Identification of glutamine chain lengths of endogenous Folypoly-γ-glytamates in rat tissues

A simplified procedure for the determination of the glutamate chain lengths of endogenous tissue folate is described.Natural pteroylpoly-γ-glutamates in tissue extracts, irrespective of their one-carbon moiety, were converted by a reductive cleavage procedure to a homologous series of p-aminobenzoylpoly-γ-glutamates, differing only in glutamate chain length. Desalting and concentration of the extracts were achieved by absorbing the derivatives on active charcoal followed by their elution with ethanol: ammonia. Aminobenzoylpolyglutamates were separated by DEAE- cellulose chromatography and quantitated by a colorimetric procedure for primary aromatic amines.The major endogenous folates in rat liver and kidney were pteroylpentaglutamate derivatives with smaller amounts of pteroyltetra- and hexaglutamate.     

Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-gamma-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver

A new reversed-phase high-performance liquid chromatographic (hplc) method is described for the separation and quantitation of picomole amounts of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates. In conjunction with our previously described procedures for the differential cleavage of one-carbon-substituted, reduced folates, this hplc method provides a rapid, sensitive, and reproducible approach to the quantitation and chain-length determination of three pools of unlabeled endogenous pteroylpolyglutamates. Analysis of rat liver (n = 9) yielded the following results ($\text{x}{}^1\text{±}\text{SE}$): total folates 14.5 ± 1.0 nmol/g; folates of pool 1 (5,10-methylenetetrahydro- and unsubstituted tetra- and dihydrofolates) 2.65 ± 0.74 nmol/g; folates of pool 2 (5-methyltetrahydrofolates) 5.30 ± 0.36 nmol/g; and folates of pool 3 (5,10-methenyltetrahydro-, 10-formyltetrahydro-, 5-formyltetrahydro-, and 5-formiminotetrahydrofolates) 6.40 ± 1.60 nmol/g. Most of the folates of rat liver occur as penta- (7.60 ± 0.69 nmol/g) and hexaglutamates (6.00 ± 0.29 nmol/g). In pool 3 the hexaglutamates predominate. We also report experiments showing that folate patterns based on the amount of radioactive label incorporated after a pulse dose of [³H]folic acid differ at all times from the true steady-state pattern of unlabeled endogenous folates.     

Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver

A new reversed-phase high-performance liquid chromatographic (hplc) method is described for the separation and quantitation of picomole amounts of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates. In conjunction with our previously described procedures for the differential cleavage of one-carbon-substituted, reduced folates, this hplc method provides a rapid, sensitive, and reproducible approach to the quantitation and chain-length determination of three pools of unlabeled endogenous pteroylpolyglutamates. Analysis of rat liver (n = 9) yielded the following results ( ): total folates 14.5 ± 1.0 nmol/g; folates of pool 1 (5,10-methylenetetrahydro- and unsubstituted tetra- and dihydrofolates) 2.65 ± 0.74 nmol/g; folates of pool 2 (5-methyltetrahydrofolates) 5.30 ± 0.36 nmol/g; and folates of pool 3 (5,10-methenyltetrahydro-, 10-formyltetrahydro-, 5-formyltetrahydro-, and 5-formiminotetrahydrofolates) 6.40 ± 1.60 nmol/g. Most of the folates of rat liver occur as penta- (7.60 ± 0.69 nmol/g) and hexaglutamates (6.00 ± 0.29 nmol/g). In pool 3 the hexaglutamates predominate. We also report experiments showing that folate patterns based on the amount of radioactive label incorporated after a pulse dose of [³H]folic acid differ at all times from the true steady-state pattern of unlabeled endogenous folates.     

Folylpolyglutamate Derivatives of Pisum sativum L. Determination of Polyglutamate Chain Lengths by High Performance Liquid Chromatography Following Conversion to p-aminobenzoylpolyglutamates

The folypolyglutamate derivatives of pea seedlings (Pisum sativumL. cv. Homesteader) were extracted in the presence of 2-mercaptoethanoland cleaved to p-aminobenzoylpolyglutamates by treatment withZn-HCl. Azo dyes were formed by reaction with naphthylethylenediamine and purified by polyacrylamide gel chromatography. p-Aminobenzoylpolyglutamateswere regenerated from these dyes by Zn treatment and then concentratedin vacuo. These derivatives were separated according to glutamylchain length by high performance liquid chromatography on WhatmanPartisil SAX columns. The folylpolyglutamates of 4 day old peacotyledons, pea leaves and isolated chloroplasts were mainlytetra- and pentaglutamates. These and folates of shorter chainlength were labelled when seeds and aerial shoots were incubatedwith p-aminobenzoate-[¹⁴C]. Labelling of the pentaglutamatewas reduced in seeds that were imbibed in the presence of 0.1mM methotrexate. Studies of cotyledon folylpolyglutamate synthetaseshowed that polyglutamate chain length was affected by incubationtime and the concentration of tetra-hydrofolate monoglutamatein the reaction system. ¹Present address: Department of Biology, University of Lethbridge,Alberta, Canada T1K 3M4 ²Present address: Department of Horticulture, Xiong-yue AgriculturalCollege, Xiong-yue, Liaoning Province, China (Received August 4, 1989; Accepted December 5, 1989)     

Oxidative and reductive cleavage of folates--a critical appraisal.

A critical analysis has been made of the oxidative and reductive techniques employedfor cleavage of the C⁹-N¹⁰ bond of folic acid and its derivaatives. The assumption has previously been made that these cleavage reactions reduce folates to a common family of p-aminobenzoylglutamate derivatives varying only in the lengths of γ-polyglutamyl peptide side chains which are readily subjected to quantitative and qualitative analysis. This assumption is incorrect. Oxidation by potassium permanganate effectively cleaved folic acid, dihydrofolic acid, tetrahydrofolic acid, and 5-formyltetrahydrofolic acid to yield p-aminobenzoylglutamate. 5-Methyltetrahydrofolic acid was merely oxidized to 5-methyldihydrofolic acid while 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid were oxidized to 10-formylfolate which was stable to further attack. Of all the folate derivatives tested only folic acid and dihydrofolic acid were cleaved to p-aminobenzoylglutamate by the zinc-hydrochloric acid reduction method. Both tetrahydrofolic acid and 5-methyltetrahydrofolic acid were stable under fully reducing conditions. 5,10-Methenyl-,10-formyl-, and 5-formyltetrahydrofolic acid yielded N-methyl-p-aminobenzoylglutamate. It is evident, therefore, that not only is the dominant mammalian tissue folate derivative, 5-methyltetrahydrofolate, resistant to cleavage by either method, but that a common family of p-aminobenzoylglutamate derivatives is not the end product of those folate compounds that are susceptible. While this may not invalidate the reports of the relative polyglutamate chain lengths of tissue folates such data should be regarded with some caution.     

Determination of three different pools of reduced one-carbon-substituted folates: I. A study of the fundamental chemical reactions

The reduced one-carbon-substituted derivatives of folic acid can be grouped in three pools according to their response to acid treatment. Pool 1 is made up of N⁵,N¹⁰-methylene-tetrahydrofolic acid and unsubstituted dihydro- and tetrahydrofolic acid which at pH 1.0 and subsequent exposure to air cleave to p-aminobenzoylglutamic acid. Pool 2 is made up by the acid-stable N⁵-methyl-tetrahydrofolic acid, and pool 3 includes N⁵,N¹⁰-methenyl-tetrahydrofolic acid, N¹⁰-formyltetrahydrofolic acid, N⁵-formyltetrahydrofolic acid, and N⁵-formiminotetrahydrofolic acid, all of which convert to the stable N⁵,N¹⁰-methenyl-tetrahydro form when acid treated. Conditions are described to selectively cleave the C⁹-N¹⁰ bond of the folates of pool 1, pools 1 + 2, and pools 1 + 2 + 3. The cleaved pools are quantitated as the Bratton-Marshall azo dyes of p-aminobenzoylglutamate. The uncleaved pools are converted to Bratton-Marshall-negative products. Pool 1 is determined by converting pool 2 to 4a-hydroxy-5-methyltetrahydrofolic acid and pool 3 to N¹⁰-formylfolic acid, both Bratton-Marshall negative, by 10% hydrogen peroxide oxidation at pH 6.0. Pools 1 + 2 are cleaved with 0.015% hydrogen peroxide and 0.1% potassium permanganate at pH 9.0 which convert the N⁵-methyltetrahydrofolic acid to the acid-cleavable N⁵-methyl-dihydrofolic acid. Pool 3 oxidizes to the Bratton-Marshall-negative N¹⁰-formylfolic acid. Pools 1 + 2 + 3 are cleaved by first reducing pool 3 to N⁵-methyltetrahydrofolic acid with sodium borohydride followed by oxidation at pH 9.0 to its acid-labile dihydro form. Determination of the poly-γ-glutamyl chain length of each pool is possible by chromatographing the azo-p-aminobenzoylpolyglutamates with authentic synthetic markers.     

The oxidative cleavage of folates. A critical study

Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C⁹N¹⁰ bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N⁵-formyl-tetrahydrofolic acid are cleaved at the C⁹N¹⁰ bond to produce p-aminobenzoylglutamic acid. N⁵, N¹⁰-methenyl-tetrahydrofolic acid, N⁵,N¹⁰-methylene-tetrahydrofolic acid, and N¹⁰-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N¹⁰-formyl-folic acid which is completely stable to the oxidative treatment employed. N⁵-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N⁵-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5-³H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-¹⁴C]folic acid to label the folates in vivo is presented.     

Polyglutamylfolate synthesis in Neurospora crassa: changes in pool size following growth in glycine- and methionine-supplemented media

The effect of media supplements on total and polyglutamylfolate concentrations has been examined in Neurospora crassa wild type (FGSC 853), an ethionine-resistant mutant (FGSC 1212), and a methionine auxotroph (FGSC 1330) which lacks folylpolyglutamate synthetase. When the culture medium contained 1 mm glycine, folate concentrations in the wild type were increased by over 90% and more p-[³H]aminobenzoate was incorporated into folates. Growth in l-methionine-supplemented media (1–5 mm) decreased folate levels and labeling in all three strains. In the wild type, this effect of l-methionine was reversed on transfer to unsupplemented media but p-[³H]aminobenzoate pulse-chase experiments suggested that exogenous methionine did not increase the turnover of labeled folates. At 1 mm, d-methionine did not affect polyglutamylfolate labeling but l-methionine reduced ³H incorporation by 65% in the wild type. Ion-exchange chromatography showed that p-[³H]aminobenzoate was incorporated in formyl- and methyltetrahydrofolates which in the wild type, were principally hexaglutamyl derivatives. Glycine-supplemented growth yielded labeled folates that were 24% heptaglutamates but these and pentaglutamates were lacking when l-methionine was supplied. The specific activity of GTP cyclohydrolase was not significantly affected by culture in l-methionine-containing media. Dialysis and gel filtration both lowered enzyme activities and product formation was not changed when up to 10 μmol of l-methionine was added to the reaction system. The data suggest that methionine or its metabolic products exerts some control over folate production which is distinct from the established inhibition of methylenetetrahydrofolate reductase by AdoMet.     

Identification of Quinoide Redox Mediators That Are Formed during the Degradation of Naphthalene-2-Sulfonate by Sphingomonas xenophaga BN6

During aerobic degradation of naphthalene-2-sulfonate (2NS), Sphingomonas xenophaga strain BN6 produces redox mediators which significantly increase the ability of the strain to reduce azo dyes under anaerobic conditions. It was previously suggested that 1,2-dihydroxynaphthalene (1,2-DHN), which is an intermediate in the degradative pathway of 2NS, is the precursor of these redox mediators. In order to analyze the importance of the formation of 1,2-DHN, the dihydroxynaphthalene dioxygenase gene (nsaC) was disrupted by gene replacement. The resulting strain, strain AKE1, did not degrade 2NS to salicylate. After aerobic preincubation with 2NS, strain AKE1 exhibited much higher reduction capacities for azo dyes under anaerobic conditions than the wild-type strain exhibited. Several compounds were present in the culture supernatants which enhanced the ability of S. xenophaga BN6 to reduce azo dyes under anaerobic conditions. Two major redox mediators were purified from the culture supernatants, and they were identified by high-performance liquid chromatography-mass spectrometry and comparison with chemically synthesized standards as 4-amino-1,2-naphthoquinone and 4-ethanolamino-1,2-naphthoquinone.     

4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase

Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.     

Synthesis of novel azo compounds containing 5(4H)-oxazolone ring as potent tyrosinase inhibitors

Six new azo dyes containing of 5(4H)-oxazolone ring were prepared by diazotization of 4-aminohippuric acid and coupling with N,N-dimethylaniline, 1-naphthol and 2-naphthol and condensation with 4-fluoro benzaldehyde or 4-trifluoromethoxy benzaldehyde. The new compounds were fully characterized by spectroscopic techniques. All synthesized compounds exhibited high tyrosinase inhibitory behavior. The results of mushroom tyrosinase inhibition assays indicate that the 4-trifluoromethoxy derivatives have high degrees of inhibition and N,N-dimethylaniline derivatives are better for tyrosinase inhibition than 1-naphthol and 2-naphthol derivatives. All synthesized azo compounds (4a–4f) showed the most potent mushroom tyrosinase inhibition, comparable to that of Kojic acid and l-mimosine, as reference standard inhibitors.     

Azo Reductase Activity of Intact Saccharomyces cerevisiae Cells Is Dependent on the Fre1p Component of Plasma Membrane Ferric Reductase

Unspecific bacterial reduction of azo dyes is a process widely studied in correlation with the biological treatment of colored wastewaters, but the enzyme system associated with this bacterial capability has never been positively identified. Several ascomycete yeast strains display similar decolorizing behaviors. The yeast-mediated process requires an alternative carbon and energy source and is independent of previous exposure to the dyes. When substrate dyes are polar, their reduction is extracellular, strongly suggesting the involvement of an externally directed plasma membrane redox system. The present work demonstrates that, in Saccharomyces cerevisiae, the ferric reductase system participates in the extracellular reduction of azo dyes. The S. cerevisiae Δfre1 and Δfre1 Δfre2 mutant strains, but not the Δfre2 strain, showed much-reduced decolorizing capabilities. The FRE1 gene complemented the phenotype of S. cerevisiae Δfre1 cells, restoring the ability to grow in medium without externally added iron and to decolorize the dye, following a pattern similar to the one observed in the wild-type strain. These results suggest that under the conditions tested, Fre1p is a major component of the azo reductase activity.     

Bacterial decolorization and degradation of azo dyes

Azo compounds constitute the largest and the most diverse group of synthetic dyes and are widely used in a number of industries such as textile, food, cosmetics and paper printing. They are generally recalcitrant to biodegradation due to their xenobiotic nature. However microorganisms, being highly versatile, have developed enzyme systems for the decolorization and mineralization of azo dyes under certain environmental conditions. Several genera of Basidomycetes have been shown to mineralize azo dyes. Reductive cleavage of azo bond, leading to the formation of aromatic amines, is the initial reaction during the bacterial metabolism of azo dyes. Anaerobic/anoxic azo dye decolorization by several mixed and pure bacterial cultures have been reported. Under these conditions, this reaction is non-specific with respect to organisms as well as dyes. Various mechanisms, which include enzymatic as well as low molecular weight redox mediators, have been proposed for this non-specific reductive cleavage. Only few aerobic bacterial strains that can utilize azo dyes as growth substrates have been isolated. These organisms generally have a narrow substrate range. Degradation of aromatic amines depends on their chemical structure and the conditions. It is now known that simple aromatic amines can be mineralized under methanogenic conditions. Sulfonated aromatic amines, on the other hand, are resistant and require specialized aerobic microbial consortia for their mineralization. This review is focused on the bacterial decolorization of azo dyes and mineralization of aromatic amines, as well as the application of these processes for the treatment of azo-dye-containing wastewaters.     

Regulation of folylpoly-gamma-glutamate synthesis in bacteria: in vivo and in vitro synthesis of pteroylpoly-gamma-glutamates by Lactobacillus casei and Streptococcus faecalis

Lactobacillus casei and Streptococcus faecalis accumulated labeled folic acid and metabolized this compound to poly-gamma-glutamates of chain lengths of up to 11 and 5, respectively. Octa- and nonaglutamates predominated in L. casei, and tetraglutamates predominated in S. faecalis. The most effective monoglutamate substrates for the L. casei and S. faecalis folylpoly-gamma-glutamate (folylpolyglutamate) synthetases were methylene- and formyltetrahydrofolate, respectively. Methylenetetrahydropteroylpoly-gamma-glutamates were the preferred poly-gamma-glutamate substrates for both enzymes and, in each case, the highest activity was observed with the diglutamate substrate. The final distribution of folylpolyglutamates in these bacteria appeared to reflect the ability of folates with various glutamate chain lengths to act as substrates for the bacterial folylpolyglutamate synthetases. The proportions of individual folylpolyglutamates were markedly affected by culturing the bacteria in medium containing adenine, whereas thymine was without effect. Adenine did not affect the level of folylpolyglutamate synthetase in either organism but caused a large increase in the proportion of intracellular folates containing one-carbon units at the oxidation level of formate, folates which are substrates for enzymes involved in purine biosynthesis. The folates with shorter glutamate chain lengths in bacteria cultured in the presence of adenine resulted from primary regulation of the de novo purine biosynthetic pathway, regulation which caused an accumulation of formyltetrahydropteroyl-poly-gamma-glutamates (folate derivatives that are ineffective substrates for folylpolyglutamate synthetases), and did not result from regulation of folylpolyglutamate synthetase per se.     

Treatment of peptidoglycan monomer with aqueous ammonia: Formation of lactoylpeptide and a saturated disaccharide

Treatment of peptidoglycan monomer (1) from Brevibacterium divaricatum with aqueous ammonia led to cleavage of the C-3 ether linkage in the N-acetylmuramoyl residue to give the d-lactoylpentapeptide and a saturated disaccharide. By using ¹³C-n.m.r. spectroscopy, the disaccharide was identified as chitobiosamine. Alkaline treatment of model compounds under similar conditions showed that N-acetylmuramoyl derivatives with C-1 unsubstituted undergo cleavage at C-3 to give the corresponding 2-acetamido-2-deoxy-d-glucopyranose derivative. The reaction of 1 with ammonia was monitored by ¹H-n.m.r. spectroscopy and, from the data obtained, rate constants and the activation energy were calculated.     

Decolorization of textile azo dye and Congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis BC01

Shewanella xiamenensis BC01 (SXM) was isolated from sediment collected off Xiamen, China and was identified based on the phylogenetic tree of 16S rRNA sequences and the gyrB gene. This strain showed high activity in the decolorization of textile azo dyes, especially methyl orange, reactive red 198, and recalcitrant dye Congo red, decolorizing at rates of 96.2, 93.0, and 87.5 %, respectively. SXM had the best performance for the specific decolorization rate (SDR) of azo dyes compared to Proteus hauseri ZMd44 and Aeromonas hydrophila NIU01 strains and had an SDR similar to Shewanella oneidensis MR-1 in Congo red decolorization. Luria-Bertani medium was the optimal culture medium for SXM, as it reached a density of 4.69 g-DCW L^?1 at 16 h. A mediator (manganese) significantly enhanced the biodegradation and flocculation of Congo red. Further analysis with UV–VIS, Fourier Transform Infrared spectroscopy, and Gas chromatography–mass spectrometry demonstrated that Congo red was cleaved at the azo bond, producing 4,4′-diamino-1,1′-biphenyl and 1,2′-diamino naphthalene 4-sulfonic acid. Finally, SEM results revealed that nanowires exist between the bacteria, indicating that SXM degradation of the azo dyes was coupled with electron transfer through the nanowires. The purpose of this work is to explore the utilization of a novel, dissimilatory manganese-reducing bacterium in the treatment of wastewater containing azo dyes.     

Folic acid and the methylation of homocysteine by Bacillus subtilis

1. Cell-free extracts of Bacillus subtilis synthesize methionine from serine and homocysteine without added folate. The endogenous folate may be replaced by tetrahydropteroyltriglutamate or an extract of heated Escherichia coli for the overall C₁ transfer, but tetrahydropteroylmonoglutamate is relatively inactive. 2. Extracts of B. subtilis contain serine transhydroxymethylase and 5,10-methylenetetrahydrofolate reductase, which are non-specific with respect to the glutamate content of the folate substrates. Methyl transfer to homocysteine requires a polyglutamate folate as methyl donor. These properties are not affected by growth of the organism with added vitamin B₁₂. 3. The synthesis of methionine from 5-methyltetrahydropteroyltriglutamate and homocysteine has the characteristics of the cobalamin-independent reaction of E. coli. No evidence for a cobalamin-dependent transmethylation was obtained. 4. S-Adenosylmethionine was not a significant precursor of the methyl group of methionine with cell-free extracts, neither was S-adenosylmethionine generated by methylation of S-adenosylhomocysteine by 5-methyltetrahydrofolate. 5. A procedure for the isolation and analysis of folic acid derivatives from natural sources is described. 6. The folates isolated from lysozyme extracts of B. subtilis are sensitive to folic acid conjugase. One has been identified as 5-formyltetrahydropteroyltriglutamate; the other is possibly a diglutamate folate. 7. A sequence is proposed for methionine biosynthesis in B. subtilis in which methyl groups are generated from serine and transferred to homocysteine by means of a cobalamin-independent pathway mediated by conjugated folate coenzymes.     

Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase

A new Trametes trogii laccase was purified and its biochemical properties were subsequently characterized. After a survey of other T. trogii laccases, this laccase showed a lower isoelectric point, different N-terminal sequence and kinetic parameters. Recently most laccase-catalyzed decolorizations of synthetic dyes are single-solute studies with commercially available dyes as model pollutants and need the employment of redox mediators. In this study, to simulate the real industry wastewaters, experiments of laccase-catalyzed decolorization of mixed dyes constituted by azo and anthraquinone dyes were carried out. The results showed that anthraquinone dyes, playing the role of mediators, dramatically promoted the degradation of azo dyes when there was no exogenous mediator in the reaction mixture. This study represents the first attempt to decolorize the mixtures of azo and anthraquinone dyes by purified T. trogii laccase, suggesting great potential for laccase to decolorize textile industry wastewaters.     

Properties of NAD (P) H Azoreductase from Alkaliphilic Red Bacteria Aquiflexum sp. DL6

Azoreductase plays a key role in bioremediation and biotransformation of azo dyes. It initializes the reduction of azo bond in azo dye metabolism under aerobic or anaerobic conditions. In the present study, we isolated an alkaliphilic red-colored Aquiflexum sp. DL6 bacterial strain and identified by 16S rRNA method. We report nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate-dependent azoreductase purified from Aquiflexum sp. DL6 by a combination of ammonium sulfate precipitation and chromatography methods. The azoreductase was purified up to 30-fold with 37 % recovery. The molecular weight was found to be 80 kDa. The optimum activity was observed at pH 7.4 and temperature 60 °C with amaranth azo dye as a substrate. The thermal stability of azoreductase was up to 80 °C. The azoreductase has shown a wide range of substrate specificity, including azo dyes and nitro aromatic compounds. Metal ions have no significant inhibitory action on azoreductase activity. The apparent K _m and V _max values for amaranth azo dye were 1.11 mM and 30.77 U/mg protein respectively. This NAD (P) H azoreductase represents the first azoreductase to be characterized from alkaliphilic bacteria.