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Background

In serious consideration of the worldwide environmental issues associated with the extensive use of the textile dyes and effluents generated thereof, the scientists across the world are in search for potential treatment technologies for their treatment. In such scenario the ligninolytic enzymes provide a potential alternative because they are cost effective, eco-friendly and can be applied to wide range of dye containing industrial effluents.

Results

Laccase produced from Pleurotus ostreatus IBL-02 during decolorization of the reactive textile dye Drimarene brilliant red K-4BL (DBR K-4BL) was purified and immobilized by hydrophobic gel entrapment. The crude laccase was 4.2-fold purified with specific activity of 573.52 U/mg after passing through the DEAE-Sepharose ion exchange and Sephadex-G-100 chromatography columns. P. ostreatus IBL-02 laccase was found to be a homogenous monomeric protein as evident by single band corresponding to 67 kDa on native and sodium dodesylsulfate polyacrylamide gel electrophoresis (PAGE). The laccase was immobilized by entrapment in Sol–gel matrix of trimethoxysilane (T) and proplytetramethoxysilane (P) prepared using different T:P molar ratios. The free and immobilized laccases were compared to investigate the effect of immobilization on catalytic efficiency and thermo-stability features. Laccase immobilized in the Sol–gel of 1:5 T:P ratio was optimally active and thermo-stable fraction at pH 5, 60°C with half-life of 3 h and 50 min. Laccases immobilized in 1:2 and 1:5 T:P ratio gels had significantly higher Km (83 and100mM) and Vmax (1000 and 1111 mM/mg) values as compared to free laccase. After 5 h reaction time varying decolorization percentages with a maximum of 100% were achieved for different dyes and effluents.

Conclusions

In summary, P. ostreatus IBL-02 laccase was immobilized by entrapping in a Sol–gel matrix with an objective to enhance its catalytic and stability properties. Sol–gel entrapped laccase presented potential efficiency as a biocatalyst when applied for decolorization of different dyes and effluents. The main benefits of the Sol–gel matrix immobilization processes are the eco-friendly approach, chemical free and energy saving reaction conditions.

The degradation of C.I. Direct red 80, a polyazo dye, was investigated using Bacillus firmus immobilized by entrapment in tubular polymeric gel. This bacterial strain was able to completely decolorize 50 mg/L of C.I. Direct red 80 under anoxic conditions within 12 h and also degrade the reaction intermediates (aromatic amines) during the subsequent 12 h under aerobic conditions. The tubular gel harboring the immobilized cells consisted of anoxic and aerobic regions integrated in a single unit which was ideal for azo dye degradation studies. Results obtained show that effective dye decolorization (97.8%), chemical oxygen demand (COD) reduction (91.7%) and total aromatic amines removal were obtained in 15 h with the immobilized bacterial cell system whereas for the free cells, a hydraulic residence time of 24 h was required for an equivalent performance in a sequential anoxic and aerobic process. Repeated-batch experiments indicate the immobilized cells could decolorize C.I. Direct red 80 and reduce medium COD in five successive batch runs with enhanced activity obtained after each consecutive run, thus suggesting its stability and potential for repeated use in wastewater treatment. UV–visible spectrophotometry and HPLC analysis were used to confirm the partial mineralization of the dye. Data from this study could be used as a reference for the development of effective industrial scale biotechnological process for the removal of dyes and their metabolites in textile wastewater.

The degradation of C.I. Direct red 80, a polyazo dye, was investigated using Bacillus firmus immobilized by entrapment in tubular polymeric gel. This bacterial strain was able to completely decolorize 50 mg/L of C.I. Direct red 80 under anoxic conditions within 12 h and also degrade the reaction intermediates (aromatic amines) during the subsequent 12 h under aerobic conditions. The tubular gel harboring the immobilized cells consisted of anoxic and aerobic regions integrated in a single unit which was ideal for azo dye degradation studies. Results obtained show that effective dye decolorization (97.8%), chemical oxygen demand (COD) reduction (91.7%) and total aromatic amines removal were obtained in 15 h with the immobilized bacterial cell system whereas for the free cells, a hydraulic residence time of 24 h was required for an equivalent performance in a sequential anoxic and aerobic process. Repeated-batch experiments indicate the immobilized cells could decolorize C.I. Direct red 80 and reduce medium COD in five successive batch runs with enhanced activity obtained after each consecutive run, thus suggesting its stability and potential for repeated use in wastewater treatment. UV–visible spectrophotometry and HPLC analysis were used to confirm the partial mineralization of the dye. Data from this study could be used as a reference for the development of effective industrial scale biotechnological process for the removal of dyes and their metabolites in textile wastewater.

This study concerns the decolorization and detoxification of the azo dye Acid Orange 51 (AO51) by crude laccase from Trametes trogii produced in solid culture using sawdust as support media. A three-level Box–Behnken factorial design with four factors (enzyme concentration, 1-hydroxybenzotriazole (HBT) concentration, dye concentration and reaction time) combined with response surface methodology was applied to optimize AO51 decolorization. A mathematical model was developed showing the effect of each factor and their interactions on color removal. The model predicted that Acid Orange 51 decolorization above 87.87 ± 1.27 % could be obtained when enzyme concentration, HBT concentration, dye concentration and reaction time were set at 1 U/mL, 0.75 mM, 60 mg/L and 2 days, respectively. The experimental values were in good agreement with the predicted ones and the models were highly significant, the correlation coefficient (R2) being 0.9. Then the desirability function was employed to determine the optimal decolorization condition for each dye and minimize the process cost simultaneously. In addition, germination index assay showed that laccase-treated dye was detoxified; however in the presence of HBT, the phytotoxicity of the treated dye was increased. By using cheap agro-industrial wastes, such as sawdust, a potential laccase was obtained. The low cost of laccase production may further broaden its application in textile wastewater treatment.

Electronic supplementary material

The online version of this article (doi:10.1007/s13205-012-0076-2) contains supplementary material, which is available to authorized users.

The studies regarding decolorization of dyes by laccase may not only inform about the possible application of this enzyme for environmental purposes, but also may provide important information about its reaction mechanism and the influence of several factors that could be involved. In this paper, decolorization of crystal violet and phenol red was carried out with different fractions of extracellular liquids from Trametes versicolor cultures, in order to describe the role of laccase in this reaction. Moreover, the possible role of the low molecular weight metabolites (LMWMs) also produced by the fungus was evaluated. The results confirm the existence of a nonenzymatic decolorization factor, since the nonprotein fraction of the extracellular liquids from cultures of T. versicolor has shown decolorization capability. Several experiments were performed in order to identify the main compounds related to this ability, which are probably low molecular weight peroxide compounds.

Trametes hirsuta and a purified laccase from this organism were able to degrade triarylmethane, indigoid, azo, and anthraquinonic dyes. Initial decolorization velocities depended on the substituents on the phenolic rings of the dyes. Immobilization of the T. hirsuta laccase on alumina enhanced the thermal stabilities of the enzyme and its tolerance against some enzyme inhibitors, such as halides, copper chelators, and dyeing additives. The laccase lost 50% of its activity at 50 mM NaCl while the 50% inhibitory concentration (IC50) of the immobilized enzyme was 85 mM. Treatment of dyes with the immobilized laccase reduced their toxicities (based on the oxygen consumption rate of Pseudomonas putida) by up to 80% (anthraquinonic dyes). Textile effluents decolorized with T. hirsuta or the laccase were used for dyeing. Metabolites and/or enzyme protein strongly interacted with the dyeing process indicated by lower staining levels (K/S) values than obtained with a blank using water. However, when the effluents were decolorized with immobilized laccase, they could be used for dyeing and acceptable color differences (ΔE*) below 1.1 were measured for most dyes.

Decolorization of six synthetic dyes using three sources of fungal laccase with the origin of Aspergillus oryzae, Trametes versicolor, and Paraconiothyrium variabile was investigated. Among them, the enzyme from P. variabile was the most efficient which decolorized bromophenol blue (100%), commassie brilliant blue (91%), panseu-S (56%), Rimazol brilliant blue R (RBBR; 47%), Congo red (18.5%), and methylene blue (21.3%) after 3 h incubation in presence of hydroxybenzotriazole (HBT; 5 mM) as the laccase mediator. It was also observed that decolorization efficiency of all dyes was enhanced by increasing of HBT concentration from 0.1 mM to 5 mM. Laccase from A. oryzae was able to remove 53% of methylene blue and 26% of RBBR after 30 min incubation in absence of HBT, but the enzyme could not efficiently decolorize other dyes even in presence of 5 mM of HBT. In the case of laccase from T. versicolor, only RBBR was decolorized (93%) in absence of HBT after 3 h incubation.

Background

Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations.

Results

A bacterial laccase (WlacD) was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ) anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG) 25 and diazo-dye Acid Red (AR) 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l) with relative decolorization values of 91.2% (3 h) and 97.1% (18 h), as well as high activity to AR18 (1 g/l) by 80.5% (3 h) and 89.0% (18 h), was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l). No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved via a subsequent 4-h cell culturing.

Conclusions

This study demonstrates, for the first time, the methodology by which the engineered P. putida with surface-immobilized laccase was successfully used as regenerable biocatalyst for biodegrading synthetic dyes, thereby opening new perspectives in the use of biocatalysis in industrial dye biotreatment.

The strain Ganoderma cupreum AG-1 (Genbank accession no. HQ328947) isolated from the decayed wood was evaluated for its ability to decolorize azo dye reactive violet 1 as well as for the production of ligninolytic enzymes. In the initial decolorization study, the strain was capable of decolorizing 19 different azo dyes. The strain was capable of decolorizing dye over a pH range of 4.5–6 at 30 °C. The optimum pH was found to be 4.5. Various other process parameters like additional carbon and nitrogen source and initial dye concentration were also optimized. The decolorization medium was supplemented with appropriate nitrogen source (yeast extract, 5 g l−1) and carbon source (mannose, 2 g l−1); the decolorization obtained was 98 %. The pattern of enzymes involved in the biodegradation was studied and laccase and MnP were found to be the major enzymes. High laccase activity shown by G. cupreum AG-1 and its ability to decolorize dyes are a good indication of its possible use in the treatment of textile effluents.

To check the suitability of enzyme entrapped beads for use in pharmaceutical industry, amylase enzyme was entrapped in agar/agarose, polyacrylamide gels and calcium alginate beads. Sodium alginate of 1% concentration was found to be best with respect to immobilization efficiency and calcium alginate beads so obtained were not much susceptible to breakage. When sodium alginate- amylase mixture was added from a height of about 20-30 cm. into CaCl2 solution, size of beads was large at higher alginate concentration due to the increase in the size of droplet formation before entering into CaCl2 solution. Enzyme entrapped polyacrylamide and agar/agarose gels were fragile and could not withstand repeated use whereas enzyme entrapped in large calcium alginate beads was used successfully for 50 cycles for the conversion of starch into product without much damage to the beads under stirring conditions. Amylase preparation was also mixed with urease, lysozyme and coimmobilized in large sized calcium alginate beads. These beads were used for 10 repeated cycles to check the conversion of substrates into their products by their respective enzymes and we concluded that an enzyme or mixture of two or three enzymes can be immobilized in the same large sized calcium alginate beads. This will save the additional cost of bioreactor, manpower, maintenance conditions required for the conversion of one drug into another using enzyme/s entrapped in large sized beads.

A new consortium of four bacterial isolates (Agrobacterium radiobacter; Bacillus spp.; Sphingomonas paucimobilis, and Aeromonas hydrophila)-(CM-4) was used to degrade and to decolorize triphenylmethane dyes. All bacteria were isolated from activated sludge extracted from a wastewater treatment station of a dyeing industry plant. Individual bacterial isolates exhibited a remarkable color-removal capability against crystal violet (50 mg/L) and malachite green (50 mg/L) dyes within 24 h. Interestingly, the microbial consortium CM-4 shows a high decolorizing percentage for crystal violet and malachite green, respectively, 91% and 99% within 2 h. The rate of chemical oxygen demand (COD) removal increases after 24 h, reaching 61.5% and 84.2% for crystal violet and malachite green, respectively. UV-Visible absorption spectra, FTIR analysis and the inspection of bacterial cells growth indicated that color removal by the CM-4 was due to biodegradation. Evaluation of mutagenicity by using Salmonella typhimurium test strains, TA98 and TA100 studies revealed that the degradation of crystal violet and malachite green by CM-4 did not lead to mutagenic products. Altogether, these results demonstrated the usefulness of the bacterial consortium in the treatment of the textile dyes.

The goal of this work was to investigate the decomposition of azo dyes by oxidative methods, such as laccase and ultrasound treatments. Each of these methods has strong and feeble sides. The laccase treatment showed high decolorization rates but cannot degrade all investigated dyes (reactive dyes), and high anionic strength led to enzyme deactivation. Ultrasound treatment can decolorize all tested dyes after 3 h at a high energy input, and prolonged sonication leads to nontoxic ionic species, which was demonstrated by ion chromatography and toxicity assays. For the first time, it was shown that a combination of laccase and ultrasound treatments can have synergistic effects, which was shown by higher degradation rates. Bulk light absorption and ion-pairing high-performance liquid chromatography (IP-HPLC) were used for process monitoring, while with reversed-phase HPLC, a lower number of intermediates than expected by IP-HPLC was found. Liquid chromatography-mass spectrometry indicated that both acid orange dyes lead to a common end product due to laccase treatment. Acid Orange 52 is demethylated by laccase and ultrasound treatment. Further results confirmed that the main effect of ultrasound is based on ˙OH attack on the dye molecules.

Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium marinum, and Mycobacterium chelonae tolerate high concentrations of the dyes malachite green and crystal violet. Cells of strains of those species decolorized (reduced) both malachite green and crystal violet. Because decolorized malachite green lacked antimicrobial activity, the resistance of these mycobacteria could be due, in part, to their ability to decolorize the dyes. Small amounts of malachite green and its reduced, decolorized product were detected in the lipid fraction of M. avium strain A5 cells grown in the presence of malachite green, suggesting that a minor component of resistance could be due to sequestering the dyes in the extensive mycobacterial cell surface lipid. The membrane fraction of M. avium strain A5 had at least a fivefold-higher specific decolorization rate than did the crude extract, suggesting that the decolorization activity is membrane associated. The malachite green-decolorizing activity of the membrane fraction of M. avium strain A5 was abolished by either boiling or proteinase exposure, suggesting that the decolorizing activity was due to a protein. Decolorization activity of membrane fractions was stimulated by ferrous ion and inhibited by dinitrophenol and metyrapone.

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.

The decolorization of direct Solophenyl red 3BL (SR), a polyazo dye extensively used in textile industry was studied. The Fomes fomentarius laccase alone did not decolorize SR. The natural redox mediator, acetosyringone (AS), was necessary for decolorization to occur. Box-Behnken design was used to evaluate the effects of three parameters, namely, enzyme concentration (0.5–2.5 U mL−1), redox mediator concentration (3–30 μM), and incubation time (1–24 h), on the SR decolorization yield. The fitted mathematical model allowed us to plot response surfaces as well as isoresponse curves and to determine optimal decolorization conditions. The results clearly indicated that the AS concentration was the main factor influencing the SR decolorization yield. The selected optimal conditions were enzyme concentration 0.8 U mL−1, mediator concentration 33 μM, and time 14 h 30 min. These conditions allowed 79.66% of SR decolorization versus 80.70% for the predicted value. These results showed a promising future of applying laccase-AS system for industrial wastewater bioremediation.

A high exhaust reactive dye, Green HE4B (GHE4B) was 98% degraded in nutrient medium by Pseudomonas desmolyticum NCIM 2112 (pd2112) within 72 h at static condition. Decolorization time in synthetic 10 g/l molasses. Addition of 5 g/l peptone to NaCl medium had reduced decolorization time from 108 to 72 h. Beef extract do not contribute more to the inducing effect of peptone, however it is a good co-substrate in sucrose or urea containing NaCl medium. Intracellular lignin peroxidase (Lip), laccase and tyrosinase activities were induced by 150, 355 and 212%, respectively till maximum dye removal took place. Aminopyrine N-demethylase (AND) and dichlorophenol indophenol reductase (DCIP-reductase) activities in pd2112 were induced by 130 and 20%, respectively at 72 h of incubation during GHE4B decolorization. By high performance liquid chromatography (HPLC) analysis, 4-hydroxybenzene sulfonic acid and 4-amino, 6-hydroxynaphthalene 2-sulfonic acids were identified as metabolites formed during 24–72 h incubation. Fourier transform infrared spectroscopy (FTIR) analysis supports the formation of these aromatic amines. pd2112, aerobically degraded GHE4B metabolites (formed at static condition) showing stationary phase of 6 days. There was no germination inhibition of Sorghum bicolor and Triticum aestivum by GHE4B metabolites at 3,000 ppm concentration however untreated dye showed germination inhibition at the same concentration. GHE4B metabolites did not show any microbial toxicity at 10,000 ppm concentration.

In the present study, the decolorization and degradation of Reactive Black 5 (RB5) azo dye was investigated by biological, photocatalytic (UV/TiO2) and combined processes. Application of Candida tropicalis JKS2 in treatment of the synthetic medium containing RB5 indicated complete decolorization of the dye with 200 mg/L in less than 24 h. Degradation of the aromatic rings, resulting from the destruction of the dye, did not occur during the biological treatment. Mineralization of 50 mg/L RB5 solution was obtained after 80 min by photocatalytic process (in presence of 0.2 g/L TiO2). COD (chemical oxygen demand) was not detectable after complete decolorization of 50 mg/L RB5 solution. However, photocatalytic process was not effective in the removal of the dye at high concentrations (≥200 mg/L). With 200 mg/L concentration, 74.9% of decolorization was achieved after 4 h illumination under photocatalytic process and the absorbance peak in UV region (attributed to aromatic rings) was not completely removed. A two-step treatment process, namely, biological treatment by yeast followed by photocatalytic degradation, was also assessed. In the combined process (with 200 mg/L RB5), absorbance peak in UV region significantly disappeared after 2 h illumination and about 60% COD removal was achieved in the biological step. It is suggested that the combined process is more effective than the biological and photocatalytic treatments in the remediation of aromatic rings.

A glucoamylase-immobilized system based on cross-linked gelatin nanoparticles (CLGNs) was prepared by coacervation method. This system exhibited characteristics of temperature-triggered phase transition, which could be used for enzyme immobilization and release. Their morphology and size distribution were examined by transmission electron microscopy and dynamic light scattering particle size analyzer. Their temperature-triggered glucoamylase immobilization and release features were also further investigated under different temperatures. Results showed that the CLGNs were regularly spherical with diameters of 155±5 nm. The loading efficiencies of glucoamylase immobilized by entrapment and adsorption methods were 59.9% and 24.7%, respectively. The immobilized enzyme was released when the system temperature was above 40°C and performed high activity similar to free enzyme due to the optimum temperature range for glucoamylase. On the other hand, there was no enzyme release that could be found when the system temperature was below 40°C. The efficiency of temperature-triggered release was as high as 99.3% for adsorption method, while the release of enzyme from the entrapment method was not detected. These results indicate that CLGNs are promising matrix for temperature-triggered glucoamylase immobilization and release by adsorption immobilization method.

Several model azo dyes are reductively cleaved by growing cultures of an ascomycete yeast species, Issatchenkia occidentalis. In liquid media containing 0.2 mM dye and 2% glucose in a mineral salts base, more than 80% of the dyes are removed in 15 h, essentially under microaerophilic conditions. Under anoxic conditions, decolorization does not occur, even in the presence of pregrown cells. Kinetic assays of azo reduction activities in quasi-resting cells demonstrated the following: (i) while the optimum pH depends on dye structure, the optimum pH range was observed in the acidic range; (ii) the maximum decolorizing activity occurs in the late exponential phase; and (iii) the temperature profile approaches the typical bell-shaped curve. These results indirectly suggest the involvement of an enzyme activity in azo dye reduction. The decolorizing activity of I. occidentalis is still observed, although at a lower level, when the cells switch to aerobic respiration at the expense of ethanol after glucose exhaustion in the culture medium. Decolorization ceased when all the ethanol was consumed; this observation, along with other lines of evidence, suggests that azo dye reduction depends on cell growth. Anthraquinone-2-sulfonate, a redox mediator, enhances the reduction rates of the N,N-dimethylaniline-based dyes and reduces those of the 2-naphthol-based dyes, an effect which seems to be compatible with a thermodynamic factor. The dye reduction products were tested as carbon and nitrogen sources. 1-Amino-2-naphthol was used as a carbon and nitrogen source, and N,N-dimethyl-p-phenylenediamine was used only as a nitrogen source. Sulfanilic and metanilic acids did not support growth either as a carbon or nitrogen source.

Acinetobacter calcoaceticus NCIM 2890 (A. caloaceticus) was found to decolorize 20 different textile dyes of various classes. Decolorization of an azo dye amaranth was observed effectively (91%) at static anoxic condition, whereas agitated culture grew well but showed less decolorization (68%) within 48 h of incubation. Induction of intracellular and extracellular lignin peroxidase, intracellular laccase, dichlorophenol indophenol (DCIP) reductase and riboflavin reductase represented their involvement in the biodegradation of amaranth. The products obtained after degradation of Amaranth were characterized as naphthalene sulfamide, hydroxyl naphthalene diazonium and naphthalene diazonium. The germination and growth of Sorghum vulgare and Phaseolus mungo seeds, and the growth of E. coli and Bacillus substilis were not inhibited by the metabolic products of the dye.

Lignin-degrading enzymes were partially purified from supernatant solutions obtained from Phanerochaete flavido-alba-decolorized olive oil mill wastewaters (OMW). The dominant enzymes, manganese peroxidases, exhibited different isoform patterns in decolorized OMW-containing cultures than in residue-free samples. Laccase induction was also detected in OMW-containing cultures but not in control cultures.

Exponentially growing cells of Bacillus subtilis and Escherichia coli were Gram stained with potassium trichloro(eta 2-ethylene)platinum(II) (TPt) in place of the usual KI-I2 mordant. This electron-dense probe allowed the staining mechanism to be followed and compared with cellular perturbations throughout the staining process. A crystal violet (CV)-TPt chemical complex was formed within the cell substance and at the cell surface of B. subtilis when the dye and Pt mordant were added. The ethanol decolorization step dissolved the precipitate from the cell surface, but the internal complex was retained by the cell wall and remained within the cell. This was not the case for E. coli; the ethanol decolorization step removed both surface-bound and cellular CV-TPt. During its removal, the outer membrane was sloughed off the cells until only the murein sacculus and plasma membrane remained. We suspect that the plasma membrane was also perturbed, but that it was retained within the cell by the murein sacculus. Occasionally, small holes within the murein and plasma membrane could be distinguished through which leaked CV-TPt and some cellular debris. Biochemical identification of distinct envelope markers confirmed the accuracy of these images.

Images

Trametes villosa laccase was used for direct azo dye degradation, and the reaction products that accumulated after 72 h of incubation were analyzed. Liquid chromatography-mass spectrometry (LC-MS) analysis showed the formation of phenolic compounds during the dye oxidation process as well as a large amount of polymerized products that retain azo group integrity. The amino-phenol reactions were also investigated by 13C-nuclear magnetic resonance and LC-MS analysis, and the polymerization character of laccase was shown. This study highlights the fact that laccases polymerize the reaction products obtained during long-term batch decolorization processes with azo dyes. These polymerized products provide unacceptable color levels in effluents, limiting the application of laccases as bioremediation agents.

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60°C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

Background

Laccases have huge potential for biotechnological applications due to their broad substrate spectrum and wide range of reactions they are able to catalyze. These include, for example, the formation and degradation of dimers, oligomers, polymers, and ring cleavage as well as oxidation of aromatic compounds. Potential applications of laccases include detoxification of industrial effluents, decolorization of textile dyes and the synthesis of natural products by, for instance, dimerization of phenolic acids. We have recently published a report on the cloning and characterization of a CotA Bacillus licheniformis laccase, an enzyme that catalyzes dimerization of phenolic acids. However, the broad application of this laccase is limited by its low expression level of 26 mg l-1 that was achieved in Escherichia coli. To counteract this shortcoming, random and site-directed mutagenesis have been combined in order to improve functional expression and activity of CotA.

Results

A CotA double mutant, K316N/D500G, was constructed by combining random and site-directed mutagenesis. It can be functionally expressed at an 11.4-fold higher level than the wild-type enzyme. In addition, it is able to convert ferulic acid much faster than the wild-type enzyme (21% vs. 14%) and is far more efficient in decolorizing a range of industrial dyes. The investigation of the effects of the mutations K316N and D500G showed that amino acid at position 316 had a major influence on enzyme activity and position 500 had a major influence on the expression of the laccase.

Conclusion

The constructed double mutant K316N/D500G of the Bacillus licheniformis CotA laccase is an appropriate candidate for biotechnological applications due to its high expression level and high activity in dimerization of phenolic acids and decolorization of industrial dyes.