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CHARACTERIZATION OF LACCASEACTIVITY PRODUCED BY CryptococcusalbidusAnjali Singhal a , Gaurav Choudhary a & Indu Shekhar Thakur aa School of Environmental Sciences, Jawaharlal Nehru University ,New Delhi , IndiaAccepted author version posted online: 31 Jan 2012.Publishedonline: 06 Mar 2012.

To cite this article: Anjali Singhal , Gaurav Choudhary & Indu Shekhar Thakur (2012)CHARACTERIZATION OF LACCASE ACTIVITY PRODUCED BY Cryptococcus albidus , PreparativeBiochemistry and Biotechnology, 42:2, 113-124, DOI: 10.1080/10826068.2011.577882

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CHARACTERIZATION OF LACCASE ACTIVITY PRODUCED BYCryptococcus albidus

Anjali Singhal, Gaurav Choudhary, and Indu Shekhar Thakur

School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India

& This study deals with the characterization of laccase enzyme activity produced byCryptococcus albidus. Industrial wastes like effluent and sludge are complex mixtures of a num-ber of chemicals. These chemicals can interfere with the proper functioning of the enzymes used forbioremediation. Thus, it is important to study the effect of such interfering solvents, detergents,metal chelators, and other chemicals on enzyme activity before industrial applications. Laccaseshowed maximum activity at pH 2.5 and temperature 20–30�C when ABTS was used as a sub-strate. The enzyme followed Michaelis–Menten kinetics: Km was 0.8158mM and Vmax was1527.74U=mg. Laccase showed good thermostability with a half-life of 81min at 25�C, 77minat 35�C, 64min at 45�C, 36min at 55�C, and 21min at 65�C. There was no effect of sodiumdodceyl sulfate (SDS) (0.1–1.0%) and EDTA (0.1–0.5%) on laccase activity. Sodium azide and2-mercaptoethanol showed complete inhibition of laccase activity at 0.1% concentration. At lowerconcentrations of acetone and acetonitrile, laccase was able to maintain its activity. However, theactivity was completely inhibited at a concentration of 50% or above of acetone, methanol,1,4-dioxan, and acetonitrile.

Keywords Cryptococcus albidus, characterization, half-life, kinetics, laccase, solvent

INTRODUCTION

Enzymes are proteins that accelerate (catalyze) biochemical reactions.They are present throughout the biological systems and have a multitudeof functions including growth, metabolism, reproduction, offense, anddefense. The catalytic potential of the enzymes has been widely used byhumans for performing various desired activities. Laccase is one suchenzyme that has been used commercially.

Laccases (EC 1.10.3.2, p-diphenol:dioxygen oxidoreductase) belong tothe blue-copper family of oxidases. They are glycoproteins, which are

Address correspondence to Dr. Anjali Singhal, School of Environmental Sciences, JawaharlalNehru University, New Delhi-110 067, India. E-mail: singhal.anjali@gmail.com; singhal_anju2002@yahoo.co.in

Preparative Biochemistry & Biotechnology, 42:113–124, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6068 print/1532-2297 onlineDOI: 10.1080/10826068.2011.577882

Preparative Biochemistry & Biotechnology, 42:113–124, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6068 print/1532-2297 onlineDOI: 10.1080/10826068.2011.577882

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ubiquitous in nature, reported in fungi, higher plants,[1,2] and bacteria.[3]

Laccase was first described by Yoshida in 1883,[4] and was later character-ized as a metal-containing oxidase by Bertrand.[5] The catalytic cycle of lac-cases involves the reduction of one molecule of oxygen to two molecules ofwater and the concomitant oxidation of four substrate molecules to pro-duce four radicals. These reactive intermediates can then produce dimers,oligomers, and polymers. The physiological functions of laccases, whichcan be extracellular or intracellular, are different in the various organismsbut they all catalyze polymerization or depolymerization processes.[6]

Laccases play diverse physiological roles in nature. They are involved infungal virulence[7] and lignification[8] and delignification of plant cellwalls.[9] Since laccases can act on a wide range of substrates, they are usedfor various commercial purposes. Their uses span from the textile to thepulp and paper industries, and from food applications to bioremediationprocesses.[6] Such applications include the detoxification of industrialeffluents, mostly from the paper and pulp, textile, and petrochemicalindustries, and using laccase as a tool for medical diagnostics and as a bior-emediation agent to clean up herbicides, pesticides, and certain explosivesin soil. Laccases are also used as cleaning agents for certain water purifi-cation systems, as catalysts for the manufacture of anticancer drugs, andeven as ingredients in cosmetics.[10,11,12]

A number of studies have characterized laccases produced by genusCryptococcus to study their role in pathogenecity. Commercial applicationof these laccases has so far been neglected. A previous study, for the firsttime, showed the potential of laccase produced by Cryptococcus albidus forbioremediation of dyes, chemicals, and pulp and paper mill effluent.[12]

Present work focuses on characterization of this enzyme. The enzyme hasbeen characterized in terms of its kinetic properties, stability at differenttemperatures, and effect of various chemical substances like detergents,metal chelators, and solvents on its activity.

EXPERIMENTAL

Microorganism and Laccase Production and Purification

Cryptococcus albidus isolate FIST3 (GenBank database accession numberEU839451) was isolated from the sediments of the Century pulp and paper milland production of laccase was optimized using the Taguchi approach. Theoptimum conditions for laccase production byCryptococcus albidus isolate FIST3are pH 6, CuSO4 2 mM, meat peptone 0.5% w=v, glucose 0.1% w=v, and bagasse1.0% w=v.[12] The enzyme was purified following the method given in the pre-vious study.[12] Characterization was done using purified enzyme.

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Estimation of Laccase and Total Protein

Laccase activity was measured by monitoring the oxidation of 2,20-azinodi-3-ethyl-benzothiazoline-6-sulfuric acid (ABTS) at 436 nm using anultraviolet–visible (UV-VIS) spectrophotometer (Varian Cary-400). Thereaction mixture contained 10 mM ABTS and 85 mM sodium tartrate buffer(pH 3.0), at temperature 30�C, and enzyme.[13] The molar extinctioncoefficient for ABTS2þ ions at 436 is 29,300 M�1cm�1.[14] One unit(IU=mg) of activity was defined as 1 mmol of ABTS oxidized to ABTS2þ ionsper minute by 1 mg enzyme under assay conditions. Estimation of totalprotein was done using the Bradford method and bovine serum albuminwas used as the standard.[15]

Effect of pH and Temperature on Laccase Activity

The effect of variation of pH on laccase activity was studied at pH 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0. The assay conditions were 10 mM ABTS,temperature 30�C, and enzyme (3 U=ml). Sodium tartrate buffer (85 mM)in the pH range 1.5–3.5 and sodium acetate buffer (85 mM) in the pHrange 4.0–5.0 were used. The effect of temperature variation was studiedat 20�C, 25�C, 30�C, 35�C, 40�C, 45�C, 50�C, 55�C, 60�C, and 65�C. Theassay conditions were 10 mM ABTS, 85 mM sodium tartrate (pH 2.5), andenzyme. The pH and temperature at which laccase showed maximumactivity were considered optimum, and reduction in activity was calculatedin comparison to this maximum activity. The experiments were conductedin triplicate and results were subjected to statistical analysis.

Residual activity ¼ 100� Initial activity � Final activityð ÞInitial acitivity

� ��100 ð1Þ

Effect of Substrate (ABTS) Concentration on Laccase Activity

Experiments were done at optimum pH (2.5) and temperature (30�C)using substrate (ABTS) range 1 mM to 10 mM. The experiments were con-ducted in triplicate and results were subjected to statistical analysis.

Calculation of Km and Vmax for Laccase

For calculating Km and Vmax, enzyme assays were done at a range ofsubstrate concentration varying from 1 mM to 5 mM. The specific enzymeactivity was calculated for each substrate concentration and a graph was

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plotted with substrate on the x-axis and specific activity on the y-axis. Curvefitting was done using SigmaPlot 2001 (SPSS, Inc., Chicago, IL). Km andVmax was calculated using nonlinear regression, single rectangularhyperbola. The equation was

y ¼ ax=b þ x ð2Þ

where x is substrate concentration, y is specific activity, a is Vmax, and b isKm. The experiments were conducted in triplicate.

Half-Life of Laccase at Different Temperatures

The enzyme was incubated along with assay buffer at different tempera-tures (25�C, 35�C, 45�C, 55�C, and 65�C) for different durations (0, 10, 20,30, and 40 min). The graph was plotted with duration on the x-axis and logof specific activity on the y-axis. The half-lives were calculated from theequation X¼X0 (1=2)t=g. This equation can also be written as log X¼log X0 – [(log 2)=g]t where X is remaining laccase activity, X0 is initiallaccase activity, t is time, and g is half-life.[16]

Effect of Various Chemicals on Laccase Activity

The effect of different chemicals on the activity of laccases was tested.The chemicals tested were detergent (sodium dodecyl sulfate, 0.1–1.0%),protein denaturants (sodium azide and 2-mercaptoethanol, 0.1–1.0%),metal chelator (ethylenediamine tetraacetic acid [EDTA], 0.1–0.5%) andsolvents (acetone, methanol, 1,4-dioxan, and acetonitrile, 10–50%). Theassay conditions were 3 mM ABTS, temperature 30�C, 85 mM sodium tar-trate buffer (pH 2.5), and enzyme. The reduction in activity was calculatedin comparison with laccase activity without any inhibitor. The experimentswere conducted in triplicate and results were subjected to statistical analysis.

Statistical Analysis

Data from the experiments were transferred to Microsoft Excel 2002spreadsheets (Microsoft Corp., Redmond, WA) and analyzed using statisti-cal functions of SPSS 10.0 and graphical functions of SigmaPlot 2001(SPSS, Inc., Chicago, IL). Residual laccase activity values (%) under differ-ent conditions of pH, temperature, and inhibitors were used in analysis ofvariance (ANOVA) at p� 0.05, that is, 95% confidence limit. FurtherDuncan test was applied between significantly different populations toevaluate the differences among them.[17]

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RESULTS AND DISCUSSION

Effect of pH and Temperature on Laccase Activity

Laccase showed maximum activity at pH 2.5 (Figure 1).Thus, in furtherexperiments the enzyme assay was done at pH 2.5. Any increase or decreasein pH leads to significant (p� 0.05) reduction in laccase activity. No activitywas detected after pH 4.5. Laccases are acidic enzymes with most of themhaving pH optima below 6.0 for ABTS. The laccase have different pHoptima for different substrates.[18] For ABTS the pH optimum is generallylow as compared to other substrates.[19] The laccase produced by Crypto-coccus albidus was very sensitive to pH change. It was observed that therewas a sharp decline in activity while moving toward basic pH as comparedto acidic pH. There was a decline of 7% at pH 2.0 and 14% at pH 3.0 incomparison with pH 2.5. The response of laccases is highly variable, withsome reports stressing laccase being more sensitive in the basic range[20]

and others saying it is more sensitive in the acidic range.[21]

The optimum temperature for laccase activity was 30�C (Figure 2).Thus,in further experiments enzyme assay was done at a temperature of 30�C.After ANOVA it was observed that there was no significant (p� 0.05) differ-ence in the activity of enzyme in the range 20–30�C. The optimum tem-perature for laccase activity varies greatly from 10 to 75�C.[19,22-24] Thelaccase produced by Chalara (syn. Thielaviopsis) paradoxa CH32 showedmaximum activity at 30�C.[25] The laccase of Pleurotus ostreatus strain RK

FIGURE 1 Variations in the activity of Cryptococcus albidus laccase at different pH. Experiments weredone in triplicate. Error bars are standard deviations. Values not followed by the same letter are signifi-cantly different at p � 0.05.

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36 showed a typical bell-shaped curve of activity with variation in tempera-ture from 10 to 70�C.[26] The optimum temperature for Pleurotus ostreatuswas 50�C and any variation, that is, increase or decrease from optimum tem-perature, adversely affected the activity of laccase. However, this was not thecase with Cryptococcus sp., where there was no significant change from 20�Cto 30�C and then the activity declined slowly with increasing temperature.

Effect of ABTS Concentration on Laccase Activity

Laccase showed substrate inhibition for ABTS. Maximum activity(1658.63 U=mg) was observed at 6 mM ABTS concentration (Figure 3).Increasing the ABTS concentration further had a negative effect on laccaseactivity. Similar results have also been reported in the case of laccase pro-duced by Cerrena unicolor, where the substrate inhibition was evident above5 mM.[21] Thus, the ABTS concentration used in enzyme assay for furtherexperiments was reduced from 10 mM to 3 mM.

Calculation of Km and Vmax for Laccase

For calculating Km and Vmax nonlinear regression, a single-rectangularhyperbola was used. This analysis was done using SigmaPlot 2001. Thevalues of Km and Vmax were 0.8158 mM and 1527.74 U=mg, respectively(Figure 4).The Km determined for laccase was quite low. This shows that

FIGURE 2 Variations in the activity of Cryptococcus albidus laccase at different temperatures. Experi-ments were done in triplicate. Error bars are standard deviations. Values not followed by the same letterare significantly different at p � 0.05.

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the laccase had good catalytic properties. Such a low Km for laccase hasbeen reported in the case of Cerrena unicolor.[21] The Km reported forPhanerochaete flavido-alba laccase is also comparable (0.74 mM) though thevalue of Vmax is very low.[22] The value of Km varies greatly with micro-organism. Very low values of Km (0.033 mM) have been reported for laccaseproduced by Trametes trogii.[27]

FIGURE 3 Effect of increasing substrate concentration on activity of Cryptococcus albidus laccase.

FIGURE 4 Kinetic constants, Km and Vmax for Cryptococcus albidus laccase. Experiments were done intriplicate. Error bars are standard deviations.

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Half-Life of Laccase at Different Temperatures

To test the stability of enzyme at different temperatures, half-lives weredetermined at different temperatures. The half-life of laccase at 25�C was81 min, at 35�C was 77 min, at 45�C was 64 min, at 55�C was 36 min, andat 65�C was 21 min (Figure 5).The fitness of the curve is shown by the r2

value written in front of each curve, which has ranged from 0.95 to 0.98.It was observed that the enzyme was quite stable in terms of variation intemperature. There was a reduction of about 40% in the activity of the lac-case with an increase in temperature by 40�C. Laccases in general showgood thermal stability. The thermostable laccase produced by P. sanguineusshowed half-lives of 200 and 170 min at 65 and 75�C.[19] Laccase producedby Trichoderma harzianum WL1 showed half-lives of 1440 min and 30 min attemperatures of 35�C and 65�C.[28] The laccase produced by Trametesversicolor showed a half-life of 24 min at 60�C.[29] This is very close to thehalf-life of laccase showed by Cryptococcus albidus. The half-life of laccaseproduced by Mauginiella sp. was 3 min at 70�C.[30]

Effect of Various Chemicals and Solvents on Laccase Activity

The effect of various chemicals and solvents on the activity of laccasewas determined. Chemicals like 2-mercaptoethanol and sodium azideshowed complete inhibition even at very low concentrations (0.1%). Thesechemicals destroy the structure of the enzyme, due to which it looses its

FIGURE 5 Half-life of laccase of Cryptococcus albidus at different temperatures. The x-axis indicatesduration in min and y-axis indicates log of residual activity.

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catalytic properties.[31,32] A study reported complete loss of activity ofOphiostoma. novo-ulmi CKT-11 laccase at 0.01% sodium azide.[18] Anotherstudy reported the complete loss of Trametes versicolor laccase activity at1 mM sodium azide concentration.[31] There was no effect of sodiumdodceyl sulfate, a detergent in the range (0.1–1.0%) and EDTA, a metalchelator in the range (0.1–0.5%) on laccase activity. Laccase is a copper-containing enzyme. Thus, it is expected that the presence of metal chela-tors like EDTA should inhibit the laccase activity. However, this was notthe case with the laccase of Cryptococcus albidus. Such results have also beenreported in the case of laccase produced by Marasmius quercophilus, wherean EDTA concentration of 10 mM had no effect on laccase activity.[23]

While laccase of Chaetomium thermophilium was inhibited by EDTA at1 mM with 100% inhibition at 25 mM.[33] The behavior of the laccase ofChalara (syn. Thielaviopsis) paradoxa CH32 was different from that of the lac-case of Cryptococcus albidus. Complete inhibition of laccase activity at 0.1%has been reported in case of C. paradoxa CH32.[24] Among all the chemicalstested, the most toxic was sodium azide. It has been reported to be toxic tolaccases of numerous fungi including Agaricus bisporus, Botrytis cinerea,Coriolus hirsutus, Daedalea quercina, and Thelephora terrestris.[34]

In the case of solvents (acetone, methanol, 1,4-dioxan, and acetoni-trile) there was reduction in enzyme activity as solvent concentrationincreased with almost complete inhibition at 50% solvent concentration(Figure 6).Among the solvents tested, methanol had the most detrimental

FIGURE 6 Effect of different solvents on the activity of laccase produced by Cryptococcus albidus. Experi-ments were done in triplicate. Error bars are standard deviations. Within each group (acetone, meth-anol, 1,4-dioxan, and acetonitrile), values not followed by the same letter are significantly different atp � 0.05.

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effect and acetonitrile the least. In a study, the effect of methanol, ethanol,acetone, acetonitrile, and 1,4-dioxan was tested on the laccase produced byMarasmius quercophilus.[35] It showed that 1,4-dioxan had the most detri-mental effect. The laccase of Marasmius quercophilus was more resistant tothe effects of solvents as complete loss of activity was found at 80% solventconcentration.[35] The laccase produced by Chalara (syn. Thielaviopsis) para-doxa CH32 was less resistant to acetonitrile, with 16% residual activity at25% solvent concentration as compared with Cryptococcus albidus laccase.[25]

CONCLUSION

The properties of laccase produced by Cryptococcus albidus isolate FIST3are as follows:

1. The optimum pH was 2.5 and optimum temperature ranged from 20�Cto 30�C. Thus, this enzyme can work in acidic conditions at roomtemperature.

2. The enzyme activity was inhibited by ABTS (substrate) at concentrationshigher than 6 mM.

3. The values of kinetic parameters were Km 0.8158 mM and Vmax

1527.74 U=mg.4. This enzyme was stable at temperatures as high as 65�C for 21 min.5. The enzyme was resistant to the adverse effect of detergents (like SDS)

and metal chelators (like EDTA).6. It can also work in the presence of various industrial solvents (acetone,

methanol, 1,4-dioxan, and acetonotrile).

ACKNOWLEDGMENTS

The authors thank the University Grants Commission (UGC), NewDelhi, India, for providing a Senior Research Fellowship to Anjali Singhaland for financial support. The authors are grateful to Department ofBiotechnology, Government of India, New Delhi, for financial support inthe form of projects.

REFERENCES

1. Thruston, C.F. The Structure and Function of 1-Fungal Laccases. Microbiology 1994, 140, 19–26.2. Levine, W.G. Laccase: A Review. In The Biochemistry of Copper; Peisach, J., Ed.; Academic Press,

New York, 1965; pp. 371–385.3. Diamantidis, G.; Effosse, A.; Potier, P.; Bally, R. Purification and Characterization of the First

Bacterial Laccase in The Rhizospheric Bacterium Azospirillum lipoferum. Soil Biol. Biochem. 2000,32, 919–927.

122 A. Singhal et al.

Dow

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ded

by [

Uni

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ity o

f W

inds

or]

at 1

9:09

20

Aug

ust 2

014

4. Yoshida, H. Chemistry of Lacquer (Urusbi) Part 1. J. Chem. Soc. 1883, 43, 472–486.5. Mayer, A.M.; Staples, R.C. Laccase: New Functions for an Old Enzyme. Phytochemistry 2002, 60,

551–565.6. Riva, S. Laccases: Blue Enzymes For Green Chemistry. Trends Biotechnol. 2006, 24, 219–226.7. Ikeda, R.; Sugita, T.; Jacobson, E.S.; Shinoda, T. Laccase and Melanization in Clinically Important

Cryptococcus Species Other Than Cryptococcus neoformans. J. Clin. Microbiol. 2002, 40, 1214–1218.8. Sterjiades, R.; Dean, J.F.D.; Eriksson, K.E. Laccase From Sycamore Maple (Acer pseudoplatanus)

Polymerizes Monolignols. Plant Physiol. 1992, 99, 1162–1168.9. Agosin, E.; Blanchette, R.A.; Silva, H.; Lapierre, C.; Cease, K.R.; Ibach, R.E.; Abad, A.R.; Muga, P.

Characterization of Palo Podrido, a Natural Process of Delignification in Wood. Appl. Environ.Microbiol. 1990, 56, 65–74.

10. Couto, S.R.; Herrera, J.L.T. Industrial and Biotechnological Applications of Laccases: A Review.Biotechnol. Adv. 2006, 24, 500–513.

11. Singhal, A.; Thakur, I.S. Decolourization and Detoxification of Pulp and Paper Mill Effluent byCryptococcus sp. Biochem. Eng. J. 2009, 46, 21–27.

12. Singhal, A.; Choudhary, G.; Thakur, I.S. Optimization of Growth Media for Enhanced Production ofLaccase by Cryptococcus albidus and Its Application for Bioremediation of Chemicals. Can. J. Civ. Eng.2009, 36, 1253–1264.

13. Niku-Paavola, M.L.; Karhunen, E.; Salola, P.; Raunio, V. Ligninolytic Enzymes of the White RotFungus Phlebia radiata. Biochem. J. 1988, 254, 877–884.

14. Mansur, M.; Arias, M.E.; Copa-Patino, J.L.; Gonzalez, M.F.A.E. The White-Rot Fungus Pleurotusostreatus Secretes Laccase Isozymes With Different Substrate Specificities. Mycologia 2003, 95,1013–1020.

15. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities OfProtein Utilizing the Principle of Protein–Dye Binding. Anal. Biochem. 1976, 72, 248–254.

16. Fukushima, Y.; Kirk, K. Laccase Component of the Ceriporiopsis subvermispora Lignin-DegradingSystem. Appl. Environ. Microbiol. 1995, 61, 872–876.

17. Singhal, A.; Thakur, I.S. Decolourization and Detoxification of Pulp and Paper Mill Effluent byEmericella nidulans var. nidulans. J. Hazard. Mater. 2009, 171, 619–625.

18. Binz, T.; Canevascini, G. Purification and Partial Characterization of the Extracellular Laccase FromOphiostoma novo-ulmi. Curr. Microbiol. 1997, 35, 278–281.

19. Litthauer, D.; Van Vuuren, M.J.; Van Tonder, A.; Wolfaardt, F.W. Purification and Kinetics of aThermostable Laccase From Pycnoporus sanguineus (SCC 108). Enzyme Microb. Technol. 2007, 40,563–568.

20. Jordaan, J.; Pletschke, B.L.; Leukes, W.D. Purification and Partial Characterization of a Thermo-stable Laccase From an Unidentified Basidiomycete. Enzyme Microb. Technol. 2004, 34, 635–641.

21. Gianfreda, L.; Xu, F.; Bollag, J.-M. Laccases: A Useful Group of Oxidoreductive Enzymes. Biorem. J.1999, 3, 1–25.

22. Perez, J.; Martinez, J.; Rubia, D.L.T. Purification and Partial Characterization of a Laccase From TheWhite Rot Fungus Phanerochaete flavido-alba. Appl. Environ. Microbiol. 1996, 62, 4263–4267.

23. Dedeyan, B.; Klonowska, A.; Tagger, S.; Tron, T.; Iacazio, G.; Gil, L.; Petit, J. Biochemical and Mol-ecular Characterization of a Laccase From Marasmius quercophilus. Appl. Environ. Microbiol. 2000, 6,925–929.

24. Robles, A.; Lucas, R.; Alvarez de Cienfuegos, G.; Galvez, A. Phenol-oxidase (Laccase) Activity inStrains of the Hyphomycete Chalara Paradoxa Isolated From Olive Mill Wastewater Disposal Ponds.Enzyme Microb. Technol. 2000, 26, 484–490.

25. Robles, A.; Lucas, R.; Martınez-Canamero, M.; Omar, N.B.; Perez, R.; Galvez, A. Characterisation ofLaccase Activity Produced by the Hyphomycete Chalara (syn. Thielaviopsis) paradoxa CH32. EnzymeMicrob. Technol. 2002, 31, 516–522.

26. Hublik, G.; Schinner, F. Characterization and Immobilization of the Laccase From Pleurotus ostreatusand Its Use for the Continuous Elimination of Phenolic Pollutants. Enzyme Microb. Technol. 2000, 27,330–336.

27. Zouari-Mechichi, H.; Mechichi, T.; Dhouib, A.; Sayadi, S.; Martınez, A.T.; Martınez, M.J. LaccasePurification and Characterization From Trametes trogii Isolated in Tunisia: Decolorization of TextileDyes by the Purified Enzyme. Enzyme Microb. Technol. 2006, 39, 141–148.

Laccase Activity Produced by C. Albidus 123

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28. Sadhasivam, S.; Savitha, S.; Swaminathan, K.; Lin, F.-H. Production, Purification and Characteriza-tion of Mid-Redox Potential Laccase From a Newly Isolated Trichoderma harzianum WL1. ProcessBiochem. 2008, 43, 736–742.

29. Roy, J.J.; Abraham, T.E. Preparation and Characterization of Cross-Linked Enzyme Crystals ofLaccase. J. Mol. Catal. Enzym. 2006, 38, 31–36.

30. Palonen, H.; Saloheimo, M.; Viikari, L.; Kruus, K. Purification, Characterization and SequenceAnalysis of a Laccase From the Ascomycete Mauginiella sp. Enzyme Microb. Technol. 2003, 33, 854–862.

31. Johannes, C.; Majcherczyk, A. Natural Mediators in the Oxidation of Polycyclic Aromatic Hydrocar-bons by Laccase Mediator Systems. Appl. Environ. Microbiol. 2000, 66, 524–528.

32. Innocenti, A.; Hiluo, M.; Slozzafava, A.; Lindfors, M.; Nordlund, H.R.; Kulomoa, M.S.; Prkkita, S.;Supuran, C.T. Carbonic Anhydrase Inhibitors: The Very Weak Inhibitors Dithiothreitol,ß-Mercaptoethanol, Tris(caroxyethyl) Phosphine and Threitol Interfere With the Binding ofSulphonamides to Isozymes II and IX. Bioorg. Med. Chem. Lett. 2008, 18, 1898–1903.

33. Chefetz, B.; Chen, Y.; Hadar, Y. Purification and Characterization of Laccase From Chaetomiumthermophilium and Its Role in Humification. Appl. Environ. Microbiol. 1998, 64, 3175–3179.

34. Baldrian, P. Fungal Laccases—Occurrence and Properties. FEMS Microbiol. Rev. 2006, 30, 215–242.35. Farnet, A.M.; Gil, G.; Ferre, E. Effects of Pollutants on Laccase Activities of Marasmius quercophilus, a

White-Rot Fungus Isolated From a Mediterranean Schlerophyllous Litter. Chemosphere 2008, 70,895–900.

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