Elevated levels of laccase synthesis by Pleurotus pulmonarius BPSM10 and its potential as a dye decolorizing agent

Laccases (EC 1.10.3.2) are a class of multi-copper oxidases that have industrial value. In the present study, forty-five isolates of wild mushrooms were screened for laccase production. Eight of the isolates exhibited exploitable levels of substrate oxidation capacity. Isolate BPSM10 exhibited the highest laccase activity of 103.50?U/ml. Internal Transcribed Spacer (ITS) rRNA gene sequencing was used to identify BPSM10 as Pleurotus pulmonarius. The response of BPSM10 to two nutritional media supplemented with various inducers was characterized and the results indicated that Malt Extract Broth (MEB) supplemented with Xylidine increased laccase production by 2.8× (349.5?U/ml) relative to the control (122?U/ml), while Potato Dextrose Broth (PDB) supplemented with xylidine increased laccase production by 1.9× (286?U/ml). BPSM10 has the ability to decolorize seven synthetic dyes in a liquid medium. Maximum decolorization was observed of malachite green (MG); exhibiting 68.6% decolorization at 100?mg/L. Fourier-transform infrared spectroscopy (FTIR) was employed to confirm the decolorization capacity. The present study indicates that P. pulmonarius BPSM10 has the potential to be used as a potent alternative biosorbent for the removal of synthetic dyes from aqueous solutions, especially in the detoxification of polluted water.     

Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii

Trametes trogii, a white rot basidiomycete involved in wood decay worldwide, produces several ligninolytic enzymes, laccase being the dominant one, with higher titers than those reported for most other white rot fungi studied up to date. The effect of copper on in vitro production of extracellular ligninolytic activities was studied. CuSO(4)·5H(2)O concentrations from 1.6 μM to 1.5 mM were tested in a synthetic medium with glucose 20 g/L and asparagine 3 g/L. The addition of copper (up to 1 mM) did not affect growth but strongly stimulated ligninolytic enzyme production; faster decolorization of the polymeric dye Poly R-478 was observed as well. Maximal production of manganese peroxidase, laccase, and glyoxal oxidase [1.28 U/mL, 93.8 U/mL (with a specific activity of 720 U/mg protein), and 0.46 U/mL respectively] was attained with 1 mM CuSO(4)·5H(2)O. However, higher copper concentrations inhibited growth and notably decreased manganese peroxidase production, although they did not affect laccase secretion. Laccase activity in the culture filtrate was maximal at 50 C and pH 3.4, and the enzyme was completely stable at pH 4.4 and above, and at 30 C for up to 5 d. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids showed two laccase activity bands (mol wt 38 and 60 kDa respectively). The pattern of isoenzyme production was not affected by medium composition but differed with culture age.     

Stereum hirsutum (Wild) Pers. action in dye degradation

Stereum hirsutum, a white rot fungus, has a good growth in solid state fermentation. This was carried on with wheat bran, soy bran and a mixture of both. Mycelia grown on soy bran showed the highest decolorization activity on Ponceau 2R (xylidine), indigo carmine and malachite green. Optimal relationship between decolorization and detoxification of malachite green was 30 g of fresh weight (mycelia plus substrate) in 500 ml malachite green solution, 42 U/l of laccase was measured in this solution. Decolorization was carried on without the addition either of nutrients or mediators. Conditions for maximal decolorization did not agree with those for maximal ligninolytic enzyme production, but effectiveness of laccase activity on decolorization was evidenced by electrophoretic analysis, that allowed laccase identification and its decolorization activity in gels stained with indigo carmine and malachite green, with ABTS as mediator. Detoxification was assayed using the sensible fungus Phanerochaete chrysosporium.     

Evaluation of Argentinean white rot fungi for their ability to produce lignin-modifying enzymes and decolorize industrial dyes

The decolorizing capacity of 26 white rot fungi from Argentina was investigated. Extracellular production of ligninolytic enzymes by mycelium growing on solid malt extract/glucose medium supplemented with different dyes (Malachite Green, Azure B, Poly R-478, Anthraquinone Blue, Congo Red and Xylidine), dye decolorization and the relationship between these two processes were studied. Only ten strains decolorized all the dyes, all ten strains produced laccase, lignin peroxidase and manganese peroxidase on solid medium. However, six of the strains could not decolorize any of the dyes; all six strains tested negative for lignin peroxidase, and produced less than 0.05 U/g agar of manganese peroxidase. Comparing the isolates with the well-known dye-degrader Phanerochaete chrysosporium, a new fungus was identified: Coriolus versicolor f. antarcticus, potentially a candidate for use in biodecoloration processes. Eighteen day-old cultures of this fungus were able to decolorize in an hour 28%, 30%, 43%, 88% and 98% of Xylidine (24 mg/l), Poly R-478 (75 mg/l), Remazol Brilliant Blue R (9 mg/l), Malachite Green (6 mg/l) and Indigo Carmine (23 mg/l), respectively. Laccase activity was 0.13 U/ml, but neither lignin peroxidase nor manganese peroxidase were detected in the extracellular fluids for that day of incubation.     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

红平菇产漆酶条件优化及对孔雀绿染料脱色的研究

采用LNAS(低氮天冬酰胺-琥珀酸)培养基添加方式,对红平菇Pleurotus djamor HP1进行培养,检测不同时间培养液对不同底物的氧化作用,进而得到光密度值的变化情况,作为漆酶的产生及活性测定的主要依据。结果表明:在含Cu2+的培养液中漆酶最大酶活为235.4 U/L。含Cu2+的培养液添加底物木屑后漆酶最大酶活为458.8 U/L。提取经优化筛选后的培养基培养出的漆酶粗酶液,对4种具有不同化学结构的染料进行了脱色试验。结果表明:三苯基甲烷类的孔雀绿在6 h时脱色率为87.5%,蒽醌类的SN4R在24 h时脱色率为49.4%,偶氮类的甲基橙在24 h时脱色率为45%,杂环类的中性红在24 h时脱色率为23.6%。因此,显示出红平菇漆酶对孔雀绿染料脱色具有较大的应用潜力,进而对废水处理具有更好的应用前景。     

Decolorization of azo and triphenylmethane dyes by Pycnoporus sanguineus producing laccase as the sole phenoloxidase

Partial decolorization of two azo dyes (orange G and amaranth) and complete decolorization of two triphenylmethane dyes (bromophenol blue and malachite green) was achieved by cultures in submerged liquid culture producing laccase as the sole phenoloxidase. Enzyme production could be correlated with dye decolorization, with sorption of dye to mycelia accounting for less than 3% of dye removal.     

裂褶菌菌株G18对孔雀石绿染料的脱色优化

通过对兔眼蓝莓幼果组织中分离得到的内生真菌G18进行形态特征、ITS序列和系统进化分析鉴定菌株G18为裂褶菌Schizophyllum commune。同时,对菌株G18产生的3种木质素降解酶进行监测,发现G18菌株可以分泌漆酶、木质素过氧化物酶和锰过氧化物酶。为明确裂褶菌G18对染料的脱色能力,利用裂褶菌G18对固体条件下8种染料进行脱色能力的检测,筛选出较易脱色的染料后,对该染料的脱色条件进行优化。结果表明,裂褶菌G18对8种染料均可以脱色,对孔雀石绿染料的脱色效果最好。裂褶菌G18对孔雀石绿的脱色优化结果为pH 7.0、20.0g/L淀粉、1.0g/L尿素、1.0g/L硫酸锌、接菌量9片（d=5.0mm）。     

Role of microalgal ligninolytic enzymes in industrial dye decolorization

Abstract

In this study, the decolorization efficiency of seven microalgae isolates; Nostoc muscorum, Nostoc humifusum, Spirulina platensis, Anabaena oryzae, Wollea saccata, Oscillatoria sp. and Chlorella vulgaris was investigated for dye decolorization. The highest decolorization percentages of Brazilwood, Orange G, and Naphthol Green B dyes (99.5%, 99.5%, and 98.5%, respectively) were achieved by Chlorella vulgaris. However, the maximum efficiency for dye decolorization percentages of CV and malachite green dyes were exhibited by A. oryzae (97.4%) and W. saccata (93.3%). Ligninolytic enzymes activity assay was carried out for laccase and lignin peroxidase enzymes, which revealed a high efficiency of the C. vulgaris, A. oryzae and W. saccata to lignin containing compound degradation. The highest laccase production recorded by C. vulgaris with Brazilwood, Orange G, and Naphthol Green B dyes (665.0, 678.6, and 659.5?U/ml, respectively). Similarly, C. vulgaris gave a high lignin peroxidase enzyme production with the above three dyes respectively (306.00, 298.34, and 311.45?U/ml). In addition, A. oryzae and W. saccata showed the highest production of the laccase enzyme (634.6 and 577.45?U/ml, respectively) with CV and malachite green dyes. The degradation products have been characterized after decolorization and verified using FTIR analysis. The high decolorization percentages achieved by C. vulgaris, A. oryzae and W. saccata make them potential candidates for bioremediation and pre-processing to remove dyes from textile effluents.     

Decolorization of synthetic dyes by solid state cultures of Lentinula (Lentinus) edodes producing manganese peroxidase as the main ligninolytic enzyme

The ability of the white-rot fungus Lentinula (Lentinus) edodes to decolorize several synthetic dyes was investigated using solid state cultures with corn cob as substrate. Cultures, containing amido black, congo red, trypan blue, methyl green, remazol brilliant blue R, methyl violet, ethyl violet and Poly R478 at 200 ppm, were completely decolorized after 18 days of incubation. Partial decolorization was observed in the cultures containing 200 ppm of brilliant cresyl blue and methylene blue. High manganese peroxidase activity (2600 U/g substrate), but very low lignin peroxidase (<10 U/g substrate) and laccase (<16 U/g substrate) activities were detected in the cultures. In vitro, the dye decolorization was markedly decreased by the absence of manganic ions and H2O2. These data suggest that manganese peroxidase appear to be the main responsible for the capability of L. edodes to decolorize synthetic dyes.     

Increased production of extracellular laccase by the white rot fungus Coriolus versicolor MTCC 138

Summary The white rot fungus Coriolus versicolor MTCC 138 has been identified as an excellent producer of the industrially important enzyme laccase. The basal medium containing glucose gave laccase activity of 155 U/ml. Screening of different media components and their effects on laccase production by Coriolus versicolor was studied using one factor at a time and L₉ (3⁴) orthogonal array method. A two-fold increase (305 U/ml) in laccase production was observed using a combination of glucose and starch as carbon source and yeast extract as nitrogen source. This activity is very high as compared to most of the reported strains. Hence this strain was explored for enhancement in laccase. The formation of extracellular laccase could be considerably stimulated by the addition of copper in the optimized medium. Addition of 1 mM copper enhanced laccase activity to 460 U/ml. Laccase production was further enhanced using different aromatic inducers. Among different inducers used 2,5-xylidine was found to be the best, and gave maximum laccase activity of 820 U/ml with 1 mM concentration. Thus, this strain could be an efficient and attractive source for laccase production.     

Purification and characterization of laccase produced by a white rot fungus Pleurotus sajor-caju under submerged culture condition and its potential in decolorization of azo dyes

An extracellular laccase was isolated and purified from Pleurotus sajor-caju grown in submerged culture in a bioreactor, and used to investigate its ability to decolorize three azo dyes. The extracellular laccase production was enhanced up to 2.5-fold in the medium amended with xylidine (1 mM). Purification was carried out using ammonium sulfate (70% w/v), DEAE-cellulose, and Sephadex G-100 column chromatography. The enzyme was purified up to 10.3-fold from the initial protein preparation with an overall yield of 53%. The purified laccase was monomeric with an apparent molecular mass of 61.0 kDa. The purified enzyme exerted its optimal activity with 2,2-azino–bis(3-ethylbenzo-thiazoline-6-sulfonate (ABTS) and oxidized various lignin-related phenols. The catalytic efficiencies k _cat/K _m determined for ABTS and syringaldazine were 9.2×10⁵ and 8.7×10⁵, respectively. The optimum pH and temperature for the purified enzyme was 5.0 and 40 °C, respectively. Sodium azide completely inhibited the laccase activity. The absorption spectrum revealed type 1 and type 3 copper signals. The purified enzyme decolorized azo dyes such as acid red 18, acid Black 1, and direct blue 71 up to 90, 87, and 72%, respectively. Decolorization ability of P. sajor-caju laccase suggests that this enzyme could be used for decolorization of industrial effluents.     

Decolorization of Victoria blue by the white rot fungus, <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Synthetic textile dyes are among the most dangerous chemical pollutants released in industrial wastewater streams. Recognizing the importance of reducing the environmental impact of these dyes, the ability of the white rot fungus Phanerochaete chrysosporium to decolorize various textile dyes was investigated. This fungus decolorized 6 of the 14 structurally diverse dyes with varying efficiency (between 14% and 52%). There was no discernable pattern of decolorization even among dyes of the same chemical class, suggesting that attack on the dyes is relatively non-specific. Among the three dyes which showed >40% decolorization, Victoria Blue B (VB) was chosen for further analysis because the ability of the fungus to decolorize VB was nearly independent over a relatively broad concentration range. Blocking lignin peroxidase (LiP) and manganese peroxidase (MnP) production by the fungus did not substantially affect VB decolorization. Inhibition of laccase production by adding various inhibitors to shaken cultures reduced VB decolorization significantly suggesting a role for laccase in VB decolorization. When sodium azide and aminotriazole were used to inhibit endogenous catalase and cytochrome P-450 oxygenase activities, there was 100% and 70% reduction in VB decolorization, respectively. Adding benzoate to trap hydrogen peroxide-derived hydroxyl radicals resulted in 50% decolorization of VB. Boiling the extracellular fluid (ECF) for 30 min resulted in approximately 50% reduction in VB decolorization. Collectively, these data suggest that laccase, and/or oxygenase/oxidase and a heat-stable non-enzymatic factor, but not Lip and MnP, play a role in VB decolorization by P. chrysosporium.     

Bioremediation of Textile Azo Dyes by Trichophyton rubrum LSK-27

Summary The potential of a recently isolated wood-degrading fungus, Trichophyton rubrum LSK-27, for effective decolorization of textile azo dyes was evaluated. Within two days of dye addition, the fungus was able to decolorize 83% of Remazol Tiefschwarz, 86% of Remazol Blue RR and 80% of Supranol Turquoise GGL in liquid cultures. The reactive dyes, Remazol Tiefschwarz and Remazol Blue, were removed by fungal biodegradation, while decolorization of the acid dye, Supranol Turquoise GGL, was accomplished mainly by bioadsorption. Therefore the fungus proved to be efficiently capable of both biodegradation and biosorption as the major dye removal mechanisms. The extent of biodegradation was associated with the levels of the extracellular ligninolytic enzymes such as manganese peroxidase and laccase.     

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.     

毛木耳对孔雀石绿染料降解条件的优化

随着我国印染工业的发展,废水对生态环境的危害日趋严重,亟需开发一种脱色明显且成本低廉的降解方法。本研究发现毛木耳Auricularia cornea菌株AC5对不同结构的染料均具有一定的降解作用,尤其是三苯甲烷类染料。利用26℃、160r/min振荡培养7d的粗酶液对染料（75.0mg/L）进行12h降解,结果显示三苯甲烷染料孔雀石绿、结晶紫,蒽醌染料活性蓝19和偶氮染料活性蓝222的降解效率分别为83.27%、71.77%、67.81%和63.92%。染料降解实验和酶活力测定结果表明,毛木耳对孔雀石绿的降解率达到最高时漆酶活性最高,为321.0U/mL,木质素过氧化物酶和锰过氧化物酶活性较低。因此,推测在降解过程中漆酶起到主要作用。研究表明利用毛木耳菌丝发酵液降解染料废水成本低且操作方便,为染料废水的降解研究提供了前期基础。     

Overproduction of lignin-degrading enzymes by an isolate of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. PSBL-1 is a mutant of this organism that generates the ligninolytic system under nonlimiting conditions during primary metabolism. Lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities for PSBL-1 under nonlimiting conditions were 4- to 10-fold higher than those of the wild type (WT) under nitrogen-limiting conditions. PSBL-1 was still in the log phase of growth while secreting the enzymes, whereas the WT had ceased to grow by this time. As in the WT, manganese(II) increased manganese peroxidase activity in the mutant. However, manganese also caused an increase in lignin peroxidase and glyoxal oxidase activities in PSBL-1. Addition of veratryl alcohol to the culture medium stimulated lignin peroxidase activity, inhibited glyoxal oxidase activity, and had little effect on manganese peroxidase activity in PSBL-1, as in the WT. Fast protein liquid chromatography (FPLC) analysis shows production of larger amounts of isozyme H2 in PSBL-1 than in the WT. These properties make PSBL-1 very useful for isolation of large amounts of all ligninolytic enzymes for biochemical study, and they open the possibility of scale-up production for pratical use.     

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.     

Overproduction of lignin-degrading enzymes by an isolate of Phanerochaete chrysosporium.

Phanerochaete chrysosporium is a white rot fungus which secretes a family of lignin-degrading enzymes under nutrient limitation. PSBL-1 is a mutant of this organism that generates the ligninolytic system under nonlimiting conditions during primary metabolism. Lignin peroxidase, manganese peroxidase, and glyoxal oxidase activities for PSBL-1 under nonlimiting conditions were 4- to 10-fold higher than those of the wild type (WT) under nitrogen-limiting conditions. PSBL-1 was still in the log phase of growth while secreting the enzymes, whereas the WT had ceased to grow by this time. As in the WT, manganese(II) increased manganese peroxidase activity in the mutant. However, manganese also caused an increase in lignin peroxidase and glyoxal oxidase activities in PSBL-1. Addition of veratryl alcohol to the culture medium stimulated lignin peroxidase activity, inhibited glyoxal oxidase activity, and had little effect on manganese peroxidase activity in PSBL-1, as in the WT. Fast protein liquid chromatography (FPLC) analysis shows production of larger amounts of isozyme H2 in PSBL-1 than in the WT. These properties make PSBL-1 very useful for isolation of large amounts of all ligninolytic enzymes for biochemical study, and they open the possibility of scale-up production for pratical use.     

Adsorption and decolorization of dyes using solid residues from Pleurotus ostreatus mushroom production

We investigated the use of solid residues from Pleurotus ostreatus mushroom production in adsorbing and decolorizing different dyes. The solid residue used in this study was composed of hemicellulose and cellulose (52.81 %), acid-insoluble lignin (25.42%), chitin (6.5%), and water extractives (14.82%). After incubating 14% (wt/vol) solid residue in distilled water for 4 h, laccase and manganese peroxidase (MnP) activities were 0.5 U/g and 12 mU/g, respectively. Enzymatic decolorization percentages were up to 100 for azure B (heterocyclic dye) and indigo carmine (indigoid dye), 74.5 for malachite green (MG) (triphenylmethane dye), and zero for xylidine (azoic dye). The optimum temperature for decolorization was in the range of 26 ∼ 36°C for all dyes. Data obtained on adsorption (enzymatic decolorization was prevented with sodium azide) at different dye concentrations and in a pH range of 3 ∼ 7 were used to plot Freundlich isotherms. The spent fungal substrate (SFS) displayed large differences in adsorption capacity, depending on the dye tested. The highest adsorption capacity was observed at pH 3 for MG, while xylidine was slightly adsorbed at pH 3 and 4 and not adsorbed at higher pH values. Laccase and MnP production were affected by the presence of the dyes. The highest enzyme levels were observed in the presence of MG, when laccase and MnP increased 1.39- and 2.13-fold, respectively. Decolorization and adsorption to SFS are both important processes in removing dyes from aqueous solutions. The application of this spent substrate for wastewater treatment will be able to take advantage of both of these dye removal processes. An important problem in bioremediation processes involving microorganisms is the amount of time required for their growth. In this report, we used the spent substrates from mushroom cultivation in wastewater treatment, thus solving the problem of waiting for microorganisms to grow.