of 2,4-DCP removal was 67% and 77% in relation to maximum values of 9.5 and 9.2 mg/l determined after 4 and 2 days for MSM+DS-1 and MSM+DS-2, respectively. The degradation kinetics of 2,4-D (50 mg/kg) in sterile soil (SS) showed different potential of tested strains to degrade 2,4-D. The times within which the initial 2,4-D concentration was reduced by 50% (DT₅₀) were 6.3, 5.0 and 9.4 days for SS+DS-1, SS+DS-2 and SS+DS-3, respectively.     

Novel Insight into the Genetic Context of the cadAB Genes from a 4-chloro-2-methylphenoxyacetic Acid-Degrading Sphingomonas

The 2-methyl-4-chlorophenoxyacetic (MCPA) acid-degrader Sphingomonas sp. ERG5 has recently been isolated from MCPA-degrading bacterial communities. Using Illumina-sequencing, the 5.7 Mb genome of this isolate was sequenced in this study, revealing the 138 kbp plasmid pCADAB1 harboring the 32.5 kbp composite transposon Tn6228 which contains genes encoding proteins for the removal of 2,4-dichlorophenoxyacetic acid (2,4-D) and MCPA, as well as the regulation of this pathway. Transposon Tn6228 was confirmed by PCR to be situated on the plasmid and also exist in a circular intermediate state - typical of IS3 elements. The canonical tfdAα-gene of group III 2,4-D degraders, encoding the first step in degradation of 2,4-D and related compounds, was not present in the chromosomal contigs. However, the alternative cadAB genes, also providing the initial degradation step, were found in Tn6228, along with the 2,4-D-degradation-associated genes tfdBCDEFKR and cadR. Putative reductase and ferredoxin genes cadCD of Rieske non-heme iron oxygenases were also present in close proximity to cadAB, suggesting that these might have an unknown role in the initial degradation reaction. Parts of the composite transposon contain sequence displaying high similarity to previously analyzed 2,4-D degradation genes, suggesting rapid dissemination and high conservation of the chlorinated-phenoxyacetic acid (PAA)-degradation genotype among the sphingomonads.     

Conversion of 2-chloromaleylacetate in Alcaligenes eutrophus JMP134

2,4-Dichlorophenoxyacetate (2,4-D) in Alcaligenes eutrophus JMP134 (pJP4) is degraded via 2-chloromaleylacetate as an intermediate. The latter compound was found to be reduced by NADH in a maleylacetate reductase catalyzed reaction. Maleylacetate and chloride were formed as products of 2-chloromaleylacetate reduction, the former being funnelled into the 3-oxoadipate pathway by a second reductive step. There was no indication for an involvement of a pJP4-encoded enzyme in either the reduction or the dechlorination reaction.Abbreviations 2,4-D 2,4-dichlorophenoxyacetate     

Identification of 9,17-Dioxo-1,2,3,4,10,19-Hexanorandrostan-5-oic Acid, 4-Hydroxy-2-Oxohexanoic Acid, and 2-Hydroxyhexa-2,4-Dienoic Acid and Related Enzymes Involved in Testosterone Degradation in Comamonas testosteroni TA441

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.     

Chemical Studies on Amino––Carbonyl Reaction

The 3-deoxy-n-pentosone (I) was isolated from the browning degradation mixture of N-n-xy1osy1-n-butylamine by the action of acetic acid at 55°C. The 3-deoxy-d-erythrohexosone (IIa) and the 3-deoxy-n-threohexosone (IIb) were also prepared by degradation of the corresponding N-glycosyl-n-butylamine. The 3-deoxy-d-pentosone was characterized as the 2,4-dinitrophenylosazone and its diacetate, and the p-nitrophenylosazone. The two 3-deoxy-d-hexosones were also characterized as the analogous derivatives. The three 3-deoxyosones gave positive color reactions with 2-thiobarbituric acid.

As one of the intermediates in 3-deoxyosone formation from N-glycoside, 1,2-eno1 form of 1-deoxy-1-n-butylamino-2-ketose (IV) was proposed.