Phenolic Azo Dye Oxidation by Laccase from Pyricularia oryzae

Laccase oxidation of phenolic azo dyes was examined with a commercially available laccase from Pyricularia oryzae as the model. Methyl-, methoxy-, chloro-, and nitro-substituted derivatives of 4-(4(prm1)-sulfophenylazo)-phenol were examined as substrates for this laccase. Only the substituents on the phenolic ring were changed. Among the dyes examined, only 2-methyl-, 2-methoxy-, 2,3-dimethyl-, 2,6-dimethyl-, 2,3-dimethoxy-, and 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol served as substrates. Preliminary kinetic studies suggest that 2,6-dimethoxy-substituted 4-(4(prm1)-sulfophenylazo)-phenol is the best substrate. Laccase oxidized the 2,6-dimethyl derivative of 4-(4(prm1)-sulfophenylazo)-phenol to 4-sulfophenylhydroperoxide (SPH) and 2,6-dimethyl-1,4-benzoquinone. The 2-methyl- and 2-methoxy-substituted dyes were oxidized to SPH and either 2-methyl- or 2-methoxy-benzoquinone. Six products were formed from laccase oxidation of the 2,6-dimethoxy-substituted dye. Three of them were identified as SPH, 4-hydroxybenzenesulfonic acid, and 2,6-dimethoxybenzoquinone. A mechanism for the formation of benzoquinone and SPH from laccase oxidation of phenolic azo dyes is proposed. This study suggests that laccase oxidation can result in the detoxification of azo dyes.     

Alkaline Phosphatase in Leucocytes by An Azo Dye Method

Alkaline phosphatase activity was demonstrated in the cytoplasm of leucocytes of man, rat and rabbit by means of naphthol AS phosphate as the substrate and fast blue BBN as the coupling agent. The reaction was effected in Tris buffer, 0.2 M at ph 9.1, in 5-10 min at 25°C. Air-dried smears, with or without fixation in 10% neutral formalin, showed activity but those heated 1 min in water at 100°C did not.     

Transformation of Azo Dye Isomers by Streptomyces chromofuscus A11

Fourteen mono-azo dyes were used to study the effects of substitution patterns on the biodegradability of dimethyl-hydroxy-azobenzene 4(prm1)-sulfonic acids by Streptomyces chromofuscus A11. Two substitution patterns were analyzed: (i) all possible substitution patterns of the two methyl and hydroxy substitution groups, 2-hydroxy (3,5; 4,5; 5,6) dimethyl and 4-hydroxy (2,3; 2,5; 2,6; 3,5) dimethyl isomers of azobenzene 4(prm1)-sulfonic acid; and (ii) replacement of the sulfonic group with a carboxylic group in these sulfonated azo dyes. The structural pattern of the hydroxy group in para position relative to the azo linkage and of two methyl substitution groups in ortho position relative to the hydroxy group was the most susceptible to degradation. Replacement of the sulfonic group with a carboxylic group enhanced overall dye degradability by S. chromofuscus A11.     

A Radioiodinated Azo Dye with Affinity for Amyloid: A Preliminary Report

Investigation was undertaken to produce a radioactive material with affinity for amyloid tissue. Studies of a number of major dyes using paraffin histological sections of an amyloid spleen as test material showed that the only compounds with marked affinity for amyloid were watersoluble disazo dyes of the type E←D→E, where D is usually benzidine, o-tolidine or o-dianisidine, and E is a naphthol or aminonaphthol sulfonic acid. Substitution of an amino or a hydroxyl group in position 1 of the naphthalene nucleus caused loss of staining affinity. It was found that trypan blue could be radioiodinated at position 8 using the Sandmeyer reaction, and the labelled dye showed excellent staining and radioautographs of the test amyloid section. Metabolism of the tagged dye using 10-200 μg./kg. in animals showed minimal organ uptake, except for the liver which accumulated 30-40% of the injected dose by the end of four days. Use of this material in the diagnosis of human amyloid disease is being explored.     

Biodegradation of Azo Dye Methyl Red by Methanogenic Microbial Communities Isolated from Volga River Sediments

Microbiology - Azo dyes are soluble xenobiotics stable under oxidizing conditions, which are widely used in human practice; they are present in liquid and solid industrial and household wastes and...     

Lignin Depletion Enhances the Digestibility of Cellulose in Cultured Xylem Cells

Plant lignocellulose constitutes an abundant and sustainable source of polysaccharides that can be converted into biofuels. However, the enzymatic digestion of native plant cell walls is inefficient, presenting a considerable barrier to cost-effective biofuel production. In addition to the insolubility of cellulose and hemicellulose, the tight association of lignin with these polysaccharides intensifies the problem of cell wall recalcitrance. To determine the extent to which lignin influences the enzymatic digestion of cellulose, specifically in secondary walls that contain the majority of cellulose and lignin in plants, we used a model system consisting of cultured xylem cells from Zinnia elegans . Rather than using purified cell wall substrates or plant tissue, we have applied this system to study cell wall degradation because it predominantly consists of homogeneous populations of single cells exhibiting large deposits of lignocellulose. We depleted lignin in these cells by treating with an oxidative chemical or by inhibiting lignin biosynthesis, and then examined the resulting cellulose digestibility and accessibility using a fluorescent cellulose-binding probe. Following cellulase digestion, we measured a significant decrease in relative cellulose content in lignin-depleted cells, whereas cells with intact lignin remained essentially unaltered. We also observed a significant increase in probe binding after lignin depletion, indicating that decreased lignin levels improve cellulose accessibility. These results indicate that lignin depletion considerably enhances the digestibility of cellulose in the cell wall by increasing the susceptibility of cellulose to enzymatic attack. Although other wall components are likely to contribute, our quantitative study exploits cultured Zinnia xylem cells to demonstrate the dominant influence of lignin on the enzymatic digestion of the cell wall. This system is simple enough for quantitative image analysis, but realistic enough to capture the natural complexity of lignocellulose in the plant cell wall. Consequently, these cells represent a suitable model for analyzing native lignocellulose degradation.     

Degradation of Sulphonated Azo Dye Red HE7B by Bacillus sp. and Elucidation of Degradative Pathways

Bacteria capable of degrading the sulfonated azo dye Red HE7B were isolated from textile mill effluent contaminated soil. The most efficient isolate was identified as Bacillus sp. Azo1 and the isolate could successfully decolorize up to 89 % of the dye. The decolorized cultural extract analyzed by HPLC confirmed degradation. Enzymatic analysis showed twofold and fourfold increase in the activity of azoreductase and laccase enzymes, respectively, indicating involvement of both reductive and oxidative enzymes in biodegradation of Red HE7B. Degraded products which were identified by GC/MS analysis included various metabolites like 8-nitroso 1-naphthol, 2-diazonium naphthalene. Mono azo dye intermediate was initially generated from the parent molecule. This mono azo dye was further degraded by the organism, into additional products, depending on the site of cleavage of R–N=N–R molecule. Based on the degradation products identified, three different pathways have been proposed. The mechanism of degradation in two of these pathways is different from that of the previously reported pathway for azo dye degradation. This is the first report of a microbial isolate following multiple pathways for azo dye degradation. Azo dye Red HE7B was observed to be phytotoxic, leading to decrease in root development, shoot length and seedling fresh weight. However, after biotreatment the resulting degradation products were non-phytotoxic.     

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.     

Kinetic Studies of Reactive Azo Dye Decolorization in Anaerobic/aerobic Sequencing Batch Reactors

The decolorization kinetics of Remazol Brilliant Violet 5R (RBV-5R) and Remazol Black B (RB-B) (mono- and diazo reactive dyes, respectively) was investigated in the first 9 h (anaerobic phase) of a 24-h cycle anaerobic/aerobic sequencing batch reactor (SBR). Two distinct, successive decolorization periods were observed for both dyes, apparently due to different decolorization mechanisms. The apparent first-order rate constants were much lower for the second periods. First-order kinetics were apparently followed for both periods of RBV-5R but not for the first decolorization period of RB-B, possibly due to the occurrence of mass transfer limitations.     

Thermally Enhanced Radiosensitivity of Cultured Chinese Hamster Cells

X-IRRADIATION of mammalian cells in culture yields a survival curve of the threshold type (for review see ref. 1). It isjnter-esting to ask how one can enhance the radiation response by small changes of the physical environment of the cells, as can be done chemically, for example, by incorporation of 5-bromo-deoxyuridine into DNA^1,2. Elevation of the temperature is a likely prospect for enhancement of radiosensitivity for the following reasons. It is known that proteins are heat labile and that temperature sensitive mutants of bacteria and phage can be obtained for many different enzymes³ which are operative at 37° C but not at 42° or 43°C. For example⁴, DNA polymerase is reversibly temperature sensitive; it is rendered inoperative above 42°C, but will be functional again when the temperature is lowered. It is not unreasonable to expect that temperature sensitive mutations for many enzymes occur frequently and that the use of temperatures somewhat higher than the normal range at which the cells grow might disclose sensitivities for specific enzymes in normal cells of higher organisms.     

Enhanced Lignin Monomer Production Caused by Cinnamic Acid and Its Hydroxylated Derivatives Inhibits Soybean Root Growth

Cinnamic acid and its hydroxylated derivatives (p-coumaric, caffeic, ferulic and sinapic acids) are known allelochemicals that affect the seed germination and root growth of many plant species. Recent studies have indicated that the reduction of root growth by these allelochemicals is associated with premature cell wall lignification. We hypothesized that an influx of these compounds into the phenylpropanoid pathway increases the lignin monomer content and reduces the root growth. To confirm this hypothesis, we evaluated the effects of cinnamic, p-coumaric, caffeic, ferulic and sinapic acids on soybean root growth, lignin and the composition of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) monomers. To this end, three-day-old seedlings were cultivated in nutrient solution with or without allelochemical (or selective enzymatic inhibitors of the phenylpropanoid pathway) in a growth chamber for 24 h. In general, the results showed that 1) cinnamic, p-coumaric, caffeic and ferulic acids reduced root growth and increased lignin content; 2) cinnamic and p-coumaric acids increased p-hydroxyphenyl (H) monomer content, whereas p-coumaric, caffeic and ferulic acids increased guaiacyl (G) content, and sinapic acid increased sinapyl (S) content; 3) when applied in conjunction with piperonylic acid (PIP, an inhibitor of the cinnamate 4-hydroxylase, C4H), cinnamic acid reduced H, G and S contents; and 4) when applied in conjunction with 3,4-(methylenedioxy)cinnamic acid (MDCA, an inhibitor of the 4-coumarate:CoA ligase, 4CL), p-coumaric acid reduced H, G and S contents, whereas caffeic, ferulic and sinapic acids reduced G and S contents. These results confirm our hypothesis that exogenously applied allelochemicals are channeled into the phenylpropanoid pathway causing excessive production of lignin and its main monomers. By consequence, an enhanced stiffening of the cell wall restricts soybean root growth.     

Localization of the Enzyme System Involved in Anaerobic Reduction of Azo Dyes by Sphingomonas sp. Strain BN6 and Effect of Artificial Redox Mediators on the Rate of Azo Dye Reduction

The effect of different artificial redox mediators on the anaerobic reduction of azo dyes by Sphingomonas sp. strain BN6 or activated sludge was investigated. Reduction rates were greatly enhanced in the presence of sulfonated anthraquinones. For strain BN6, the presence of both cytoplasmic and membrane-bound azo reductase activities was shown.     

Azo Dye Colony Staining Technique To Detect Fungal Mutants Lacking β-Glucosidase Activity

A simple, rapid, convenient, easily interpreted azo dye colony-staining technique was developed to detect fungal mutants lacking β-glucosidase activity on a 6-bromo-2-naphthyl substrate. The procedure includes net replication of colonies and chemical restriction of colony growth to allow testing of many colonies per culture dish.     

Bioremediation of Textile Azo Dyes by Aerobic Bacterial Consortium Aerobic Degradation of Selected Azo Dyes by Bacterial Consortium

An aerobic bacterial consortium consisting of two isolated strains (BF1, BF2) and a strain of Pseudomonas putida (MTCC1194) was developed for the aerobic degradation of a mixture of textile azodyes and individual azodyes at alkaline pH (9-10.5) and salinity (0.9-3.68 g/l) at ambient temperature (28 +/- 2 degrees C). The degradation efficiency of the strains in different media (mineral media and in the Simulated textile effluent (STE)) and at different dye concentrations were studied. The presence of a H2O2 independent oxidase-laccase (26.5 IU/ml) was found in the culture filtrate of the organism BF2. The analysis of the degraded products by TLC and HPLC, after the microbial treatment of the dyes showed the absence of amines and the presence of low molecular weight oxidative degradation products. The enzymes present in the crude supernatant was found to be reusable for the dye degradation.     

Taurine Release Is Enhanced in Cell-Damaging Conditions in Cultured Cerebral Cortical Astrocytes

The release of preloaded [³H]taurine from cultured cerebral cortical astrocytes was studied under various cell-damaging conditions, including hypoxia, ischemia, aglycemia and oxidative stress, and in the presence of free radicals. Astrocytic taurine release was enhanced by K⁺ (50 mM), veratridine (0.1 mM) and the ionotropic glutamate receptor agonist kainate (1.0 mM). Metabotropic glutamate receptor agonists had only weak effects on taurine release. Similarly to the swelling-induced taurine release the efflux in normoxia seems to be mediated mainly by DIDS-(diisothiocyanostilbene-2,2-disulphonate) and SITS-(4-acetamido-4-isothiocyanostilbene-2,2-disulphonate) sensitive CI^– channels, since these blockers were able to reduce both basal and K⁺ -stimulated release. The basal release of taurine was moderately enhanced in hypoxia and ischemia, whereas the potentiation in the presence of free radicals was marked. The small basal release from astrocytes signifies that taurine release from brain tissue in ischemia may originate from neurons rather than glial cells. On the other hand, the release evoked by K⁺ in hypoxia and ischemia was greater than in normoxia, with a very slow time-course. The enhanced release of the inhibitory amino acid taurine from astrocytes in ischemia may be beneficial to surrounding neurons, outlasting the initial stimulus and counteracting overexcitation.     

Manganese-Mediated Lignin Degradation by Pleurotus pulmonarius

Pleurotus pulmonarius produced the strongest degradation of lignin during solid-state fermentation of [(sup14)C]lignin wheat straw with different fungi. A manganese-oxidizing peroxidase seemed to be involved in lignin attack, since the addition of Mn(sup2+) to the culture increased lignin mineralization by ca. 125%. This enzyme was purified and characterized from both solid-state fermentation and liquid cultures.     

Lignin degradeation by white rot fungi

Abstract. The wood-degrading white-rot fungus Phanerochaete chrysosporium , has been the subject of intensive research in recent years and, based upon isolation of the extracellular enzyme ligninase, major advances have now been made toward elucidating the mechanism by which this fungus degrades lignin. From these developments, a model emerges which could explain the process by which wood-degrading fungi in general, attack lignin.     

Dye removal by immobilised fungi

Dyes are widely used within the food, pharmaceutical, cosmetic, printing, textile and leather industries. This has resulted in the discharge of highly coloured effluents that affect water transparency and gas solubility in water bodies. Furthermore, they pose a problem because of their carcinogenicity and toxicity. Therefore, removal of such dyes before discharging them into natural water streams is essential. For this, appropriate treatment technologies are required. The treatment of recalcitrant and toxic dyes with traditional technologies is not always effective or may not be environmentally friendly. This has impelled the search for alternative technologies such as biodegradation with fungi. In particular, ligninolytic fungi and their non-specific oxidative enzymes have been reported to be responsible for the decolouration of different synthetic dyes. Thus, the use of such fungi is becoming a promising alternative to replace or complement the current technologies for dye removal. Processes using immobilised growing cells seem to be more promising than those with free cells, since the immobilisation allows using the microbial cells repeatedly and continuously. This paper reviews the application of fungal immobilisation to dye removal.     

Lignin gel with unique swelling property

Lignin gels were prepared from acetic acid lignin by use of polyethylene glycol diglycidyl ether as cross-linker. The gels were found to swell in aqueous ethanol solution, in particular 50% (v/v) solution. In addition, they also swelled in alkaline solution and shrank upon heating. A literature search showed that investigation on gel swelling in aqueous ethanol has not been reported so far. Gels prepared from the cross-linker alone and its analogues did not show such swelling characteristics in aqueous ethanol. Therefore, the unique swelling property must be attributable to an intrinsic property of lignin.