Purification of
1. Introduction
Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are enzymes that catalyze the oxidation of phenolic compounds and aromatic amines with the simultaneous reduction of molecular oxygen to water [1]. They are widely distributed in many plants and fungi, some insects and bacteria, being particularly abundant in white-rot basidiomycetes [2]. Typical fungal laccases are described as glycosylated multicopper proteins, which are produced as extracellular monomeric forms of around 60-80 kDa, containing four copper atoms and 15-20% carbohydrates. Operatively, they are moderately thermotolerant, showing optima activity at 50-55 °C, and under acidic conditions (pH 3-5); although their maxima stability occurs in the alkaline zone (pH 8-9) [3]. Their copper atoms are distributed in three different sites bringing unique spectroscopic properties: The type 1 copper (CuT1) atom, is responsible of the intense blue color of enzymes by light absorption around 610 nm; The type 2 copper (CuT2) atom exhibits a weak absorption in the visible region; and the two type 3 copper (CuT3) atoms are present as a binuclear center, which has an absorption maximum about 330 nm. Moreover, CuT2 and CuT3 copper atoms are structural and functionally arranged as a trinuclear cluster. The four copper atoms form part of the active site of enzyme contributing directly to reaction. CuT1 is involved in the initial electron subtraction from reducer substrates, while trinuclear CuT2 and CuT3 cluster is responsible of the electron transference, from CuT1 to diatomic oxygen [4].
According to their redox potential, most of blue laccases belong to class II (-500 to -600 mV) or class III (-700 to -800 mV) laccases [5]. This is a disadvantage when compared to the ability of lignin peroxidase (LiP) and manganese peroxidase (MnP) to attack compounds with higher redox potential, including non-phenolic lignin units. To overcome this limitation laccases have evolutively developed a synergistic catalytic strategy, which combines a flexible ability to recognize a great variety of chemical compounds, with an extended capability to act at the distance through the activation of diffusible low molecular substances which serve as redox mediators. From a biological stand point this strategy let laccases to become one of the most versatile enzymes in nature, adaptable to multiple functions in plants, insects, fungi and bacteria. Another interesting possibility arises from the properties of atypical “yellow” and “white” laccases, which have shown the ability to catalyze the direct oxidation of high redox potential non-phenolic lignin model substrates or polyaromatic hydrocarbons [6, 7]. It has been proposed that the improved redox capabilities of these laccases come up either, by substituting some copper atoms for zinc, iron, or manganese in the metal clusters or by a change of the redox state of the CuT1(due to the interaction with a lignin-derived ligand) at the active site of, otherwise normal laccase protein structures. So, evolution and prevalence of laccases as a part of the lignin modifying enzyme (LME) system in white-rot basidiomycetes could also be the result of a “biochemical spring-up” mechanism acting under a short term ecophysiological selective pressure.
Whether directly or by mediation, laccases are able to oxidize a broad range of natural or xenobiotic compounds, including: mono, poly or methoxy- amine- and chloro-substituted phenols as well as aromatic heterocyclic and inorganic/organometallic substances; some of them recognized among the most recalcitrant industrial pollutants, for example; polycyclic aromatic hydrocarbons (PAH), pentachlorophenols (PCP), polychlorinated biphenyls (PCB), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), trinitrotoluene (TNT), and many azo, triarylmethane, anthraquinonic, indigoid and heterocyclic textile dyes [8,9]. Therefore, laccases are considered enzymes with a great potential for the development of environmental and industrial applications. Current and potential laccase applications include biobleaching of pulp and bioremediation of pulp and paper industries, bioremediation of olive mill wastewater, bioremediation of effluents of the textile and dye manufacturing industries, biocatalytic synthesis of antibiotics and novel polymeric materials, development of biosensors, clarification and stabilization of beer, juices and wines, and panification [1, 10, 11]. Some laccase formulations have already reached a commercial significance, but general thought is that their biotechnological applications and performances could be greatly improved or expanded with the development and finding of new enzyme variants with desirable functional properties, such as higher redox potential, optimum activity at neutral or alkaline pH and thermal stability [12, 13, 14]. It has been proposed that these new laccases could be obtained by protein engineering or through the exploration of the natural biodiversity. The importance of prospective studies in natural biodiversity applying an ecophysiological approach is illustrated by reports about isolation of new thermostable laccases from fungi, either from thermophilic compost [15] or tropical environments [13,16], or by the finding of novel laccases with improved ability to oxidize substrates with a higher than normal redox potential culturing under solid phase conditions [6, 7,17]. Northeast Mexico shelters a high diversity of white-rot basidiomycetes as a result of its particular combination of physiography and climate, including species associated to pine, oak and mixed forests, sub-mountain and semi-desert scrublands, and grass-land. In this work we first present information on the isolation, identification and selection of a northeast Mexico native strain of
2. Materials and methods
2.1. Chemicals
All chemicals used as buffers, enzyme substrates, culture media ingredients and electrophoresis reagents, were reactive grade and commercially available through local distributors of Difco, Sigma-Aldrich and Fluka, or BioRad products: PDB (potato dextrose broth), bacteriological agar, yeast extract, malt extract, peptone and dextrose, were from Difco. Acrylamide, bis-acrylamide, TEMED (N,N,N’,N’- tetramethylethylenediamine), 2-mercaptoethanol, SDS (sodium dodecyl sulfate), trizma-base, glycine, Coomassie blue, and low range markers kit, were from Bio Rad. Enzyme substrates and dyes: 2,6-dimethoxyphenol (2,6-DMP);
2.2. Isolation and identification of fungal strain
The
2.3. Enzyme and protein assays
Laccase activity was determined by triplicate at 25 °C in 3 ml cuvettes, monitoring the increase in absorbance at A468 (ε=49,600 M-1cm-1), using a Shimadzu UV-VIS mini 1240 spectrophotometer and 2,6-DMP as substrate. The assay mixture contained 0.01ml enzymatic extract, 0.1 ml of 60 mM 2.6-DMP in 2.89 ml of 200 mM citrate-phosphate buffer at pH 4.0 [21]. One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 μmol of 2, 6-DMP per minute at 25 °C. In some cases, it was necessary to assess the presence of lignin peroxidase (LiP) and manganese peroxidase (MnP). LiP activity was estimated by the H2O2-dependant veratryl alcohol oxidation to veratraldehyde as in reference [22] MnP activity was measured by the formation of Mn3+-tartrate complex during the oxidation of MnSO4 in tartrate buffer as in [23]. The protein concentration was estimated by the Bradford assay (Protein Assay Bradford of BioRad) with bovine serum albumin as standard.
2.4. Strain selection and enzyme production
Isolated
Enzyme production was evaluated in submerged liquid cultures on the natural containing laccase-inductors BF or a modified Kirk medium (MK) [25], with the following composition: 10 g l-1 dextrose, 1.0 g l-1 yeast extract, 5.0 g l-1 peptone, 2.0 g l-1 ammonium tartrate, 1.0 g l-1 KH2PO4, 0.5 g l-1 MgSO4, 0.5 g l-1 KCl, and 1.0 ml of 100 X trace element solution (0.5 g EDTA, 0.2 g FeSO4, 0.01 g ZnSO4, 0.003 g MnCl2, 0.03 g H3BO4, 0.02 g CoCl2, 0.001 g CuCl2, and 0.003 g NaMoO4 in 100 ml); amended with 350 μM CuSO4 and 3% ethanol [26]. Cultures were performed at 28 °C and 150 rpm for 14 days. 50 μL aliquots were taken every two days to determine the laccase activity.
2.5. Laccase purification
All the procedures were performed at 4 °C, unless otherwise stated. Extracellular liquid from 14 day-old submerged cultures was separated from mycelium by filtration through a cotton-polyester 50:50% cloth. Then, water-soluble polysaccharides were removed from sample solution by freezing (- 20 °C for 24 h), thawing and filtration (Whatman # 1). Culture filtrate was concentrated to approximately 200 ml by 10 kDa ultrafiltration (Millipore prep/scale TFF cartridge). The obtained fluid was further reduced to 20 ml by using a stirred ultrafiltration system equipped with an YM10 membrane (Amicon, Millipore). The reddish-brown enzyme concentrate was equilibrated by diafiltration with 20 mM potassium phosphate, pH 6.0, and applied to a pre-equilibrated anion-exchange DEAE-Sepharose column (2.5 × 17 cm). Once on the column, unadsorbed protein and most of the pigment were removed by washing with two volumes of equilibrium buffer. Retained proteins were eluted with a linear gradient of potassium phosphate pH 6.0 from 20 to 300 mM, and the eluted fractions were assayed for laccase activity and the A280 nm monitored. Fractions with laccase activity were pooled, concentrated, equilibrated by diafiltration with 100 mM potassium phosphate, and applied on a pre-equilibrated Biogel P-100 column (2.6 x 65 cm). The loaded proteins were eluted with the same buffer. Active fractions were pooled, concentrated and diafiltrated against 20 mM potassium phosphate buffer pH 6.0. Enzyme was further purified by anion-exchange on a pre-equilibrated Q-Sepharose column (2.5 x 17 cm). Once set the sample, active fractions were eluted with a lineal gradient of potassium phosphate from 20 to 300 mM. These fractions were pooled, concentrated and diafiltrated against water, and stored at - 20 °C.
2.6. Electrophoresis analysis
Protein purity and molecular mass were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as in reference [27] with 4% stacking gel and 12% resolving gel. Protein bands were stained with Coomassie brilliant blue and the molecular mass (
2.7. UV-Vis absorbance spectra
As a part of the characterization of the physicochemical properties of the laccase, its absorbance spectrum from 200 to 800 nm was obtained in a UV-Vis Shimadzu-Mini 1240 spectrophotometer. The assay was performed with 25 μM of protein diluted in 1 ml of bidistilled water.
2.8. Effect of pH on enzymatic activity
Optimum pH of activity was determined in McIlvine buffer (consisting of a combination of 100 mM citrate/50 mM potassium phosphate) adjusted in a range from 3.0 to 7.0. Activity determination was made according to described method with 2,6-DMP using 0.2 M citrate phosphate buffer, at pH 4.0.
2.9. Effect of temperature on enzyme activity and stability
The effect of temperature on reaction rate was determined using 2, 6-DMP in 0.2 M citrate/phosphate buffer, at pH 4.0. The temperature of reaction mixture was adjusted to indicate value and then the reaction was started by the addition of enzyme. The assays were done by triplicate and data in graphics appear as relative activity as a function of temperature, considering as 100% the average of maxima obtained. The activation energy of the system was calculated by the Arrhenius model, according to the expression: Log
2.10. Determination of kinetic parameters
Kinetic analysis was performed on some common substrates of laccase: 2, 6-DMP, ABTS,
2.11. Decolorization assays
The decolorizing ability of laccase was evaluated with two recalcitrant dyes, the non-phenolic azo Methyl Red (MR), and the diazo reactive black 5 (RB 5). The reaction mixture consisted of 0.890 ml of 0.2 M citrate/phosphate buffer, pH 4.0, 0.1 ml of 250 μM MR or RB5 (final concentration 25 μM), and 0.01 ml of pure laccase (final concentration 5 U/ml). Assays were performed at 25 °C and reaction was initiated with the addition of enzyme. Decolorization was estimated by the decreasing of absorbance at 530 nm for MR or 597 nm for RB5. The results are expressed as the percent of remaining color as a function of incubation time according to the relationship: remaining color (%) = [(
3. Results and discussion
3.1. Strain identification
In this study an autochthonous strain of
3.2. Production and purification of laccase
Guzmán (2003), considers that
Laccase purification was started from about 1850 ml of mycelium-free filtrates from 14 day-old submerged cultures. After 10K ultraconcentration and sequential steps of anionic exchange chromatography on DEAE- Sepharose, gel filtration on Biogel P-100, and anionic exchange on Q-Sepahrose, laccase activity eluted as an apparently single protein peak with 100-140 mM phosphate (Figure 3). When aliquots of pooled laccase from this last chromatographic step were analyzed by denaturing SDS-PAGE, multiple protein bands were detected by Coomassie staining (not shown). A similar effect was reported in a work with a
|
|
|
|
|
|
Culture filtrate | 1551 | 6477 | 4.18 | 100.0 | 1.0 |
Ultraconcentration 10 K | 422 | 3724 | 8.8 | 57.4 | 2.1 |
DEAE-Sepharose FF | 64 | 2308 | 35.8 | 35.6 | 8.5 |
Biogel P-100 | 32 | 1924 | 59.2 | 29.7 | 14.1 |
Q-Sepharose | 22 | 1522 | 69.8 | 23.5 | 16.7 |
3.2. Biochemical properties
Electrophoresis analysis indicated that
In addition to blue laccases, other “atypical” forms of the enzyme named “yellow” laccases and “white” laccases have been reported. In the first case, it has been proposed that a variation in the redox state of Cu-T1 centers, by the presence of endogenous ligands, decreases the absorbance at 600 nm, without altering the spectral characteristics of the Cu-T2 and Cu-T3 centers, resulting in a yellow color [6]. In white laccases, like the one produced by
3.3. pH and temperature dependence
Enzyme was further characterized for its pH and temperature dependence. The effect of pH on laccase activity was studied using some of the most common laccase substrates, including the phenolic 2, 6-DMP,
The influence of temperature on
Thermostability is a desirable property of industrial enzymes. Curves of temperature-stability of
In comparison to laccases isolated from other
3.4. Kinetic properties
The kinetic properties of enzyme were studied with some typical substrates. The values of the Michaelis constant (Km), catalytic constant (Kcat) and specificity constant (Kcat/Km), were calculated by the Lineweaver-Burk method. Laccase showed the highest affinity and molecular activity, on ABTS (Km = 23 mM, Kcat = 221 s-1) compared to
|
|
1 | EAVVVNGITPAPLIAGKK |
2* | GPFVVYDPNDPQASLYDIDNDDTVITLADWYHLAAKVGQR |
3* | FPLGADATLINGLGR |
4* | TPGTTSADLAVIKVTQGK |
5 | YSFVLDASQPVDNYWIRANPPFGNVGFAGGINSAILR |
6 | SAGSSEYNYDNPVFR |
|
|
|
|
|
2,6-DMP | 41 | 500 | 88 | 2.16 x 106 |
o-dianisidine | 44 | 1111 | 197 | 4.49 x 106 |
ABTS | 23 | 1250 | 221 | 9.38 x 106 |
Among these substrates this laccase showed preference for ABTS and this characteristic was consistent with most of fungal laccases [32, 41, 42]. Unexpectedly the substrate saturation graphics with SYR showed a sigmoidal-like behavior instead of the common hyperbolic one (not shown). This result could be explained considering a kinetic mechanism of positive cooperativity as that described for monomeric mnemonical enzymes [43], where a conformational change of interacting enzyme at the end of the first catalytic cycle, reacts more readily with a second substrate molecule than other free-enzyme. Other factor contributing to this result could be the presence of ethanol in routinely SYR assay affecting the substrate solubility and/or enzyme activity. Whether mechanistic on phenomenological, this observation must be taken into account in future works, considering the relevance of this substrate in laccase characterization.
3.5. Dye decolorization
As revealed by the activity staining of native gels shown above,
While
4. Conclusion and future prospects
Its thermostability and ability for acting on high redox substrates and recalcitrant dyes, makes
Acknowledgments
Authors thank the financial support provided by the Sistema de Fondos INNOVAPYME-CONACYT (Proyecto No. 139352). We also tank to Unidad de Proteómica, IBT-UNAM for assistance in peptide sequencing.References
- 1.
Mayer AM, Staples RC. Laccase: New Functions for an Old Enzyme. Phytochemistry 2002;60 551-565. - 2.
Baldrian P. Fungal Laccases-Occurrence and Properties. FEMS Microbiological Reviews 2005;20 1-28. - 3.
Morozova OV, Shumakovich GP, Gorbacheva MA, Shleev SV, Yaropolov AI. ‘‘Blue’’ Laccases. Biochemistry (Moscow) 2007;72 1136–1412. - 4.
Thurston CF. The Structure of Fungal Laccases. Microbiology 1994 ;140 19-26. - 5.
Eggert C, Temp U, Eriksson K-EL. The Ligninolytic System of the White- rot Fungus Pycnoporus cinnabarinus : Purification and Characterization of the Laccase. Applied Environmental Microbiology 1996;62 1151–1158. - 6.
Leontievsky AA, Vares T, Lankinen P, Shergill JK, Pozdnyakona NN, Myasoedova NM. Blue and Yellow Laccases of Ligninolytic Fungi. FEMS Microbiological Letters 1997;156 9-14. - 7.
Palmieri G, Giardina, P, Bianco C, Sacloni A, Capasso A, Sannia G.A A Novel White Laccase from Pleurotus ostreatus . Journal of Biological Chemistry 1997;50 31301-31307. - 8.
Pointing SB. Feasibility of Bioremediation by White–rot Fungi. Applied Microbiology and Biotechnology 2001;57 20-33. - 9.
Reddy CA. The Potential of White-rot Fungi in the Treatment of Pollutants. Current Opinion in Biotechnology 1995;6 320-328. - 10.
Rodríguez-Couto S, Toca-Herrera JL. Laccases in the Textile Industry. Biotechnology and Molecular Biolology Reviews 2006a;1 115-120. - 11.
Rodríguez-Couto S, Toca-Herrera JL. Industrial and Biotechnological Applications of Laccases: A review. Biotechnology Advances 2006b;24 500-513. - 12.
Xu F, Berka RM, Wahleithner JA, Nelson BA, Shuster JR, Brown SH, Palmer AE, Solomon EI. Site-directed Mutations in Fungal Laccase: Effect on Redox Potential, Activity and pH Profile. Biochemistry Journal 1998;334(1) 63-70. - 13.
Uzan E, Nousiainen P, Balland V, Sipila J, Piumi F, Navarro D, Asther M, Record E, Lomascolo A. High Redox Potential Laccases from the Ligninolytic Fungi Pycnoporus coccineus andPycnoporus sanguineus Suitable for White Biotechnology: from Gene Cloning to Enzyme Characterization and Applications. Journal of Applied Microbiology 2010;108 2199-2213. - 14.
Rodgers CJ, Blanford CF, Giddens SR, Skamnioti P, Armstrong FA Gurr SJ. Designer Laccases: A Vogue for High-potential Fungal Enzymes?. Trends in Biotechnology 2009;28(2) 63-72. - 15.
Jordaan J, Leukes WD. Isolation of a Thermostable Laccase with DMAB and MBTH Oxidative Coupling Activity from a Mesophilic White-rot Fungus. Enzyme and Microbial Technology 2003;33 212-219. - 16.
Dantán-González E, Vite-Vallejo O, Martínez-Anaya C, Méndez-Sánchez M, González MC, Palomares LA, Folch-Mallol J. Production of Two Novel Laccase Isoforms by a Thermotolerant Strain of Pycnoporus sanguineus Isolated from an Oil-polluted Tropical Habitat. International Microbiology 2008;11 163–169. - 17.
Pozdnyakova NN, Turkovskaya OV, Yudina EN Rodakiewicz-Nowak Y. Yellow Laccase from the Fungus Pleurotus ostreatus D1: Purification and Characterization. Applied Biochemistry and Microbiology 2006;42(1) 56-61. - 18.
Hernández-Luna CE, Gutiérrez-Soto G, Salcedo-Martínez SM. Screening for Decolorizing Basidiomycetes in Mexico. World Journal of Microbiology and Biotecnology 2008;24 465-473. - 19.
Ryvarden L. Genera of Polypores Nomenclature and Taxonomy. Synopsis fungorum Vol. 5. Fungiflora A/S Oslo; 1991. - 20.
Guzmán G. Identificación de los Hongos: Comestibles, Venenosos, Alucinantes y Destructores de la Madera: Editorial Limusa S.A. México; 1980. - 21.
Abadulla E, Tzanov T, Costa S, Robra K, Gübitz G. Decolorization and Detoxification of Textile Dyes with a Laccase from Trametes hirsuta . Applied and Environmental Microbiology 2000;66 3357-3362. - 22.
Tien M, Kirk TK. Lignin Peroxidase of Phanerochaete chrysosporium . Methods in Enzymology 1988;161 238-248. - 23.
Kuan C, Johnson J, Tien M. Kinetic Analysis of Manganese Peroxidase. The Reaction with Manganese Complexes. Journal of Biological Chemistry 1993;268 20064–20070 - 24.
Pickard MA, Vandertol H, Roman R, Vazquez-Duhalt R. High Production of Ligninolytic Enzymes from White-rot Fungi in Cereal Bran Flakes Liquid Medium. Canadian Journal of Microbioliology 1999;45 627-631. - 25.
Dhouib A, Hamza M, Zouari H, Mechichi T, Hmidi R, Labat M, Martinez MJ, Sayadi S. Screening for Ligninolytic Enzyme Production by Diverse Fungi from Tunisia. World Journal of Microbiology and Biotechnology 2005 ;21 1415-1423. - 26.
Zouari-Mechichi H, Mechichi T, Dhouib A, Sayadi S, Martínez AT, Martínez MJ. Laccase Purification and Characterization from Trametes trogii isolated in Tunisia: Decolorization of Textile Dyes by the Purified Enzyme. Enzyme and Microbial Technology 2006;39 141-148. - 27.
Laemmli U. Cleavage of Structural Proteins During the Assembly of the Head of the Bacteriophage T4. Nature 1970;227 680-685. - 28.
Garfin DE. Methods in Enzymology. Academic Press Inc. 1990. - 29.
Guzmán G. Los Hongos del Edén Quintana Roo. ( Introducción a la Micología Tropical de México ). INECOL y CONABIO, Xalapa ; 2003. - 30.
Wang ZX, Caia YJ, Liao XR, Taoc GJ, Lia YY, Zhanga F, Zhang DB. Purification and Characterization of Two Thermostable Laccases with High Cold Adapted Characteristics from Pycnoporus sp. SYBC-L1. Process Biochemistry 2010;45 1720-1729. - 31.
Litthauer D, Jansen van Vuuren M, van Tonder A, Wolfaardt FW. Purification and Kinetics of a Thermostable Laccase from Pycnoporus sanguineus (SCC 108). Enzyme Microbial Technology 2007;40 563–568. - 32.
Lu L, Zhao M, Zhang BB, Yu SY, Bian X-J, Wang W, Wang Y. Purification and Characterization of Laccase from Pycnoporus sanguineus and Decolorization of an Anthraquinone Dye by the Enzyme. Applied Microbiology and Biotechnology 2007;74 1232-1239. - 33.
Hernández-Fernaud JR, Marina A, González K, Vázquez J, Falcón MA. Production, Partial Characterization and Spectroscopic Study of the Extracellular Laccase Activity from Fusarium proliferatum. Applied Microbiology and Biotechnology 2006 ;70 212-221. - 34.
Madzak C, Mimmi MC, Caminade E, Brault A, Baumberger S, Briozzo P, Mougin C, Jolivalt C. Shifting the Optimal pH of Activity for a Laccase from the Fungus Trametes versicolor by Structure-based Mutagenesis. Protein Engineering 2006;334 63-70. - 35.
Hildén K, Hakala TK, Lundell T. Thermotolerant and Thermostable Laccases. Biotechnological Letters 2009;31 1117–1128. - 36.
Holm KA, Nielsen DM, Eriksen J. Automated Colorimetric Determination of Recombinant Fungal Laccase Activity in Fermentation Samples Using Syringaldazine as Chromogenic Substrate. Journal of Automated Chemistry 1998;20 199 –203. - 37.
Schneider K, Caspersen MB, Mondorf K, Halkier T, Skov LK, Ostergaard PR, Brown KM, Brown SH, Xu F. Characterization of a Coprinus cinereus Laccase. Enzyme and Microbial Technology 1999;25 502–508. - 38.
Murugesan K, Nam IH, Kim YM, Chang YS. Decolorization of Reactive Dyes by a Thermostable Laccase Produced by Ganoderma lucidum in Solid-state Culture. Enzyme and Microbial Technology 2007;40 1662-1672. - 39.
García TA, Santiago MF, Ulhoa CJ. Studies on the Pycnoporus sanguineus CCT-4518 Laccase Purified by Hydrophobic Interaction Chromatography. Applied Microbiology and Biotechnology 2007;75 311–318. - 40.
Antorini M, Herpoel-Gimbert I, Choinowski T, Sigoillot JC, Asther M, Winterhalter K, Piontek K. Purification, Crystallization and X-ray Diffraction Study of Fully Functional Laccases from Two Ligninolytic Fungi. Biochemistry and Biophysics Acta 2002;1594 (1) 109-114 - 41.
Ko EM, Leem YE, Choi HT. Purification and Characterization of Laccase Isoenzymes from the White-rot Basidiomycete Ganoderma lucidum . Applied Microbiology and Biotechnology 2001;57 98-102. - 42.
Zang H, Zang Y, Huang F, Gao P, Chen J. Purification and Characterization of a Thermostable Laccase with Unique Oxidative Characteristics from Tramentes hirsuta . Biotechnology Letters 2009;31 837-843. - 43.
Ricard J, Noat G. Kinetic Co-operativity of Monomeric Mnemonical Enzymes ‘The Significance of the Kinetic Hill- coefficient. European Journal of Biochemistry 1985;152 557-564. - 44.
Haibo Z, Yinglong Z, Feng H, Peiji G, Jiachuan C. Purification and Characterization of a Thermostable Laccase with Unique Oxidative Characteristics from Trametes hirsuta . Biotechnological Letters 2009;31 837-843. - 45.
Nagai M, Sato T, Watanabe H, Saito K, Kawata M, Enei H. Purification and Characterization of an Extracellular Laccase from the Edible Mushroom Lentinula edodes , and Decolorization of Chemically Different Dyes. Applied Microbiology and Biotechnology 2002;60 327-335. - 46.
Camarero S, Ibarra D, Martínez MJ, Martínez AT. Lignin-Derived compounds as Efficient Laccase Mediators for Decolorization of Different Types of Recalcitrant Dyes. Applied and Environmental Microbiology 2005;71(4) 1775-1784. - 47.
Zeng X, Cai Y, Liao X, Zeng X, Li W, Zhang D. Decolorization of Synthetic Dyes by Crude Laccase from a Newly Isolated Trametes trogii Strain Cultivated on Solid Agro-industrial Residue. Journal of Hazardous Materials 2011;189 517-525.