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.     

Laccase-mediator system in the decolorization of different types of recalcitrant dyes

Phloroglucinol, thymol, and violuric acid (VIO) were selected as laccase mediators after screening 14 different compounds with indigo carmine (indigoid dye) as a substrate. With the presence of these three mediators, a nearly complete decolorization (90-100%) was attained in 1 h. Thus, these three compounds were used as mediators for the decolorization of other four dyes. The results indicated that VIO was effective mediator in decolorization of Remazol brilliant blue R (RBBR, anthraquinoid dye) and Coomassie brilliant blue G-250 (CBB, triphenylmethane dyes), and Acid red (diazo dye). In presence of VIO, the four dyes described above attained 70% decolorization. Thymol was able to mediate decolorization of RBBR and Azure A (heterocyclic dye). Phloroglucinol has no mediating capability in decolorization of the four dyes analyzed. Mediator concentration, pH, and copper ion have an effect on the decolorization of the RBBR. Our data suggested that the decolorization capabilities of laccase/mediator system were related to the types of mediator, the dye structure and decolorization condition.     

Lignin-derived compounds as efficient laccase mediators for decolorization of different types of recalcitrant dyes

Ten phenols were selected as natural laccase mediators after screening 44 different compounds with a recalcitrant dye (Reactive Black 5) as a substrate. Their performances were evaluated at different mediator/dye ratios and incubation times (up to 6 h) by the use of Pycnoporus cinnabarinus and Trametes villosa laccases and were compared with those of eight known synthetic mediators (including -NOH- compounds). Among the six types of dyes assayed, only Reactive Blue 38 (phthalocyanine) was resistant to laccase-mediator treatment under the conditions used. Acid Blue 74 (indigoid dye), Reactive Blue 19 (anthraquinoid dye), and Aniline Blue (triarylmethane-type dye) were partially decolorized by the laccases alone, although decolorization was much more efficient and rapid with mediators, whereas Reactive Black 5 (diazo dye) and Azure B (heterocyclic dye) could be decolorized only in the presence of mediators. The efficiency of each natural mediator depended on the type of dye to be treated but, with the only exception being Azure B (< 50% decolorization), nearly complete decolorization (80 to 100%) was attained in all cases. Similar rates were attained with the best synthetic mediators, but the reactions were significantly slower. Phenolic aldehydes, ketones, acids, and esters related to the three lignin units were among the best mediators, including p-coumaric acid, vanillin, acetovanillone, methyl vanillate, and above all, syringaldehyde and acetosyringone. The last two compounds are especially promising as ecofriendly (and potentially cheap) mediators for industrial applications since they provided the highest decolorization rates in only 5 to 30 min, depending on the type of dye to be treated.     

Amelioration of the ability to decolorize dyes by laccase: relationship between redox mediators and laccase isoenzymes in Trametes versicolor

The effect of redox mediators in the dye decolorization by two laccase isoenzymes from Trametes versicolor cultures supplemented with barley bran has been investigated. All the redox mediators tested, 1-hydroxybenzotriazole (HBT), promazine (PZ), para-hydroxybenzoic acid (pHBA) and 1-nitroso-2-naphthol-3,6-disulfonic acid (NNDS), led to higher dye decolorization than those obtained without mediator addition. Among the different tested mediators, PZ was the most effective one at a low range of concentration (0.5–50 μM) and the natural mediator employed, pHBA did not improve significantly the degree of decolorization, and was slightly inhibitory.The two laccase isoenzymes, LacI and LacII, showed different decolorization capability depending on the mediator used. No significant differences were detected for NNDS, however LacII was more effective than LacI in the presence of PZ, while in the presence of HBT LacI was the fastest and the most effective isoenzyme.     

Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization

Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick two-step procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccase was found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity of laccase was found to be highest for catechol and lowest for hydroquinone, however, highest K_cat and K_cat/K_m were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0 and 80 °C, respectively. Mn²⁺, Cd²⁺, Fe²⁺, Cu²⁺ and Na⁺ activated laccase while Ca²⁺ treatment increased laccase activity up to 3 mM, beyond which it inhibited laccase. Co²⁺, Hg²⁺, DTT, SDS and EDTA showed an inhibition of laccase activity. The Leucaena laccase was found to be fairly tolerant to organic solvents; upon exposure for 1 h individually to 50% (v/v) each of ethanol, DMF, DMSO and benzene, more than 50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol and chloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized synthetic dyes such as indigocarmine and congo red in the absence of any redox mediator.     

Capacity of Irpex lacteus and Pleurotus ostreatus for decolorization of chemically different dyes

The rate and efficiency of decolorization of poly R-478- or Remazol Brilliant Blue R (RBBR)-containing agar plates (200 μg g⁻¹) were tested to evaluate the dye degradation activity in a total of 103 wood-rotting fungal strains. Best strains were able to completely decolorize plates within 10 days at 28 °C. Irpex lacteus and Pleurotus ostreatus were selected and used for degradation of six different groups of dyes (azo, diazo, anthraquinone-based, heterocyclic, triphenylmethane, phthalocyanine) on agar plates. Both fungi efficiently degraded dyes from all groups. Removal of RBBR, Bromophenol blue, Cu-phthalocyanine, Methyl red and Congo red was studied with I. lacteus also in liquid medium. Within 14 days, the following color reductions were attained: RBBR 93%, Bromophenol blue 100%, Cu-phthalocyanine 98%, Methyl red 56%, Congo red 58%. The ability of I. lacteus to degrade RBBR spiked into sterile soil was checked, the removal being 77% of the dye added within 6 weeks. The capacity of selected white rot fungal species to remove efficiently diverse synthetic dyes from water and soil environments is documented.     

Polysaccharide conjugated laccase for the dye decolorization and reusability of effluent in textile industry

Nine different polysaccharides were screened for conjugation with laccase and evaluated for pH and thermal stability. All the polysaccharides decreased the thermal and pH stability of laccase at 50 °C and 60 °C, where conjugation with gum Arabic showing the most pronounced effect. Thermal instability of gum Arabic conjugated laccase was affirmed by differential scanning calorimeter while the structural changes in the conjugated laccase responsible for thermal instability was analysed by fluorescence spectrophotometer. The gum Arabic conjugated laccase showed an unusually high tolerance to sodium chloride, thermal instability and lower stability in alkaline conditions. Gum Arabic conjugated laccase was found to decolorize Remazol brilliant blue R in the textile effluent at a slower rate without any microbial growth which was unlike that observed in effluent treated with free laccase. Further, effluent treated with conjugated laccase enabled its reuse as liquor for the dyeing to get desired shade.     

Cloning,expression, and characterization of a thermostable and pH-stable laccase from Klebsiella pneumoniae and its application to dye decolorization

A thermostable and pH-stable laccase from Klebsiella pneumoniae was cloned and expressed in Escherichia coli. The recombinant laccase (rLac) achieved a specific activity of 7.12 U/mg after purification by Ni-affinity chromatography. Optimal enzyme activity was observed at pH 4.0 and 35 °C for 2,2′-azino-bis (3-ethylbenzthiazoline sulfonic acid) (ABTS) oxidization and pH 8.0 and 70 °C for 2,6-dimethoxyphenol (2,6-DMP) oxidization. Thermostability and pH stability studies showed that the rLac was stable over the range of 30–70 °C and pH 5.0–9.0 using 2,6-DMP as substrate. Circular dichroism analysis suggested that the secondary structure of the rLac mainly consisted of α-helix that played a vital role in maintaining laccase activity and revealed the potential mechanisms for the changes in laccase activity under varying pHs (3.0–11.0) and temperatures (20–90 °C). Finally, the rLac could decolorize the tested dyes with high decolorization efficiency.     

Decolorization of reactive dyes by a thermostable laccase produced by Ganoderma lucidum in solid state culture

Dye decolorizing potential of the white rot fungus Ganoderma lucidum KMK2 was demonstrated for recalcitrant textile dyes. G. lucidum produced laccase as the dominant lignolytic enzyme during solid state fermentation (SSF) of wheat bran (WB), a natural lignocellulosic substrate. Crude enzyme shows excellent decolorization activity to anthraquinone dye Remazol Brilliant Blue R (RBBR) without redox mediator whereas diazo dye Remazol Black-5 (RB-5) requires a redox mediator. Polyacrylamide gel electrophoresis (PAGE) of crude enzyme confirms that the laccase enzyme was the major enzyme involved in decolorization of either dyes. Native and SDS-PAGE indicates that the presence of single laccase with molecular weight of 43 kDa. N-Hydroxybenzotriazole (HBT) at a concentration of 1 mM was found as the best redox mediator. RB-5 (50 mg l^−l) was decolorized by 62% and 77.4% within 1 and 2 h, respectively by the crude laccase (25 U ml⁻¹). RBBR (50 mg l^−l) was decolorized by 90% within 20 h, however, it was more efficient in presence of HBT showing 92% decolorization within 2 h. Crude laccase showed high thermostability and maximum decolorization activity at 60 °C and pH 4.0. The decolorization was completely inhibited by the laccase inhibitor sodium azide (0.5 mM). Enzyme inactivation method is a good method which averts the undesirable color formation in the reaction mixture after decolorization. High thermostability and efficient decolorization suggest that this crude enzyme could be effectively used to decolorize the synthetic dyes from effluents.     

New and different lignocellulosic materials from Turkey for laccase and manganese peroxidase production by Trametes versicolor

In the present study, the production of laccase (Lac) and manganese‐dependent peroxidase (MnP) by the white‐rot fungus Trametes versicolor grown in submerged cultures with different agricultural residues was investigated. The lignocellulosic materials studied were almond shells, hazelnut husks, sunflower stems, clover straw and hazelnut cobs, because they are common agricultural wastes in Turkey. Among the different lignocellulosic materials studied, hazelnut cobs provided the highest Lac and MnP activities (47.09 and 109.21 U/L, respectively). The optimum conditions were determined for Lac and MnP production in submerged cultures of T. versicolor by using hazelnut cobs as substrate. For Lac production, the optimum incubation time, hazelnut cob concentration, pH, and shaking rate were found as 4 days, 2% w/v, 6.0 and 130 rpm, respectively. For MnP production, the optimum incubation time, hazelnut cob concentration, pH and shaking rate were found as 5 days, 2% w/v, 6.0 and 90 rpm, respectively.     

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.     

Influence of NaCl and Na2SO4 on the kinetics and dye decolorization ability of crude laccase from Ganoderma lucidum

In a solid state medium using yellow passion fruit waste as substrate, the basidiomycete Ganoderma lucidum produced a laccase as the main ligninolytic enzyme. This crude enzyme presented Michaelian behavior with both substrates tested, namely 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and the anthraquinone dye remazol brilliant blue R (RBBR). The K_M’s for these substrates were, respectively, 0.232 × 10⁻³ and 0.602 × 10⁻³ M. The actions of NaCl and Na₂SO₄, two important salts usually found in textile wastewaters, were investigated. The enzyme was inhibited by NaCl, but not by Na₂SO₄. Inhibition by NaCl was of the mixed type with two different inhibition constants. The enzyme was able to completely decolorize RBBR in the presence of 1.0 M Na₂SO₄ and 50% decolorization was found in the presence of 0.1 M NaCl. Such properties certainly make the enzyme a good agent for textile dye effluent treatment considering the fact that wastewaters of this industry usually contain high concentrations of NaCl and Na₂SO₄.     

毛栓菌产漆酶条件优化及该酶对合成染料脱色的特性

【目的】提高菌株Trametes hirsuta SYBC-L19漆酶产量,并研究该酶对合成染料脱色的性质。【方法】通过单因素和响应面设计,对产漆酶培养基进行优化。【结果】最优培养基为:玉米粉20.0 g/L、马铃薯淀粉32.4 g/L、酒石酸铵2.9 g/L、吐温80 0.5 g/L、CuSO4.5H2O 2.0 mmol/L、香兰素0.54 mmol/L、NaH2PO4.2H2O 2.0 g/L、MgSO4.7H2O0.5 g/L、MnSO4.H2O 0.1 g/L;最佳培养条件为:培养温度30°C,初始pH 6.0,装液量40 mL/250 mL,接种量8%。【结论】培养8 d酶活达35 U/mL,是优化前的39倍。对漆酶催化合成染料脱色进行了考察,发现该酶在60°C下对偶氮类染料AR1和RB5能迅速脱色,5 min内即可完成。     

Purification and characterization of laccase from <Emphasis Type="Italic">Pycnoporus sanguineus</Emphasis> and decolorization of an anthraquinone dye by the enzyme

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65°C, respectively. The enzyme was stable up to 40°C, and high laccase activity was maintained at pH 2.0–5.0. Sodium azide, l-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.     

Effect of copper,nutrient nitrogen,and wood-supplement on the production of lignin-modifying enzymes by the white-rot fungus Phlebia radiata

Production of the oxidoreductive lignin-modifying enzymes – lignin and manganese peroxidases (MnPs), and laccase – of the white-rot basidiomycete Phlebia radiata was investigated in semi-solid cultures supplemented with milled grey alder or Norway spruce and charcoal. Concentrations of nutrient nitrogen and Cu-supplement varied also in the cultures. According to extracellular activities, production of both lignin peroxidase (LiP) and MnP was significantly promoted with wood as carbon source, with milled alder (MA) and low nitrogen (LN) resulting with the maximal LiP activities (550 nkat l⁻¹) and noticeable levels of MnP (3 μkat l⁻¹). Activities of LiP and MnP were also elevated on high nitrogen (HN) complex medium when supplemented with spruce and charcoal. Maximal laccase activities (22 and 29 μkat l⁻¹) were obtained in extra high nitrogen (eHN) containing defined and complex media supplemented with 1.5 mM Cu²⁺. However, the nitrogen source, either peptone or ammonium nitrate and asparagine, caused no stimulation on laccase production without Cu-supplement. This is also the first report to demonstrate a new, on high Cu²⁺ amended medium produced extracellular laccase of P. radiata with pI value of 4.9, thereby complementing our previous findings on gene expression, and cloning of a second laccase of this fungus.     

Induction of a white laccase from the deuteromycete Myrothecium verrucaria NF-05 and its potential in decolorization of dyes

Myrothecium verrucaria NF-05 is a deuteromycete fungus capable of producing a white laccase. The optimal concentration of Cu²⁺ for laccase production by this strain is 0.2 mM (43.23 ± 1.16 U mL^{? 1}). A comprehensive investigation of the induction demonstrated that NF-05 laccase production could be synergistically enhanced by various inducers, including aromatic phenols, amines and recalcitrant dyes, in the presence of 0.2 mM Cu²⁺. Sixteen phenols, fourteen amines and four dyes exhibited significant inductive effects on laccase production. The best inducer was 3, 3’-dimethylbenzidine, which increased laccase production to 258.1 ± 11.1 U mL^{? 1}. These results suggest that M. verrucaria NF-05 is a promising industrial laccase producer. Based on the increased production, purified NF-05 laccase was used to decolorize dyes of various structural types in the presence of six redox mediators. Among the 26 tested dyes, the decolorization rate of six azo dyes, chromotrope 2R, orange G6, Congo red, Ponceau S, amaranth and reactive yellow 135 and two arylmethane dyes, fast green 3 and neutral red, were significantly increased by each of the six mediators. These results demonstrate the potential use of the NF-05 laccase for the decolorization of recalcitrant dyes in dye bleaching and effluent detoxification.     

Fungal laccase, cellobiose dehydrogenase, and chemical mediators: combined actions for the decolorization of different classes of textile dyes

Dyes belonging to the mono-, di-, tri- and poly-azo as well as anthraquinonic and mono-azo Cr-complexed classes, chosen among the most utilized in textile applications, were employed for a comparative enzymatic decolorization study using the extracellular crude culture extracts from the white rot fungus Funalia (Trametes) trogii grown on different culture media and activators able to trigger different levels of expression of oxidizing enzymes: laccase and cellobiose dehydrogenase. Laccase containing extracts were capable to decolorize some dyes from all the different classes analyzed, whereas the recalcitrant dyes were subjected to the combined action of laccase and the chemical mediator HBT, or laccase plus cellobiose dehydrogenase. Correlations among the decolorization degree of the various dyes and their electronic and structural diversities were rationalized and discussed. The utilization of cellobiose dehydrogenase in support to the activity of laccase for the decolorization of azo textile dyes resulted in substantial increases in decolorization for all the refractory dyes proving to be a valid alternative to more expensive and less environmentally friendly chemical treatments of textile dyes wastes.     

白囊耙齿菌产锰过氧化物酶条件优化及其对染料的脱色

锰过氧化物酶（manganese peroxidase,MnP）是白腐真菌降解多种异生物质的主要降解酶之一。本研究对白囊耙齿菌Irpex lacteus产MnP的酶活曲线进行监测,利用单因素和正交试验对I. lacteus产MnP的发酵条件进行优化,同时检测了I. lacteus的MnP粗酶液对5种染料的脱色效果。结果显示,I. lacteus在培养5d时MnP活性较大;I. lacteus产MnP较优的条件为：可溶性淀粉20g/L、尿素1g/L、pH 6.3、CaCl₂ 1mmol/L、FeCl₃ 1mmol/L,该条件下MnP活性达29.24U/L,与优化前MnP活性相比提高了1.25倍;I. lacteus的MnP粗酶液对5种染料均可脱色,其中对直接大红和活性红的脱色效果更为明显,脱色5d后的脱色率分别达到82%和81%。     

Biochemical characterization of laccase from hairy root culture of <Emphasis Type="Italic">Brassica juncea</Emphasis> L. and role of redox mediators to enhance its potential for the decolorization of textile dyes

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2′-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg⁻¹ of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg⁻¹ specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0–6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and l-cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.