Appl Biochem Biotechnol (2013) 169:660–672 DOI 10.1007/s12010-012-0031-z
Decolorization of Synthetic Dyes by Crude and Purified Laccases from Coprinus comatus Grown Under Different Cultures: The Role of Major Isoenzyme in Dyes Decolorization Man Jiang & Zhen Ten & Shaojun Ding
Received: 4 September 2012 / Accepted: 10 December 2012 / Published online: 28 December 2012 # Springer Science+Business Media New York 2012
Abstract Coprinus comatus laccase isoenzyme induction and its effect on decolorization were investigated. The C/N ratio, together with aromatic compounds and copper, significantly influenced laccase isoenzyme profile and enzyme activity. This fungus produced six laccase isoenzymes in high-nitrogen low-carbon cultures but much less in low-nitrogen high-carbon (LNHC) cultures. The highest laccase level (3.25 IU/ml), equivalent to a 12.6fold increase compared with unsupplemented controls (0.257 IU/ml), was recorded after 13 days in LNHC cultures supplemented with 2.0 mM 2-toluidine. Decolorization of twelve synthetic dyes belonging to anthraquinone, azo, and triphenylmethane dyes, by crude laccases with different proportion of isoenzymes produced under selected culture conditions, illustrated that the LacA is the key isoenzyme contributed to dyes decolorization especially in the presence of 1-hydroxybenzotriazol, which was further confirmed by dyes decolorization with purified LacA in the same condition. The crude laccase only was able to decolorize over 90 % of Reactive Brilliant Blue K-3R, Reactive Dark Blue KR, and Malachite Green, and higher decolorization for broader spectrum of synthetic dyes was obtained in presence of redox mediator, suggesting that C. comatus had high potential to decolorize various synthetic dyes as well as the recalcitrant azo dyes. Keywords Decolorization . Coprinus comatus . Crude laccase . Isoenzyme . Synthetic dye
Introduction More than 10,000 different synthetic dyes are extensively used in various industrial dyeing and printing processes. The textile industry is the largest user of synthetic dyes consuming about 56 % of the total annual world production (7×105 tons) [1, 2]. However, it was estimated that approximately 10–15 % of these were released into the environment during manufacturing and usage [3], which cause serious environmental pollution due to their high M. Jiang : Z. Ten : S. Ding (*) Department of Biological Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, People’s Republic of China e-mail:
[email protected]
Appl Biochem Biotechnol (2013) 169:660–672
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stable to light, temperature, and microbial attack. Conventional wastewater treatment systems, such as activated sludge and trickling filters, are often inefficient. Several physicochemical methods, including coagulation/flocculation, membrane filtration, and activated carbon adsorption have high operating costs and of limited applicability [4]. Currently, one of the possible alternatives for treatment of colored effluents is the use of white-rot fungi, which can oxidize a wide spectrum of organic pollutants including synthetic dyes [5, 6]. Biodegradative ability of white-rot fungi is generally assumed to be associated with the production of extracellular ligninolytic enzymes, among them laccase being the most intensively studied [7]. Laccases (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) are copper-containing enzymes that catalyze the oxidation of a broad spectrum of phenolic compounds and non-phenolic substrates using molecular oxygen as the electron acceptor. Dye decolorization with laccase attracted considerable attention, recently, laccase from several microbial sources has been reported to efficiently degrade/decolorize various categories of dyes [8, 9]. In many fungal species, laccases occur as groups of isoenzymes encoded by gene families [10, 11]. Laccase isoenzymes formation in a number of white-rot fungi is known to be influenced by various physiological factors [12, 13], and therefore, their isoenzyme proportion can be altered by the changing of C/N ratio and the addition of organic compounds and metal ions. This gene redundancy usually caused troublesome in enzyme production and purification. Crude laccase without purification is cheaper and more stability comparing to purified or recombinant laccase [5, 14, 15], nevertheless, using crude laccase would reduce the cost of enzymatic-based decolorization process in industrial scale. However, the influence of culture conditions and relative contribution of different isoenzymes in crude laccases to decolorization are not yet completely understood and this knowledge could be helpful for process design and optimization. Coprinus comatus was widely cultivated in many countries as a delicious and highly nutritious edible mushroom. It produced multiple extracellular laccase isoenzymes [16]. In this study, we compared the synthetic dyes decolorization capacity of crude and purified laccases from C. comatus grown under different cultures. The aims of the work presented here were: (1) to further determine its laccase isoenzyme induction and its effect on decolorization and (2) to evaluate its capability and the contribution of a major isoenzyme in crude cultures to the decolorization of synthetic dyes.
Materials and Methods Microorganism and Media C. comatus was obtained from the Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, People’s Republic of China and maintained on potato dextrose agar (PDA) at 4 °C with periodic transfer. Four different media with different C/N ratio were used in this study. The high-nitrogen high-carbon (HNHC) medium contained (per liter): 1.0 g KH2PO4, 0.4 g K2HPO4, 0.5 g MgSO4 7H2O, 0.013 g CaCl2·2H2O, 0.1 g yeast extract, 1.0 g NH4NO3, 1.876 g asparagines, 10.8 g glucose, and 2 ml Tween 80. After autoclaving and cooling to room temperature, 2.5 mg/l thiamine and 1 ml/l of a trace elements solution consisting of (per liter): 4.8 g FeC6H5O7·5H2O, 2.64 g ZnSO4⋅4H2O, 2.0 g MnCl2 4H2O, 0.4 g CoCl2·6H2O, and 0.4 g CuSO4·5H2O was added. Other media are the same as HNHC except the concentration of carbon and nitrogen sources was adjusted. High-nitrogen low-carbon (HNLC) contains same concentration nitrogen source as HNHC, but the glucose is reduced to 1.08 g/l. Low-
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nitrogen high-carbon (LNHC) and low-nitrogen low-carbon (LNLC) contain same concentration of nitrogen (0.10 g/l NH4NO3 and 0.1876 g/l asparagine) but with 10.8 or 1.08 g/l glucose, respectively. Effect of C/N Ratio and Aromatic Compounds on Laccase Isoenzymes Formation and Production The effect of C/N ratio on isoenzymes formation was carried in 250-ml Erlenmeyer flasks with 50 ml of medium (pH 6.0), which was inoculated with four agar discs (1 cm diameter), cut from the growing edge of a 7-day PDA culture. Flasks were incubated at 25 °C and 150 rpm on an orbital shaker for up to 22 days. Cultures were harvested periodically, aliquots of the supernatant were collected aseptically, or the whole culture was filtered through a filter paper using a Büchner funnel. The culture supernatants were centrifuged at 10,000×g for 20 min at 4 °C and used for native-polyacrylamide gel electrophoresis (PAGE) and activity analysis. The effect of aromatic compounds as well as Cu2+ on laccase isoenzyme formation was determined by adding different test compounds (0.05–3.0 mM final concentration) to 4-day-old cultures grown on HNLC or LNHC media. Purification of a Major Laccase Isoenzyme from LNHC Culture Laccase from C. comatus was purified from LNHC media supplemented with 2.0 mM 2toluidine. The culture liquid from 13 days was separated from mycelia by filtration on Whatman paper, concentrated and dialyzed against 20 mM Tris–HCl (pH 8.0) by ultrafiltration with the Pellicon ultrafiltration system (Millipore) using a 10-kDa molecular mass cut-off membrane. The concentrated enzyme solution was applied to a column (1.6×30 cm) of DEAE-Sephrose™ CL-6B pre-equilibrated with the same buffer. After washing with 200 ml same buffer to remove unbound laccase isoforms and protein, bound laccase was subsequently eluted from the column with a linear salt gradient (0–1.0 M NaCl in the same buffer) with flow rate of 3 ml/min. Elution was simultaneously monitored at 280 nm. The active fractions were pooled, concentrated to
