Extracellular laccase production and its optimization from Arthrospira maxima catalyzed decolorization of synthetic dyes

In the present study laccase production potential of a photosynthetic, non nitrogen fixing cyanobacteria Arthrospira maxima (SAE-25780) was investigated for their probable use in synthetic dye decolorization which poses environmental pollution problem in aquatic bodies. A. maxima (SAE-25780) showed a constitutive production of laccase which increased up to 80% in the presence of inducer guaiacol. The optimal condition for laccase was 30 °C, 10 mM sucrose as a carbon source, 10 mM sodium nitrate as a nitrogen source, and 2 mM copper as metal activator. The partially purified laccase showed 84% and 49% decolorization potential for the two anthroquinonic dyes-Reactive Blue 4 and Remazol Brilliant Blue R, respectively (RBBR) within 96 h without any mediator. Therefore the laccase extracted from A. maxima (SAE-25780) can be used efficiently in bioremediation of synthetic dyes from paper, pulp and textile industries.     

Potential of Trametes trogii culture fluids and its purified laccase for the decolorization of different types of recalcitrant dyes without the addition of redox mediators

Trametes trogii BAFC 463 culture fluids (containing 110 U ml⁻¹ laccase; 0.94 U ml⁻¹ manganese peroxidase), as well as its purified laccase were capable of decolorizing azoic, indigoid, triphenylmethane, anthraquinonic and heterocyclic dyes, in the absence of redox mediators. Six dyes: RBBR, Indigo Carmine, Xylidine, Malachite Green, Gentian Violet and Bromophenol Blue were almost completely degraded (more than 85% decolorization after 1 d) by either laccase or T. trogii itself in culture, proving the role of the enzyme in dye decolorization. The purified laccase also decolorized 65% of Fast Blue RR and 30% of Azure B and Methylene Blue after 24 h. The use of redox mediators significantly increased the decolorization rates (90% decolorization of Azure B after 1 h). 1-hydroxybenzotriazole resulted the best redox mediator, but the natural mediator p-hydroxybenzoic acid also demonstrated its efficiency for dye decolorization. Due to their ability to decolorize recalcitrant dyes without addition of redox mediators, high laccase activities, high thermostability and efficient decolorization at 70 °C and pH 7.0, even in the presence of high concentrations of heavy metals (100 mM Cu⁺², Pb⁺² or Cd⁺²) or in a synthetic dyebath, T. trogii culture fluids could be effectively used to decolorize synthetic dyes from effluents.     

Textile dye degrading laccase from Pseudomonas desmolyticum NCIM 2112

A laccase requiring optimum temperature 60 °C, pH 4.0 for the activity and having apparent molecular weight 43,000 Da was purified from Pseudomonas desmolyticum NCIM 2112 by three steps, including heating, anion exchange, and molecular sieve chromatography. The purification fold and yield of laccase obtained through Biogel P100 were 45.75 and 19%, respectively. Staining of native gel with L-dopa showed dark brown color band indicating the presence of laccase. In relation to hydroquinone, the substrate specificity of laccase was in the following order: DAB > o-tolidine > ABTS > L-dopa. The absence of monophenolase activity in eluted fractions conformed that the purified protein is laccase. This laccase showed substrate dependent optimum pH character. Effect of inhibitor and metal ion on enzyme activity was analyzed. UV–vis analysis showed the decolorization of Direct Blue-6, Green HE4B and Red HE7B in the presence of laccase. The FTIR spectral comparison between the control dye sample and the metabolites extracted after decolorization by purified laccase have confirmed degradation of these dyes. This study contributes for the structural requirement of a dye to be degradable by P. desmolyticum laccase and is important in order to optimize potential bioremediation systems for industrial textile process water treatment.     

Decolorization of textile azo dyes by aerobic bacterial consortium

The decolorization potential of two bacterial consortia developed from a textile wastewater treatment plant showed that among the two mixed bacterial culture SKB-II was the most efficient in decolorizing individual as well as mixture of dyes. At 1.3 g L^?1 starch supplementation in the basal medium by the end of 120 h decolorization of 80–96% of four out of the six individual azo dyes Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC (10 mg L^?1) was noted. The culture exhibited good potential ability in decolorizing 50–60% of all the dyes (Congo red, Bordeaux, Ranocid Fast Blue and Blue BCC) when present as a mixture at 10 mg L^?1. The consortium SKB-II consisted of five different bacterial types identified by 16S rDNA sequence alignment as Bacillus vallismortis, Bacillus pumilus, Bacillus cereus, Bacillus subtilis and Bacillus megaterium which were further tested to decolorize dyes. The efficient ability of this developed consortium SKB-II to decolorize individual dyes and textile effluent using packed bed reactors is being carried out.     

Detection,quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

Efficient decolorization of an anthraquinone dye by recombinant dye-decolorizing peroxidase (rDyP) immobilized in silica-based mesocellular foam

A recombinant dye-decolorizing peroxidase (rDyP) produced from Aspergillus oryzae was immobilized in synthesized silica-based mesocellular foam (MCF: average pore size 25 nm) and used for decolorization of the anthraquinone dye, Remazol Brilliant Blue R (RBBR). The adsorption yields of rDyP immobilized in MCF increased as the pH decreased from 6 to 3. However, the activity yields of the immobilized rDyP decreased with decreasing pH. The overall efficiency, defined as adsorption yield × activity yield, reached its maximum of 83% at pH 5. In repeated dye-decolorization tests, 20 batches of RBBR could be decolorized by the MCF-immobilized rDyP. MCF showed significantly better performance for rDyP immobilization in term of retaining enzyme activity and dye-decolorization ability compared to previous studies using other mesoporous materials.     

Detoxification of Indigo carmine using a combined treatment via a novel trimeric thermostable laccase and microbial consortium

For the first time, the investigation of Indigo carmine decolorization was done using an atypical Scytalidium thermophilum laccase. Crude and purified laccases required high temperatures and slight acidic pH to achieve maximum Indigo decolorization. Kinetic parameters (K_m and k_cat) of the homotrimeric laccase toward Indigo carmine were determined and laccase efficacy toward repeated dye decolorization process was studied. For the first time, 5 g l⁻¹ as initial Indigo carmine concentration were efficiently transformed up to 50% within 6 h of incubation using 0.1 U ml⁻¹ of laccase and without presence of any mediators. In this study, we showed that the atypical laccase transformed the indigoid dye structure, confirmed by the color changing from blue to red. This intermediate (red) was a subject to an efficient microbial consortium treatment monitored by measuring the decrease in optical density and the total organic carbon removal efficiencies. Toxicological studies via micro-toxicity test showed that the released enzymatic and adapted consortium degradation products were both non-toxic while the initial product was toxic.     

Decolorization of synthetic and real textile wastewater by the use of white-rot fungi

Batch and continuous reactors inoculated with white-rot fungi were operated in order to study decolorization of textile dyes. Synthetic wastewater containing either Reactive Blue 4 (a blue anthraquinone dye) or Reactive Red 2 (a red azo dye) was used during the first part of the study while real wastewater from a textile industry in Tanzania was used in the later part. Trametes versicolor was shown to decolorize both Reactive Blue 4 and Reactive Red 2 if glucose was added as a carbon source. Reactive Blue 4 was also decolorized when the fungus was allowed to grow on birch wood discs in a continuous biological rotating contactor reactor. The absorbance at 595 nm, the wavelength at which the dye absorbs at a maximum, decreased by 70% during treatment. The initial dye concentration in the medium was 200 mg/l and the hydraulic retention time in the reactor 3 days. No glucose was added in this experiment. Changes of the absorbance in the UV range indicated that the aromatic structures of the dyes were altered. Real textile wastewater was decolorized by Pleurotus flabellatus growing on luffa sponge packed in a continuous reactor. The reactor was operated at a hydraulic retention time of 25 h. The absorbance at 584 nm, the wavelength at which the wastewater absorbed the most, decreased from 0.3 in the inlet to approximately 0.1 in the effluent from the reactor.     

Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye

The white-rot fungus Pleurotus ostreatus strain 32 is an excellent producer of the industrially important enzyme laccase. Laccase was the only ligninolytic activity detected in the supernatant when the fungus was grown in liquid culture with or without shaking. Growth and laccase production in static cultivation were superior to that in agitated cultivation, and N-limited culture is of benefit to laccase production. When using cellobiose and peptone as carbon and nitrogen source, a higher activity level was obtained. 2,2′-Azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) (1 mM) was shown to be the best inducer of laccase production, reaching maximum values of about 400 U/ml. Cu²⁺ (1 mM) also had a positive effect on laccase production, activity being enhanced to 360 U/ml. In addition, anthraquinone dye SN4R can be effectively decolorized by crude laccase (30 U/ml), the rate of which was 66%. The decolorization rate was increased by 90% with ABTS (0.16%) addition as a mediator of laccase.     

Overproduction of Trametes versicolor laccase by making glucose starvation using yeast

In the present paper, overproduction of laccase by microbe interaction was studied. When Trametes versicolor was co-cultured with Candida sp. HSD07A in submerged fermentation, laccase activity could be improved significantly and reached 10500 ± 160 U/l, 11.8 times more than that of the contrast group. Fermentation tests of the yeast indicated that it could produce amylase and cellulase, but couldn’t excrete laccase and the overproductive laccase was produced by T. versicolor; the interaction mechanism between T. versicolor and Candida sp. HSD07A was investigated and the results showed that amylase and cellulose could hydrolyze cell walls of T. versicolor; however, the degree of hydrolysis was at a very low level, could not lead to overproduction of laccase; glucose starvation state made by the yeast was the real reason why T. versicolor could overproduce laccase; moreover, this study also proved that making glucose starvation using the yeast was a novel and effective method.     

Immobilization of fungal (Trametes versicolor) laccase onto Amberlite IR-120 H beads: Optimization and characterization

Laccase from Trametes versicolor was immobilized on Amberlite IR-120 H beads. Maximum immobilization obtained was 78.7% at pH = 4.5 and temperature T = 45 °C. Kinetic parameters, K_m and V_max values, were determined respectively as 0.051 mM and 2.77 × 10^?2 mM/s for free and 4.70 mM and 5.27 × 10^?3 mM/s for immobilized laccase. The Amberlite–laccase system showed a 30% residual activity after 7 cycles. On the other hand, the loss of activity for free laccase after 7 days of storage at 4 °C was 18.5% in comparison to Amberlite–laccase system with a loss of 1.4%, during the same period. Improved operational, thermal and storage stabilities of the immobilized laccase were obtained compared to the free counterpart. Therefore, the use of low-cost matrices, like Amberlite for enzyme immobilization represents a promising product for enzymatic industrial applications.     

Differential decolorization of textile dyes in mixtures and the joint effect of laccase and cellobiose dehydrogenase activities present in extracellular extracts from Funalia trogii

The largest part of the bio-decolorization investigations have been performed to date on a single dye without exploring the behavior in complex mixtures as the real dyeing baths. Therefore, mixtures of dyes belonging to azo and anthraquinonic classes, chosen among the most utilized in textile wool dyeing, were employed for comparative enzymatic decolorization studies using the extracellular extracts from the white rot fungus Funalia trogii, to understand how the concomitant presence of more than one dye could influence their degradation course and yield.Fungal extracts containing laccase activity only were capable to partially decolorize dyes mixtures from the different classes analyzed. The deconvolution of the decolorization with time allowed to monitor the degradation of the single dyes in the mixtures evidencing a time dependent differential decolorization not observed for the singles alone. Some dyes in the blend were in fact decolorized only when the most easily converted dyes were largely transformed. These experiments would allow to help the dyeing factories in the selection of the most readily degraded dyes.Since F. trogii grown on different media and activators shows diverse levels of expression of the redox enzymes laccase and cellobiose dehydrogenase (CDH), the dyes mixtures recalcitrant to decolorization by laccase activity alone, were subjected to the combined action of extracts containing laccase and CDH. The use of CDH, in support to the activity of laccase, resulted in substantial decolorization increases (>84%) for all the refractory dyes mixtures.     

The evaluation of pre-grown mycelial pellets in decolorization of textile dyes during repeated batch process

This study was undertaken for the possibility of application of pre-grown pellets for biotechnological treatment of dyes and textile industry waste waters. Mycelial pellets of five different white rot fungi were tested for their dye decolorization activity. The pellets of Funalia trogii, Phanerochaete chrysosporium and Trametes versicolor were determined as the most effective ones. The decolorization ability of viable pellets was compared with the decolorization (adsorption) ability of dead pellets during repeated batch studies. Astrazon Black dye was decolorized effectively, about 90%, by viable pellets of all fungi during the first use. Viable F. trogii pellets were found as the most effective pellets. Upon pellet treatment not only a high decolorization but also reduced toxicity (antimicrobial activity) of the Astrazon Black dye was recorded. This type of decolorization activity with commercial or crude laccase was partially observed. Growing cells of F. trogii in batch system showed lower efficiency in color removal of mixed dyes compared to the pre-grown pellets in repeated batch system. The results in this study showed that mycelial pellets could effectively be used as an alternative to traditional physicochemical processes.     

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.     

A protein from Pleurotus eryngii var. tuoliensis C.J. Mou with strong removal activity against the natural steroid hormone,estriol: Purification,characterization, and identification as a laccase

A protein with strong removal activity against the natural estrogen estriol was purified from a culture supernatant of Pleurotus eryngii var. tuoliensis C.J. Mou. The protein was characterized as a laccase and had a molecular mass of 60 kDa on SDS-PAGE. The enzyme was most active at pH 7.0 and 50 °C. The partial N-terminal amino acid sequence of the enzyme showed homology with laccases from mushrooms, such as Pleurotus ostreatus, Coriolus versicolor (current name: Trametes versicolor), Pycnoporus cinnabarinus, and P. eryngii. A recombinant yeast assay confirmed that laccase treatment was very efficient for removing the estrogenic activity of steroid estrogens. Our results suggest that the enzyme may be applicable as a potential factor for removing natural steroid hormones.     

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.     

Trametes versicolor: Potential for atrazine bioremediation in calcareous clay soil,under low water availability conditions

This study examined the feasibility of Trametes versicolor to actively degrade atrazine (0.5 μg g^?1) in non-sterile calcareous clay soil (Algarve, Portugal) microcosms for up to 24 weeks (20 °C), under low water availability (soil water potentials of ?0.7 and ?2.8 MPa). Soil respiration, laccase activity, and atrazine quantification by high-performance liquid chromatography (HPLC) were assessed. Respiration was significantly (p < 0.05) enhanced in soil containing the inoculant, particularly in the presence of atrazine, indicating that it remained metabolically active throughout the study. Furthermore, up to 98% and 85% (at ?0.7 and ?2.8 MPa, respectively) of atrazine was degraded in soil containing both the atrazine and the inoculant, compared to 96% and 50% in soil containing atrazine only. The contribution of T. versicolor to atrazine degradation was only significant (p < 0.005) under the driest soil treatment conditions. The strategies used for enhancing colonisation and biodegradation capabilities of the inoculant, as well as the selection of sawdust as carrier, were thus effective. However, there were no differences (p > 0.05) in quantified laccase activity in soil containing the inoculant and the control. Overall, this study demonstrated that T. versicolor was a strong candidate for atrazine bioremediation in soil with low moisture and organic matter contents, such as that found in semi-arid and Mediterranean-like ecosystems.     

Characterization of a laccase gene from the white-rot fungi Trametes sp. 5930 isolated from Shennongjia Nature Reserve in China and studying on the capability of decolorization of different synthetic dyes

Laccase belongs to a family of multi-copper oxidases which is especially useful for biotechnological and industrial applications. A laccase-producing white-rot fungi strain designated as Trametes sp. 5930 was nearly isolated from Shennongjia Nature Reserve in China. Trametes sp. 5930 had the high yield of laccase and was capable of decolorizing different dyes efficiently. Laccase played a very important role in the decolorization of different dyes by this fungus. The laccase gene lac5930-1 and its corresponding full-length cDNA were then cloned and characterized from Trametes sp. 5930. The 1563 bp full-length cDNA of lac5930-1 encoded a mature laccase protein consisting of 499 amino acids preceded by a signal peptide of 21 amino acids. lac5930-1 gene was successfully expressed in Pichia pastoris, which verified the function of lac5930-1 encoding active laccase by means of gene expression. The recombinant laccase produced by the yeast transformant in which lac5930-1 was efficiently expressed, conferred the ability to decolorize different dyes. The capability of decolorizing different dyes was positively related to the laccase activity, which provided strong evidence for the important function of laccase used in decolorizing industrial dyes.     

Comparison of the potential of Ficus sycomorus latex and horseradish peroxidases in the decolorization of synthetic and natural dyes

The aim of this study was to compare the potential of Ficus sycomorus latex peroxidase (POL) and horseradish peroxidase (HRP) in the decolorization of a wide spectrum of eight synthetic dyes and two natural dyes, hibiscus flower color and pomegranate juice. We study for the first time the decolorization of natural dyes enzymatically. The highest decolorization percent was reported at 20 mg/l for all dyes treated with POL and HRP. Both the enzymes had lower decolorization % for azo-carmin (30–33%). During decolorization treatment, both natural dyes and titan yellow formed precipitates which settled down and were removed by centrifugation. The enhancement of the decolorization % of the most tested dyes by treatment with POL and HRP was reported in the presence of some redox mediators. The rate of decolorization was enhanced by increasing the time and the most significant changes were observed during the first 6 h of incubation. One hundred percent enhancement in decolorization was reported for azo-carmine in the presence of histidine and α-naphthol as redox mediators. A few of redox mediators caused no significant effect or decreases the decolorization % for a little number of tested dyes. The decolorization of dyes by POL and HRP in the presence of redox mediators appeared without the formation of precipitate. A similar decolorization % for all the tested dyes by POL and HRP was detected. The data suggested that the peroxidase/mediator system was an effective biocatalyst for the decolorization of synthetic and natural dyes, and POL could be used as a potential option for the application of dye decolorization.