Production of laccase from newly isolated Pseudomonas putida and ...

Biocatalysis and Agricultural Biotechnology 2 (2013) 333–338

Contents lists available at ScienceDirect

Biocatalysis and Agricultural Biotechnology journal homepage: www.elsevier.com/locate/bab

Original Research Paper

Production of laccase from newly isolated Pseudomonas putida and its application in bioremediation of synthetic dyes and industrial effluents Mohammed Kuddus a,n, Babu Joseph b, Pramod Wasudev Ramteke c a

Department of Biochemistry, University of Hail, Hail, Saudi Arabia College of Applied Medical Science, Shaqra University, Shaqra, Saudi Arabia c Department of Biological Sciences, SHIATS, Allahabad, India b

art ic l e i nf o

a b s t r a c t

Article history: Received 9 May 2013 Received in revised form 5 June 2013 Accepted 18 June 2013 Available online 26 June 2013

A novel laccase enzyme producing bacterium, Pseudomonas putida MTCC 7525, was isolated and subjected to optimization of laccase production. Maximum production (94.10 U/ml) was achieved at 30 1C and pH 8 (108 h incubation) with 10% skim milk and 1 mM sodium nitrate as additional nitrogen source. The laccase was purified by salt precipitation followed by ion exchange chromatography which showed 6.44 fold purification. The purified enzyme had optimal activity at pH 8.0 and 40 1C, and showed stability in DMSO retaining more than 85% of original activity. In presence of manganese and cadmium, enzyme retained 495% and 92% activity, respectively. The molecular weight of laccases was 39.5 kDa and activity was inhibited by pCMB (93%). The synthetic dyes (0.02%) and industrial effluents (10%) were decolorized to 74–93 and 58–68%, respectively when treated with culture of P. putida. However, the culture supernatant of P. putida showed about 36–94 and 16–86% decolorization of synthetic dyes and effluents, respectively within 24 h of incubation. & 2013 Elsevier Ltd. All rights reserved.

Keywords: Laccase Pseudomonas putida Synthetic dyes Industrial effluents Decolorization

1. Introduction The laccase enzyme (EC 1.10.3.2) is a dimeric/tetrameric glycoprotein, which catalyzes the oxidation of ortho- and para-diphenols, aminophenols, polyphenols, polyamines, lignins and aryl diamines as well as some inorganic ions also (Yaropolov et al., 1994; Solomon et al., 1996; Gianfreda et al., 1999). Although oxidation reactions are vital in several industries, most of the conventional oxidation technologies have the drawbacks such as non-specific or undesirable side-reactions and use of environmentally hazardous chemicals. Laccases have received much attention from researchers in last decades due to their ability to oxidize both phenolic and nonphenolic lignin related compounds as well as environmental pollutants, which makes them very useful in biotechnological processes such as detoxification of industrial effluents and in bioremediation (Couto and Herrera, 2006). Laccase-mediated systems have been applied to various processes such as dye decolourization (Claus et al., 2002) pulp delignification (Bourbonnais et al., 1998; Li et al., 1999), oxidation of organic pollutants (Collins et al., 1996) and the development of biosensors (Kuznetsov et al., 2001) or biofuel cells (Palmore and Kim, 1999). Interest in laccase has increased recently because of their potential use in the detoxification of pollutants such as detoxification of

n

Corresponding author. Tel.: +966 504984419. E-mail address: [email protected] (M. Kuddus).

1878-8181/$ - see front matter & 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bcab.2013.06.002

industrial effluents, mostly from the paper and pulp, textile and petrochemical industries. The use of bacterial laccases in treatment of effluents from paper and textile industry and removal of toxic wastes have to be studied widely. Therefore, the present study was concerned to obtain a novel stable bacterial laccase with low cost and to find its applications in degradation of toxic synthetic dyes and industrial effluents. 2. Materials and methods 2.1. Chemical Solvents and enzyme inhibitors viz. sodium azide, IAA, pCMB, EGTA and EDTA were obtained from SRL chemicals, India. DEAEcellulose was purchased from Hi-media Laboratory, India. Standard protein markers were purchased from Bangalore Genei India. 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and syringaldazine were purchased from Sigma-Aldrich, USA. All chemicals used were of the highest purity available and of the analytical grade. 2.2. Isolation and screening of laccase producing bacteria Soil samples containing sawdust and dairy effluents were collected from around the saw mills and dairy plant near SHIATS, Allahabad, India. All the samples (10 g each) were added to 100 ml

334

M. Kuddus et al. / Biocatalysis and Agricultural Biotechnology 2 (2013) 333–338

of nutrient broth supplemented with 0.2 mM CuSO4 and 2 mM guiacol as an inducer; and incubated at 37 1C for 48 h. The bacterial isolated were obtained by a standard serial dilution plate technique using nutrient agar medium containing 0.2 mM CuSO4. Plates were incubated at 37 1C for 48 h. Screening of laccaseproducing isolates was performed by adding 2–3 drops of 1 mM ABTS laccase substrate to the bacterial colonies. Positive colonies were visualized by the appearance of green color formation (Coll et al., 1993). Potential laccase producing bacteria based on maximum laccase activity were selected for detail study. 2.3. Optimization of enzyme production To investigate optimum incubation time for laccase production, Guaiacol broth media was inoculated with bacterial culture (1%) and incubated at 27 1C for 132 h in shaking condition (120 rpm). The samples were withdrawn at different time intervals and laccase activity was assayed as per standard protocol. The effect of temperature on enzyme production was determined by incubating inoculated broth media at different temperatures (10–50 1C) for optimized period of time. The effect of pH of broth media on laccase production was investigated by varying of the initial pH of the culture media from pH 5–10 at optimized temperature and incubation period. Chitinase assay was performed as per standard protocol. Influence of additional nitrogen sources (1 mM) was studied as additional supplement in media for maximum enzyme production. The supplemented media was inoculated with 1% inoculums and fermented at optimized condition. Simultaneously media without any nitrogen source was used as control. Production of laccase was also studied by employing skim milk at different concentrations (5–25%). All the experiments were conducted in triplicate under shaking condition (120 rpm) and the results are average of three independent experiments. 2.4. Laccase assay and protein concentration Laccase activity was determined as per method of Benny et al. (1998) by using syringaldazine as a substrate. One unit of enzyme activity was defined as 1 μmol of product formed per minute. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin (BSA) as standard. 2.5. Identification of bacterial strains The potential laccase producing bacterial strains were identified by studying morphological and biochemical characteristics as per Bergey's Manual of Determinative Bacteriology (Holt et al., 1989). The identity of strains was further confirmed by Microbial Type Culture Collection (MTCC), Chandigarh, India. 2.6. Extraction and purification of enzyme The most potential isolate was grown in broth media under optimized conditions and cells were removed by centrifugation at 10,000 g for 10 min at 4 1C. Cell-free supernatant was subjected to salt precipitation and protein precipitate was resuspended in 0.05 M sodium phosphate buffer (pH 8) that was dialyzed against the same buffer for 24 h. The partially purified protein was subjected to ion exchange chromatography on a DEAE cellulose column that was pre-equilibrated with sodium phosphate buffer (0.02 M, pH 8). The protein was eluted (flow rate 60 ml/h) with a linear gradient of NaCl (0.1–1 M) in the same buffer. A total of 40 fractions were collected and assayed for protein and enzyme activity. Fractions having highest laccase activity was desalted and concentrated by dialysis and lyophilization, respectively.

2.7. Characterization of enzyme 2.7.1. Effect of pH on enzyme activity and stability To determine the effect of pH on enzyme activity, enzymesubstrate mixture was incubated with the buffers of different pH such as citrate phosphate (pH 5–6), sodium phosphate (pH 7), Tris–HCl (pH 8) and glycine–NaOH (pH 9–11) and residual enzyme activity was determined by standard assay methods. The effect of pH on the stability of enzyme was determined by pre-incubating the laccase at different pH values (pH 5–11) for 1 h and the relative enzyme activity at each exposure was measured as per standard assay procedure. 2.7.2. Effect of temperature on enzyme activity and stability The effect of temperature on laccase activity was determined by performing the standard assay procedure within a temperature range from 10–40 1C. To determine the enzyme stability with changes in temperature, purified enzyme was pre-incubated at different temperatures (10–50 1C) and relative laccase activity was assayed as standard assay conditions. 2.7.3. Effect of inhibitors on enzyme activity Some of the most common laccase inhibitors such as EDTA, SDS, EGTA, pCMB, KI, and DTT were included in this study. The activity in presence of 1 mM and 10 mM salts solutions was carried out. After 1 h incubation in particular solution, residual activity of enzyme was measured. 2.7.4. Effect of organic solvents on enzyme activity To determine the effect of organic solvents on laccase activity, the enzyme was incubated in presence of various organic solvents such as methanol, DMFO, iso-propanol, ethanol, acetone and DMSO at final concentration of 5% and 10% (v/v). Enzyme was removed after 1 h by centrifugation and the enzyme activity was measured under the standard condition. 2.7.5. SDS polyacrylamide gel electrophoresis The homogeneity and molecular weight of purified laccase was determined by SDS-PAGE that was performed by the method of Laemmli (Laemmli, 1970). The gel was stained with Coomassie Brilliant Blue R-250 and the relative molecular mass of the protein was calculated using standard protein markers run simultaneously. 2.8. Decolorization of dyes and effluents The isolated bacterium was tested for its ability to decolorize various synthetic dyes and industrial effluents over a period of 5 days. The final concentration of the dye and effluent in the medium on day zero were considered as a control. The extent of decolorization was recorded as residual color. Various dyes such as Brilliant green, bromophenol blue, crystal violet and congo red were monitored at their absorbance maxima at 623, 591, 589 and 486 nm, respectively. Paper effluents (A and B) were also monitored at their absorbance maxima of 347 nm. Paper effluent ‘A’ contained mixture of rodhamine, violet and blue dye and effluent ‘B’ contained mainly brown dye (2 kg/1500 kg of pulp) along with resin and alum. The culture filtrate with maximum laccase activity was also used as a source of enzyme to test its efficiency in decolorization of various dyes and effluents. In brief this is carried out by incubating the enzyme with dyes and effluents for 6–24 h at room temperature. The percentage of decolorization achieved was calculated with reference to the control samples that were not treated with the enzyme. The supernatant was also treated with the inhibitor


pCMB and tested for decolorization to check whether decolorization is due to enzyme or other metabolites. 2.9. Statistical analysis All the experiments were carried out in triplicate and the findings are average of three independent experiments.

3. Results and discussion 3.1. Isolation of laccase producing bacteria Laccase producing bacteria isolated from soil and effluent samples are given in Table 1. On the basis of primary screening seven potential isolates designated as LAC-1, LAC-2, LAC-3, LAC-4, LAC-5, LAC-6 and LAC-7, were selected for quantification of enzyme. Out of seven laccase positive isolates, Lac-5 was found to be most potential isolate producing 4.25 U/ml laccase followed by LAC-4 (3.65 U/ml) and LAC-3 (3.47 U/ml) in Guaiacol broth. LAC-1, LAC-2, LAC-6 and LAC-7 produced only 0.9, 2.62, 2.65 and 2.87 U/ml, respectively. Guaiacol broth was used for laccase production as it is the best carbon source as suggested by Jhadav et al. (2009). Some other bacteria producing laccase enzyme are reviewed by Shraddha et al. (2011).

335

(8.759 and 8.845 U/ml, respectively) at 30–40 1C. However, at 40–50 1C LAC-1 produced the maximum amount of enzyme (7.636 U/ml). The other isolates produced insignificant amount of enzyme at temperature ranging from 10 to 50 1C (Fig. 2). Our results are in accordance with other published report that concluded that the optimum temperature range for laccase production is between 25 1C and 50 1C (Pointing and Jones, 2000; Farnet et al., 2000). Additionally, the composition and concentration of media components and pH were also optimized for two most potential isolates (LAC-4 and LAC-5). The pH of the culture media strongly affects many enzymatic reactions and transport of compounds across the cell membrane as they are sensitive to the concentration of hydrogen ions present in the medium. However, Thurston (Thurston, 1994) concluded that the effect of pH is limited in case of laccase enzyme production. The results showed that LAC-4 and LAC-5 produce maximum laccase, 23.14 and

3.2. Optimization of laccase production Seven potential isolates were subjected to enzyme production optimization with respect to incubation time and temperature. The results of different incubation periods showed that all the isolates producing enzyme maximally in between 108–120 h. LAC5 produces maximum enzyme (4.25 U/ml) at 108 h followed by LAC-4 (3.65 U/ml) at 120 h (Fig. 1). Both the isolates were obtained from Saw mill effluent samples. It is well known that temperature has a profound influence on enzyme production. In this study maximum laccase production was obtained by LAC-4 and LAC-5

Fig. 2. Production of laccase enzyme at different temperature (pH 7, time 120 h).

Table 1 Number of presumptive laccase producers from different sources. S. no. Sampling site

Total viable count Presumptive laccase producers

1 2 3 4

2.0 104 4.0 104 1.8 105 2.5 104

Saw mill effluent Dairy effluent Contaminated soil (saw dust) Contaminated soil (cow dung)

1.2 104 2.0 103 1.2 105 2.0 104 Fig. 3. Effect of pH on laccase enzyme production (temp. 30 1C, time 120 h).

Fig. 1. Production of laccase enzyme at different time duration (pH 7 and temp. 27 1C).

Fig. 4. Effect of additional nitrogen sources on enzyme production (temp 301 C, pH 8 for LAC 4 and 9 for LAC 5, time 120 h).

336


34.45 U/ml, at pH 9 and 8, respectively (Fig. 3). Nitrogen sources are necessary for the proper growth and metabolism of microorganisms. The use of economical nitrogen sources is important for production of laccase as these can significantly reduce the cost. Among the tested nitrogen source, both the isolates LAC-4 and LAC-5 gave maximum yield, 27.79 and 49.71 U/ml, respectively with sodium nitrate (Fig. 4). Also, the literature suggested that sodium nitrate is the commonly used nitrogen sources for laccase production (Shraddha et al., 2011). In addition, when the skim milk is used as a nutrient, the laccase production was increased tremendously. With 10% of skim milk LAC-5 produced 94.1 U/ml of laccase enzyme.

3.3. Identification of bacteria The most potential isolate, LAC-5, was identified as Pseudomonas. putida on the basis of its morphological and biochemical characteristics as mentioned in Table 2. The organism was a gramnegative, motile and rod-shaped bacterium. The identity of the strain was further confirmed by Microbial Type Culture Collection (MTCC), Chandigarh, India, and given an accession number MTCC 7525.

3.4. Purification and characterization of laccase The elution profile of purified laccase revealed that laccase was eluted as a well-resolved single peak of enzyme activity. Approximately 6.44-fold purification was achieved with the specific activity of 84.26 U/mg (Table 3). Purified laccase migrated as a single band in SDS-PAGE suggested its homogeneity with a molecular mass of 39.5 kDa (Fig. 5). The enzyme was active over a broad pH range (pH 6–11) with maximum activity at pH 8. It was relatively stable over a pH range of 8–10 and retained more than 75% of the original enzyme activity. The optimum temperature for laccase activity was 40 1C and it was stable with 90% activity at 10–50 1C. A stronger inhibitory effect was observed in the presence

Table 2 Morphological and biochemical characteristics of P. putida. Tests

LAC 5

Configuration, margin, surface

Round, entire, smooth Rods, moderate, single Fluorescent Negative Positive 10–37 1C pH 5-11 2.5–7 Negative Positive

Cell shape, size, arrangement

Pigments Gram's reaction and endospore Motility and fluorescence (UV) Growth at temperatures Growth at pH Growth on NaCl (%) Growth under anaerobic condition Growth on MacConkey agar, cytochrome oxidase test, catalase test, arginine dihydrolase test indole test, methyl red test, Voges Proskauer test, citrate Negative utilization, gas production from glucose, casein hydrolysis, gelatin hydrolysis, starch hydrolysis, urea hydrolysis, nitrate reduction, H2S production, ornithine decarboxylase, lysine decarboxylase Acid production from adonitol, arabinose, cellobiose, dulcitol Negative inositol, inulin, lactose, maltose, raffinose, rhamnose, salicin, sorbitol, sucrose, trehalose Acid production from dextrose, galactose, fructose, mannose, Positive melibiose, xylose

Table 3 Purification of laccase from P. putida MTCC 7525. Purification step

Sp. activity (U/mg)

Purified (fold)

Yield (%)

2063.3 35.63

13.08 16.83

1.0 1.28

100 2.2

14.24

84.26

6.44

4.4

Total activity Total (units) protein (mg)

Crude enzyme 27,000 (NH4)2SO4 600 precipitation DEAE-cellulose 1200

Table 4 Decolorization of dye/effluent with culture media inoculated with P. putida MTCC 7525 and by using culture supernatant. Dye/effluent

Decolorization (%) Pseudomonas putida

Fig. 5. Molecular weight of laccases (Lane 1: purified laccase from P. putida MTCC 7525; Lane 2: crude laccase from P. putida MTCC 7525; Lane 7: protein molecular weight marker).

Brilliant green Bromophenol blue Crystal violet Congo red Paper effluent A Paper effluent B

Culture supernatant of P. putida

1 day 2 day 3 day 4 day 5 day 6 h

12 h

24 h

79 40 40 55 49 34

88 36 68 60 78 42

94 36 70 54 86 41

88 46 62 68 68 52

90 81 68 85 62 58

93 82 73 92 62 54

93 80 74 90 61 51

86 30 52 42 55 37


337

Fig. 6. Decolorization of dye and effluents by using culture supernatant of P. putida. (a) Bromo. Blue, (b) Congo red, (c) Brilliant green, Crystal violet, (ai) Paper effluent A, (bi) Paper effluent B and (ci) Textile effluent.

of Hg2+ ions at 10 mM concentration retaining only 27% of total enzyme activity. In case of inhibitors, pCMB was able to inhibit enzyme activity almost completely at 10 mM concentration, while IAA exhibited 80% inhibition. However, other tested inhibitors had insignificant effect. The purified enzyme having more stability in DMSO (10%) retaining more that 90% activity followed by acetoneb (76%). 3.5. Decolorization of synthetic dyes and industrial effluents The synthetic dyes (0.02%) were decolorized by 74–93% within 4–5 days when treated with P. putida MTCC 7525. On the other hand about 58–68% decolorization of paper mills effluent (used at 10% concentration) was achieved by day 2–3 (Table 4). Also the culture supernatant of P. putida showed about 36–94 and 16–86% decolorization of dyes and effluents, respectively within 12–24 h (Fig. 6). It was observed that when culture supernatant was treated with laccase enzyme inhibitor (pCMB, 10 mM), there was no change in color indicating that the decolorization of dyes/ effluents was due to laccase enzyme produced by P. putida not by other metabolic products. Enzymatic oxidation techniques have potential within a great variety of industrial fields including the pulp and paper, textile and food industries. Thus, laccase is a particularly promising enzyme for the above mentioned purposes. The industrial preparation of paper requires separation and degradation of lignin in wood pulp. Environmental concerns urge to replace conventional and polluting chlorine based delignification/bleaching procedure. Oxygen delignification processes have been introduced industrially (Carter et al., 1997), but pretreatments of wood pulp with ligninolytic enzymes might provide milder and cleaner strategies for delignification that are also respectful of the integrity of cellulose (Kuhad et al., 1997). The textile industry accounts for two-thirds of the total dyestuff market (Riu et al., 1998) and consumes large volumes of water and chemicals for wet processing of textiles. The chemical reagents used are very diverse in chemical composition, ranging from inorganic compounds to polymers and organic products. Due to their chemical structure, dyes are resistant to fading on exposure

to light, water and different chemicals and most of them are difficult to decolorize due to their synthetic origin. Concern arises, as several dyes are made from known carcinogens such as benzidine and other aromatic compounds (Baughman and Perenich, 1988). Most currently existing processes to treat dye wastewater are ineffective and uneconomical. Therefore, the development of processes based on laccases seems an attractive solution due to their potential in degrading dyes of diverse chemical structure (Hou et al., 2004), including synthetic dyes currently employed in the industry (Rodriguez et al., 2005). Laccase can also be applied to certain processes that enhance or modify the color appearance of food or beverage.

4. Conclusion In the present study purified enzyme from P. putida gave maximum activity and stability at 40 1C and pH 8. The synthetic dyes and industrial effluents were decolorized to 58–93% when treated with culture. However, the culture supernatant showed about 16–94% decolorization within 24 h of incubation. Other worker also reported decolorization of synthetic dyes by laccase but in most of the studies fungi were used (D'Souza et al., 2006; Mechichi et al., 2006). To the best of our knowledge this is the first report on bacteria P. putida producing laccase and its use along with organism for decolorization of synthetic dyes and industrial effluents. Therefore, this strain can be used to decolorize and detoxify the industrial effluents and help in wastewater treatment. It can also be used efficiently in paper and pulp industries, textile industries, and in bioremediation.

Acknowledgment Financial support by Council of Scientific and Industrial Research, New Delhi, India (Grant no. 38 (1093)/04-EMR-II) is gratefully acknowledged. Work done at SHIATS, Allahabad, India.

338


References Baughman, G.L., Perenich, T.A., 1988. Fate of dyes in aquatic systems: the solubility and partitioning of some hydrophobic dyes and related compounds. Environ. Toxicol. Chem. 7, 183–199. Benny, C., Yona, C, Yitzhak, H., 1998. Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl. Environ. Microbiol. 64 (9), 3175–3179. Bourbonnais, R., Leech, D., Paice, M.G., 1998. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim. Biophys. Acta 1379, 381–390. Carter, D.N., McKenzie, D.G., Johnson, A.P., Idner, K., 1997. Performance parameters of oxygen delignification. Tappi. J. 80, 111–117. Claus, H., Faber, G., König, H., 2002. Redox-mediated decolorization of synthetic dyes by fungal laccases. Appl. Microbiol. Biotechnol. 59, 672–678. Coll, P.M., Abalos, J.M.F., Villanueva, J.R., Santamaria, R., Perez, P., 1993. Purification and characterization phenoloxidase (laccase) from the lignin-degrading Basidiomycete PM1 (CECT 2971). Appl. Environ. Microbiol. 59, 2607–2613. Collins, P.J., Kotterman, M.J.J., Field, J.A., Dobson, A.D.W., 1996. Oxidation of anthracene and benzopyrene by laccases from Trametes versicolor. Appl. Environ. Microbiol. 62, 4563–4567. Couto, S.R., Herrera, J.L.T., 2006. Industrial and biotechnological applications of laccase: a review. Biotech. Adv. 24, 500–513. D'Souza, D.T., Tiwari, R., Sah, A.K., Raghukumar, C., 2006. Enhanced production of laccase by a marine fungus during treatment of colored effluents and dyes. Enz. Microb. Technol. 38, 504–511. Farnet, A.M., Criquet, S., Tagger, S., Gil, G., Le Petit, J., 2000. Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17. Can. J. Microbiol. 46, 189–194. Gianfreda, L., Xu, F., Bollag, J.M., 1999. Laccases: a useful group of oxidoreductases enzymes. Biorem. J. 3, 1–25. Holt, J.G., Krieg, H.R., Sneath, P.H.A., Staley, J.T., Williams, S.T., 1989. Bergey's Manual of Systematic Bacteriology. vol. 1–4. Williams and Wilkins, Baltimore p. 2648. Hou, H., Zhou, J., Wang, J., Du, C., Yan, B., 2004. Enhancement of laccase production by Pleurotus ostreatus and its use for the decolorization of anthraquinone dye. Process Biochem. 39, 1415–1419. Jhadav, A., Vamsi, K.K., Khairnar, Y., Boraste, A., Gupta, N., Trivedi, S., Patil, P., Gupta, G., Gupta, M., Mujapara, A.K., Joshi, B., Mishra, D., 2009. Optimization of production and partial purification of laccase by Phanerochaete chrysosporium using submerged fermentation. Int. J. Microbiol. Res. 1 (2), 09–12.

Kuhad, R.C., Singh, A., Eriksson, K.E.L, 1997. Microorganism and enzymes involved in the degradation of plant fiber cell wall. In: Eriksson, K.E.L. (Ed.), Biotechnology in the Pulp and Paper Industry. Advances in Biochemical Engineering Biotechnology. Springer Verlag, Berlin. (Chapter 2). Kuznetsov, B.A., Shumakovich, G.P., Koroleva, O.V., Yaropolov, A.I., 2001. On applicability of laccase as label in the mediated and mediatorless electroimmunoassay: effect of distance on the direct electron transfer between laccase and electrode. Biosens. Bioelectron. 16, 73–84. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Li, K., Xu, F., Eriksson, K.E., 1999. Comparison of fungal laccases and redox mediators in oxidation of a nonphenolic lignin model compound. Appl. Environ. Microbiol. 65, 2654–2660. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randal, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Mechichi, H.Z., Mechichi, T., Dhouib, A., Sayadi, S., Martinez, A.T., Martinez, M.J., 2006. Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme. Enz. Microb. Technol. 39, 141–148. Palmore, G.T.R., Kim, H.H., 1999. Electro-enzymatic reduction of dioxygen to water in the cathode compartment of a biofuel cell. J. Electroanal. Chem. 565, 110–117. Pointing, S.B., Jones, E.B.G., 2000. Vrijmoed LLP. Optimization of laccase production by Pycnoporus sanguineus in submerged liquid culture. Mycologia 92, 139–144. Riu, J., Schonsee, I., Barcelo, D., 1998. Determination of sulfonated azo dyes in ground water and industrial effluents by automated solid phase extraction followed by capillary electrophoresis/mass spectrometry. J. Mass Spectrom. 33, 653–663. Rodriguez, C.S., Sanroman, M.A., Gubitz, G.M., 2005. Influence of redox mediators and metal ions on synthetic acid dye decolorization by crude laccase from Trametes hirsute. Chemosphere 58, 417–422. Shraddha, Shekher R., Sehgal, S., Kamthania, M., Kumar, A., 2011. Laccase: microbial sources, production, purification, and potential biotechnological applications. Enzy Res. , http://dx.doi.org/10.4061/2011/217861. Solomon, E.I., Sundaram, U.M., Machonkin, T.E., 1996. Multicopper oxidases and oxygenases. Chem. Rev. 96, 2563–2605. Thurston, C.F., 1994. The structure and function of fungal laccases. Microbiology 140, 19–26. Yaropolov, A.I., Skorobogat'ko, O.V., Vartanov, S.S., Varfolomeyev, S.D., 1994. Laccase properties, catalytic mechanism, and applicability. Appl. Biochem. Biotechnol. 49, 257–280.