SP. FIELD OF THE INVENTION

This invention relates to a method for obtaining laccase enzyme from Arthrographis sp.MTCC5479 and applications thereof.

BACKGROUND OF THE INVENTION

Laccase (benzenediol: oxygen oxidoreductase EC 1.10.3.2) is a polyphenol oxidase and belongs to a group of enzymes known as multi-copper oxidases. Laccases have one type-1 copper atom, which shows absorbance at 600 nm and imparts blue color to the enzyme. Apart from one type-1 copper atom, laccase have one type-2 and two type-3 copper atoms per enzyme molecule. Laccase catalyze the four electron reductions of molecular oxygen to water accompanied by the oxidation of a substrate [ 1 ] .

Laccase enzyme was first reported to occur in the Japanese lacquer tree, Rhus vernicifera in 1883. The enzyme was later found to occur in fungi known as 'white rot fungi' belonging to the class Basidiomycetes. It includes genera Agaricus, Cyathus, Lentinus, Phlebia, Partus, Pleurotus and Trametes. A few fungi belonging to the class Ascomycetes such as Curvularia, Gaeumannomyces, Mauginella, and Melanocarpus also produce laccase [2].

Laccase is an economically important enzyme because of its ability to oxidize range of xenobiotic compounds. Laccase exhibit broad substrate specificity and can catalyze oxidation of polyphenols, methoxy-substituted phenols, aminophenol and their derivatives [3]. Laccase catalyze various oxidation reactions, which are useful in paper and pulp industry [4], in food and beverage industry [5], bioremediation [6], biosensors [7,8] and bio-fuel cells [9]. Economical availability of purified laccase is an important factor for usage of laccase in industry [5]. White rot fungi belonging to the class Basidiomycetes are the major source of laccase enzyme. However, extracellular level of laccase enzyme produced by these fungi is low [10]. Cloning and expression of laccase of Basidiomycetes in a recombinant host is beset by problems, such as different codon usage, missing chaperone and post translational modifications [1 1]. Specific activity of laccase enzyme is dependent on the redox potential of laccase enzyme. Laccase produced in a recombinant host tends to have a lower redox potential than their counterparts produced by the wild strain [12]. Hence, it is important to explore microbial diversity for novel laccase producing strains having higher laccase yield and laccase enzyme with higher specific activity and higher redox potential. If, such a laccase producer is from Ascomycetes class of fungi, laccase genes from such fungus can also preferably be cloned and expressed efficiently in fungal hosts, such as Pichia or Aspergillus for their large scale production.

United states Patent No.7,927,849B2 has disclosed a method for production of laccase enzyme from Thielavia species [13]. The highest activity obtained reported in the patent document is 20 nkatal ml ^"1 within six days. The specific activity of the enzyme using ABTS as substrate is 1020 nkatl/mg.

This application seeks to disclose a method for production of laccase enzyme with high specific activity and yield. The laccase enzyme is produced by a fungal strain belonging to the class Ascomycetes. The laccase produced by the novel fungus according to the method disclosed herein can be applied for various laccase catalyzed biochemical reactions in diverse areas of applications, such as degradation of textile dyes in the effluent, treatment of the denim for bleaching and removal of back staining and bioremediation in general, such as degradation of some pollutants like polyaromatic hydrocarbons (PAH) and xenobiotics. The applications of laccase also exist in bakery, brewery and wine industry, in synthesis of chemicals, fabrication of cathode for fuel cell.

OBJECTIVES OF THE INVENTION

The objective of the present invention is to provide a method for obtaining laccase enzyme from Arthrographis sp.MTCC5479. SUMMARY OF THE INVENTION

Accordingly, the present invention provides a method for obtaining laccase enzyme from Arthrographis sp. MTCC5479 (Figure 1 and 2). The novel laccase enzyme is secreted into the medium by the producer strain belonging to the genus, Arthrographis. The yield of the enzyme under the conditions described herein is 9-14 IU ml ^"1 or 150- 250 nktal ml ^"1 when assayed using 1 mM ABTS in 50 mM Mcllvain buffer, pH 4.0 at 30°C. (Figure 3). The extracellular laccase produced by Arthrographis sp. can be isolated and purified easily from the culture filtrate. The enzyme is active in the wide pH range from 3-8. However, the pH optimum lies in the range of 3 to 6 for various common substrates, such as 2,2'azinobis-3-ethylbenzthiazole-6-sulphonate (ABTS), guaiacol, 2-methoxyphenol(guaiacol), 2,6 dimethoxyphenol (DMP), syringaldazine and indigo. The enzyme is active in the range of 25°C to 70°C; however, the optimum temperature of the enzyme is in the range of 40°C to 50°C for various substrates. The molecular weight of the enzyme is about 63 kDa, as determined by SDS-PAGE (Figure 4 & 5). The molecular weight of the enzyme by MALDI-ToF is 58.33 kDa (Figure 6). The isoelectric point of the enzyme (pi) is about 3.5 as determined by isoelectric focusing (Figure 7). The N-terminal amino acid sequence of the enzyme is Gly-Ile- Gly-Pro-Val-Thr-Asp-Leu-Thr-Ile-Ser-Asn-Ala-Glu-Val . The specific activity of Arthrographis laccase is 347 IU mg ^"1 (5784 nkatal mg ^"1 ) against ImM ABTS in 50 mM Mcllvain buffer, pH 4.0 at 30 °C. Specific activity of Arthrographis laccase measured by kinetic method using 0.02 mM syrigaldazine as the substrate in 50 mM Mcllvain buffer, pH 5.5 at 30 °C is 6380 IU mg ^"1 (10635 nkatal mg^.The enzyme can efficiently degrade various textile dyes belonging to anthroquinone group, azo group, triarylmethane group, eurhodin group, and reactive dyes. Presence of specific mediators, such as 1 -hydroxybenzotriole, violuric acid, methylsyringate and acetosyringone, the rate of degradation of dyes is significantly enhanced. Degradation of indigo (vat blue) requires acetosyringone or 2,2'azinobis-3-ethylbenzthiazole-6- sulphonate (ABTS) as a redox mediator. The enzyme may also be employed in applications which exists in food industry, in brewery and wine industry, in paper and pulp industry, in synthesis of chemicals, and fabrication of cathode for fuel cell and where, the laccase enzyme is known to have been used. Arthrographis laccase is capable of oxidizing indigo dye (Vat Blue) in the presence of mediators such as acetosyringone (4'-Hydroxy-3', 5'-dimethoxyacetophenone) and 2, 2'azinobis-3-ethylbenzthiazole-6-sulphonate (ABTS). This property of Arthrographis laccase can be exploited for the treatment of denim for obtaining fading effect on denim or removing back staining of denim caused by the stonewashing process. Use of different mediators in the process can bring about different grades of bleaching effects. Bleaching of denim by laccase is a desired alternative to the hypochlorite method because it offers ecological benefit as well as it does not weaken the fabric strength.

In an embodiment of the present invention provides a method for obtaining laccase enzyme from Arthrographis sp.MTCC5479 strain comprising of :

a. growing Arthrographis sp.MTCC5479 strain in sterile liquid culture

medium as herein described,

b. innoculating production medium containing flasks with strain obtained in step a,

c. incubating the medium obtained in step b, at 30°C on rotary shaker at the speed of about 180 rpm for nearly 72 hrs.

d. inducing the culture obtained in step c after 72 hrs with Xylidine 0.00001 to 0.0001% (v/v), CuS0 ₄ .5H ₂ 0, 0.0025 to 0.025% (w/v) or 100 μΜ to 1000 μΜ,

e. incubating the culture obtained in step d at nearly 30°C for about 12-15 days.

f. withdrawing of culture obtained in step e, and separating Laccase enzyme excreted into the medium from cell mass by centrifugation at nearly 8000 X g for nearly 10 minutes.

g. concentrating and purifying the enzyme obtained in step f by methods described herein.

In another embodiment of the present invention wherein the yield of laccase enzyme ranges from 9-14 IU/ml.

In another embodiment of the present invention wherein the laccase enzyme is characterized in having : I. Molecular weight nearly 63kDa, by SDS-PAGE, or 58.33 by MALDI-

ToF method.

II. pi of about 3.5,

III. Specific activity is nearly 347 IU mg ^"1 Or 5784 nkatal mg ^"1 against ImM ABTS in 50 mM Mcllvain buffer, pH 4.0 at 30 °C, Specific activity of

6380 IU mg ^"1 or 10635 nkatal mg ^"1 using 0.02 mM syrigaldazine as the substrate in 50 mM Mcllvain buffer, pH 5.5 at 30 °C.

IV. N-terminal amino acid sequence having Seq ID. No.l In another embodiment of the present invention wherein the enzyme functions at pH 2.5 to 8.5, preferably at pH 3 to 5.5.

In yet another embodiment of the present invention wherein the enzyme is effective in dye degradation treatment at temperatures 30 to 70°C, preferably at temperature 35 to 50°C.

In yet another embodiment of the present invention wherein the enzyme preparation is in the form of liquid, powder or granulate after blending or mixing with inert substances or salts in order to increase its shelf life.

In yet another embodiment of the present invention wherein the enzyme is useful for degradation of textile dyes including azo, anthraquinone, triphenylmethane, indigoid, triazine and eurhodin groups of dyes in individual or in a mixture form of these dyes.

In yet another embodiment of the present invention wherein the laccase enzyme is isolated from Arthrographis sp., and is useful for removing stains, for bleaching of pulp, for treating of fibers for whitening, for coloring of animal hairs including wool for treating of textile dye or dyes in effluents, for fabrication of cathode in a fuel cell, for formulations of coatings and adhesives based on polymerization of phenolic monomers etc.

BRIEF DESCRIPTION OF FIGURES AND TABLES:

FIGURE 1. Colony of Arthrographis sp.MTCC5479 on YPD agar,

Figure 2. Microscopic morphology of Arthrographis sp.MTCC5479 (1000X) Figure 3 Enzyme activity profile for Arthrographis sp.

Figure 4. Analysis of steps in purification of laccase. Figure 5. Estimation of molecular weight of Arthrographis laccase by SDS-PAGE

Molecular weight markers used were Phosphorylase b (97kDa), Bovine Serum Albumin(67kDa), Ovalbumin (45kDa) and Carbonic anhydrase. (30kDa).

Figure 6. MALDI-ToF analysis of Arthrographis Laccase

Figure 7. Isoelectric focusing of Arthrographis laccase

Figure 8. Bleaching of denim for light fading effect

Figure 9. Bleaching of denim for heavy fading effect

Figure 10. Arthrographis laccase treatment of Denim for removing back staining

DETAILED DESCRIPTION OF THE INVENTION

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention

EXAMPLES

Example 1

Production of laccase from the fungal Arthrographis sp.MTCC5479 strain.

Laccase enzyme was produced by growing Arthrographis strain in sterile liquid culture medium comprising

Peptone 0.1 to 2%, (w/v),

Yeast extract 0.25 to 0.5% (w/v)

KH ₂ P0 ₄ 0.01% to 0.25%

MgCl ₂ 0.0001 to 0.05% (w/v)

Glucose separately autoclaved 1-5% (w/v).

The fungal strain Arthrographis sp.MTCC5479 was grown on yeast, peptone and dextrose (YPD) agar plates and incubated for 6-7 days. Mycelial mass and spores were collected from the plate by scrapping surface of agar plate wth a sterile loop and inoculated into the 500 ml flask containing 100 ml YPD medium and was incubated at 30 °C for 3 days. The cell mass obtained was used as inoculum to inoculate 10 flasks containing 500 ml of production medium. Innoculated production medium containing flasks were incubated at 30°C on rotary shaker at the speed of 180 rpm for 72 hrs. After 72 hrs Xylidine 0.00001 to 0.0001% (v/v) CuS0 ₄ .5H ₂ 0, 0.0025 to 0.025% (w/v) or 100 μΜ to 1000 μΜ were added as inducers for laccase production. Production flasks were incubated further under stationory condition at 30°C. The onset and the progress of the enzyme synthesis was monitored by withdrawing of culture and the assay of the culture supernatant every 24 hrs interval as described in the example 3.

Laccase enzyme excreted into the medium was separated from cell mass after peak of enzyme activity was reached after about 12-15 days of incubation at 30°C. Extra cellular enzyme activity obtained is in the range of 9-14 IU ml ^"1 of the culture broth (Figure 3).

Though the production of the laccase from Arthrographis laccase was demonstrated in shake flasks, other suitable containers for the production of the laccase such as trays or fermenters may be used for this purpose by those skilled in the art of microbial fermentation.

Example 2

Isolation and purification of laccase from Arthrographis sp. culture supernatant.

Fungal mycelial mass was separated from the culture broth by centrifugation at 8000 X g for 10 minutes. The supernatant obtained (4.1L) was concentrated about 20 fold using ultrafiltration membrane of 30kD MWCO size.

The ultrafiltrate (180 ml) was loaded on to Q-Sepharose column (40 ml) equilibrated at pH 5.5 using 50 mM sodium acetate buffer and eluted using 0-50% linear gradient of 500 mM sodium chloride in the same buffer. Active fractions were pooled and precipitated by 100% saturation of ammonium sulphate. The precipitate was loaded on Sephacryl S-200

Column (200ml) and was eluted by 0.15 M NaCl in 50 mM sodium acetate buffer. Active fractions were pooled and analyzed by for determining molecular weight, isoelectric point, specific activity and other properties of the enzyme.

Example 3

Assay of laccase activity using 2, 2 ^, azinobis-3-ethylbenzthiazole-6-sulphonate (ABTS) as the chromogenic substrate [10]

Laccase activity was measured by end point spectrophotometric method. Conditions for assay were as given below. The assay was conducted in the temperature controlled cuvette chamber of spectrophotometer (Analytica Jena SPECORD 600).

Incubation buffer 975 μΐ 50 mM Mcllvaine buffer, pH 4.0, Chromogenic substrate 2, 2'azinobis-3-ethylbenzthiazole-6-sulphonate (ABTS), 1 raM

Laccase solution 25 μΐ (diluted)

Incubation time 5 min

Incubation temperature 30 °C

Wavelength 420 nm

Calculation On the basis of extinction coefficient of oxidized cation of

ABTS

36 mM ^"1 cm ^"1 . One unit of enzyme activity was defined as the amount of enzyme oxidizing 1 μΜ ABTS per minute under these conditions.

Example 4

Assay of laccase activity using chromogenic substrate syringaldazine.

Laccase activity was measured by kinetic spectrophotometric method. Conditions for assay were as given below. The assay was conducted in the temperature controlled cuvette chamber of spectrophotometer (Analytica Jena SPECORD 600).

Incubation buffer 2745 μΐ 25 mM Mcllvaine buffer, pH 5.5, containing 48% ethanol

Chromogenic substrate Syringaldazine, Stock solution, 0.28 mM in 48% ethanol Syringaldazine solution 225_μ1

Laccase solution 30 μΐ diluted

Incubation time 5 min

Incubation temperature 30 °C

Wavelength 530 nm

Calculation On the basis of Δ A (U- ), extinction coefficient ε ₅₃₀ nm of oxidized

Syrigaldazine, 65 mM ^"1 cm ^"1 . One unit of enzyme activity was defined as the amount of enzyme oxidizing 1 μΜ syringaldazine per minute under these conditions. Example 5

Estimation of protein in the enzyme preparation.

Protein content of enzyme preparations were estimated according to the method of Bradford using Bradford reagent (B-6916, Sigma chemicals, USA) and solution of known concentration of bovine serum albumin as the protein standard.

Example 6

Estimation of specific activity of the purified laccase from Arthrographis laccase.

Specific activity of purified enzyme preparation was determined in terms of International Units per milligram of protein IU mg ^"1 . Enzyme activity was measured as given in example 3 and 4. Protein content was estimated as given in example 5. Specific activity in terms of derived SI units nkatal can be calculated by multiplying the international units by the factor 16.67.

Example 7

Application of the enzyme for dye degradation.

Following dyes were dissolved in 50 mM Mcllvaine buffer (Citrate-Phosphate) buffer at the concentration of 50 mg/L. These dyes include dyes belonging to azo group, anthroquinone group, acridine group, eurhodin group, and reactive dye. 20 ml solution of dye was taken in a flask of 100 ml volume.

1. Brilliant Blue

2. Cibacron blue

3. Crystal violet

4. Rose Bengal

5. Erythrosine

6. Acridine orange

7. Malachite green

8. Neutral red

9. Remazol brilliant blue

10. Bromophenol blue

1 1. Congo red 12. Methyl red

13. Cotton blue (Acid Blue)

14. Rhodamine B

Crude enzyme in form of ultrafiltrate was added to the dye solution to make final enzyme activity about 100 Units/mL. One more set of dye solution was also set. In the second set Hydroxybenzotriazole (HOBT), a mediator molecule was added to the concentration of 5.0 mM.

Flasks were incubated at 30 °C. Residual dye contents were estimated after 12 hrs and 24 hrs. of incubation.

Flasks containing the mediator 1-HOBT, showed more than 90% decolorization of Acridine orange, Brilliant blue, Cibacron Blue, Rose Bengal, Erythrosine, Malachite green, Neutral red, Bromophenol blue Remazol brilliant blue, Crystal violet, Congo red Rhodamine B and Cotton Blue within 12 hrs.

Within 24 hrs, 90% degradation of Acridine orange, Brilliant blue, Cibacron Blue, Rose Bengal, Erythrosine, Malachite green, Neutral red, Remazol brilliant blue, Congo red was observed in flasks in which, mediator (HOBT) was not added.

Application of laccase from Arthrographis sp. for degradation of textile dyes is not limited only the number of dyes listed above. The above experiment only demonstrates the versatility of Arthrographis laccase to degrade textile dyes belonging to different groups. The enzyme from Arthrographis sp can be applied to the degradation of dyes other than being mentioned herein by a person skilled in the art of use of laccase enzyme and different mediators for the reaction carried out by the enzyme. The enzyme may also be applied for the purpose of degradation of dyes in a suitable reactor where contact of dyes and enzymes can take place.

Example 8

Treatment of denim by Arthrographis laccase for bleaching in order to obtain faded look.

A piece of desized denim cloth was obtained from Rossari Biotech India Pvt. Ltd, Mumbai, India. The cloth was rinsed with tap water and cut into approximately 1 square inch swatches (dry weight about 800-900 mg) Denim swatch in were submerged in 25 ml Mcllvaine buffer, pH 3.0 contained in 100 ml conical flask. Redox mediator acetosyringone (4'-Hydroxy-3', 5'-dimethoxyacetophenone) and 2, 2'azinobis-3-ethylbenzthiazole-6-sulphonate (ABTS) were added in two flasks separately at the concentration of 1-5 mg ml ^"1 . ABTS was added as dry powder whereas, acetosyringone was added as solution in ethanol (50 mg/ml). The denim swatches in flasks were allowed to imbibe the buffer and mediator for 5 minutes. Arthrographis laccase 25 IU was added to each flask in order to initiate the reaction. The flasks were incubated in a rotary shaker at 45°C, 180-rpm speed. After 60 min the denim swatches were removed form the flasks, rinsed with tap water and immersed in flasks containing fresh buffer, and mediator in the same quantity as stated before. Addition of the enzyme (25 U) was done and incubation at 45°C 180-rpm speed was carried out for another 90 min. At the end of 90 minutes, the denim swatches were removed from the flask, rinsed with water blot dried using filter paper ironed. The results were photographed (Figure 8, 9). Example 9

Treatment of stone washed denim by Arthrographis laccase for removing back staining. Stone washed denim fabric was obtained from local market. Denim was cut into about 1 square inch. The stone washed (dry weight about 800-900.mg) Denim swatches in were submerged in 25 ml Mcllvaine buffer, pH 3.0 contained in 100 ml conical flask. Redox mediator acetosyringone (4'-Hydroxy-3', 5'-dimethoxyacetophenone) and 2,2'azinobis-3-ethylbenzthiazole-6-sulphonate (ABTS) were added in two flask separately at the concentration of 1 -5 mg ml ^"1 . ABTS was added as dry powder whereas, acetosyringone was added as solution in ethanol (50 mg/ml). The denim swatches in flasks were allowed to imbibe the buffer and mediator for 5 minutes. Arthrographis laccase 25 IU was added to each flask in order to initiate the reaction. The flasks were incubated in a rotary shaker at 45°C, 180-rpm speed. After 90 min the denim swatches were removed form the flasks, rinsed with water blot dried using filter paper ironed. The results were photographed (Figure 10).

Examples given in 8 and 9 demonstrate efficacy of Arthrographis laccase in bleaching denim for obtaining faded look effect or enhancing contrast in the stonewashed denim However, laccase from Arthrographis laccase can also be applied for treatment of garments made from denim fabric by a person skilled in the art of use of laccase enzymes for bleaching.

Example 10

Immobilization of laccase on O-Sepharose.

Enzyme partially purified by ion exchange chromatography method as described in example 2 were dialyzed against 50 mM citrate phosphate buffer pH 6.0. The enzyme solution was concentrated to reduce its volume using Amicon centrifugal ultrafiltration devices. The concentrated enzyme solution had activity of 1200 IU per ml. Q- Sepharose was packed in a column and equilibrated at pH 6.0 and then transferred into a 25 ml flask. 20 ml of concentrated solution of laccase containing 1220U ml ^"1 was added to 2 ml Q-Sepharose equilibrated at pH 6.0 and allowed to bind it for 30 min. The unbound enzyme was removed from the Q-Sepharose by filtering was the passed through Whatman number 1 filter paper. The Sepharose with enzyme adsorbed on it was suspended in the buffer pH 6.0 containing ImM 1,3-Propylenediamine and was incubated for 1 hour at room temperature. After 1 hr 1, 4-Butanediol Diglycidyl Ether (BDDGE) was added in the suspension and was mixed by inverting tube several times. The quantity of 1, 4-Butanediol diglycidyl ether (BDDGE) may range from 0.05 mM to 10 mM. The suspension was then kept at room overnight and then washed with citrate phosphate buffer pH 4.0 The sepharose was then suspended in phosphate buffer pH 6.0 containing 1 mM Glycyl-glycine and incubated for 6 hrs at room temperature. After this treatment for blocking free oxirane groups, the suspension was again washed with 50 mM Citrate phosphate buffer pH 4.0. Final activity of the immobilized laccase preparation was 10,000 IUgm ^"1 .

Example 11

Use of Immobilized laccase for removal of phenolics from must and wine.

Use of immobilized laccase from Arthrographis strain for clarification of must and wine

Must was prepared by mashing locally available grapes. About 100 ml clarified must was obtained by centrifugation at 10,000Xg for lOminutes. The pH of the must was about 3.5. 2000 units of laccase enzyme immobilized on Q-sepharose as per method given in example 10 was added to 30 ml must and was kept at 15 C for 48 hrs. The must was then spinned at 1 OOOXg for 2 minutes for recovering the immobilized enzyme and then at 10,000rpm for 10 min for precipitation of phenolics formed. Initial and residual phenolics in the must were estimated by Denis method using Tannic acid as standard. The laccase treatment resulted in 5.8 fold reduction in phenolics content. The grape must of locally available grapes was allowed to ferment using its own flora. The fermented wine was clarified by spinning at 1 OOOXg. 2000 units of immobilized laccase was added to wine and was allowed to stand at 15°C for 48 hrs.

The wine was then first filtered through Whatman Number 1 filters paper and then through 0.5-micron filter. Initial and residual phenolics content were determined by Denis method, using tannic acid as model phenolic compound. Reduction in phenolics contents by laccase treatment was 3 fold.

ADVANTAGES OF THE INVENTION

1. Laccase produced using Arthrographis strain is not repressible by glucose or nitrogen sufficiency.

2. Laccase production is under submerged fermentation as well as solid-state culture.

3. Laccase produced is extracellular, with a high yield of 10,000 to 20,000 IU per liter of the culture supernatant and can be easily purified from the culture supernatant. 4. Laccase produced offers broad specificity in dye degradation. The cell free enzyme is capable of degradation of dyes belonging to diverse groups of dyes such as azo dyes, triphenylmethane dyes, reactive dyes, anthroquinone dyes, eurhodin dyes and acridine dyes.

5. Degradation of such diverse groups of dyes by a single extracellular laccase has not been reported so far.

6. The Arthrographis laccase treatment of Denim is capable of creating a heavy faded look on denim when, ABTS is used as a mediator for denim bleaching. This provides an alternative to the use of hypochlorite treatment for creating heavy faded look on denim. References:

1. Gianfreda, L., Xu, F., and Bollag, J-M. Laccases: A useful group of oxidoreductive enzymes. 1999; Bioremediat. J; 3:125.

2. Baldrian, P. Fungal laccases occurrence and properties. 2006; FEMS Microbiol. Rev. 30:215-242.

3. Couto, S. R., and Harrera, J.L.T. Industrial and biotechnological applications of Laccases: a review. 2006; Biotechnol. Adv. 24:500-13.

4. Archibald, F.S, Bourbonnais, R., Jurasek, L., Paice, M.G., Reid, I.D. Kraft pulp bleaching and delignification by Trametes versicolor. J. Biotechnol.1997. 53:215-36.

5. Minussi, R. C, Pastore, G.M. and Duran, N. Potential applications of laccase in the food industry. 2002; Trends Food Sci Technol 13:205-16.

6. Dec, J., and Bollag, J.-M. Dehalogenation of chlorinated phenols during oxidative coupling. 1994; Environ. Sci.Technol. 28:484-490.

7. Lisdat F., Wollenberger U, Makower A, Hortnagl H, Pfeiffe, D and Scheller, F.W. Catecholamine detection using enzymatic amplification. Biosens. Bioelectron. 1997; 12: 1199-211.

8. Kulys, J., Vidziunaite, R. Amperometric biosensor based on recombinant laccases for phenols determination. 2003; Biosens. Bioelectron. 18, 319-325.

9. Barriere, F., Kavanagh P. Leech, D. A laccase-glucose oxidase biofuel cell prototype operating in a physiological buffer Electrochimica Acta, 2006; 51 :5187-5192

10. Revankar, M.S. and Lele, S.S. Enhanced production of laccase using a new isolate of white rot fungus WR-1, Process Biochem. 2006; 41, pp. 581-588.

11. Bulter, T., Alcalde, M., Sieber, V., Meinhold, P., Schlachtbauer, C, Arnold, F.H.

Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl. Environ. Microbiol. 2003; 69:987-995.

12. Sigoillot, C, Record, E., Belle, V., Robert, J.L., Levasseur, A., Punt, P. J., Van Den Hondel, C.A., Foumel, A., Sigoillot, J. C, Asther, M., Natural and recombinant fungal laccases for paper pulp bleaching. Appl. Microbiol. Biotechnol. 2004; 64:346-352.

aloheimo, M., Valtakari, L., Puranen, T., Kruus, K., Kallio, J., Mantyla, A., Fagerstrom, R., Ojapalo, P., Vehmaanpera, J. 2011; US Patent No. 7,927,849B2 Niku-Paavola, M.-L., Karhunen, E., Salola, P., Raunio, V. Ligninolytic enzymes of the white-rot fungus Phlebia radiata. Biochem. J. 1988; 254, 877- 884.