Decolorization of Azo, Triphenylmethaneand Anthraquinone Dyes by Laccase of a NewlyIsolated Armillaria sp. F022

Tony Hadibarata & Abdull Rahim Mohd Yusoff &Azmi Aris & Salmiati & Topik Hidayat &Risky Ayu Kristanti

Received: 23 June 2011 /Accepted: 5 August 2011# Springer Science+Business Media B.V. 2011

Abstract A newly isolated white-rot fungus, Armillariasp. strain F022, was isolated from the decayed wood in atropical rain forest. Strain F022 was capable ofdecolorizing a variety of synthetic dyes, including azo,triphenylmethane, and anthraquinone dyes, with anoptimal efficiency of decolorization obtained when dyesadded after 96 h of culture, with the exception of

Brilliant Green. All of the tested dyes were decolorizedby the purified laccase in the absence of any redoxmediators, but only a few were completely removed,while others were not completely removed even whendecolorization time was increased. The laccase, withpossible contributions from unknown enzymes, played arole in the decolorization process carried out byArmillaria sp. F022 cultures, and this biosorptioncontributed a negligible part to the decolorization bycultures. The effect of dye to fungal growth was alsoinvestigated. When dyes were added at 0 h of culture,the maximum dry mycelium weight (DMW) values inthe medium containing Brilliant Green were 1/6 of thatachieved by the control group. For other dyes, the DMWwas similar with control. The toxic tolerance of dye forthe cell beads was excellent at least up to a concentrationof 500 mg/l. The optimum conditions for decolorizationof three synthetic dyes are at pH 4 and 40°C.

Keywords Armillaria sp. F022 . Brilliant Green .

Laccase activity. Microbial decolorization .

Reactive Black 5 . Remazol Brilliant Blue R

1 Introduction

The textile industry, by far the most avid user ofsynthetic dyes, is in need of ecologically efficient

Water Air Soil PollutDOI 10.1007/s11270-011-0922-6

T. Hadibarata (*) :A. R. M. Yusoff :A. Aris : SalmiatiInstitute of Environmental and Water Research Management,Universiti Teknologi Malaysia,81310 Skudai, Johor, Malaysiae-mail: hadibarata@utm.my

T. HidayatDepartment of Biological Science,Faculty of Bioscience and Bioengineering,Universiti Teknologi Malaysia,81310 Skudai, Johor, Malaysia

T. HidayatDepartment of Biology Education,Faculty of Mathematic and Natural Science,University of Education (UPI) Bandung,Jalan Dr. Setiabudhi No. 229,Bandung 40154, Indonesia

R. A. KristantiDepartment of Research, Interdisciplinary Graduate Schoolof Medicine and Engineering, University of Yamanashi,4-3-11, Takeda,Kofu, Yamanashi 400-8511, Japan

solutions for its colored effluents. Effluents from textileindustries are a complex mixture of many pollutingsubstances such as heavy metals, organochlorine-basedpesticides, pigments and dyes. The wastewater contain-ing dyes are highly colored which can cause waterpollution (Zouari-Mechichi et al. 2006). Based on thechemical structure of the chromophoric group, dyes areclassified as azo, triphenylmethane, anthraquinone,heterocyclic, and polymeric dyes, among which theversatile azo and triphenylmethane dyes account formost textile dyestuffs produced. Because these dyes aremutagenic, carcinogenic, and also cannot be completelyremoved by conventional wastewater treatment sys-tems, before disposal and discharge of dye-containingeffluents, they need to be treated to reduce theirlevels of toxicity, which will minimize their pollutionimpact. White-rot basidiomycetes are well known fortheir natural ability to decompose lignin, a highlycomplex non-phenolic polymer, which also givesthem the potential capacity to degrade a wide varietyof complex organopollutants. This degradative abilityhas opened up new prospects for the development ofbiotechnological processes aimed at the degradationof complex polymers such as xenobiotics for effluentdecolorization and biobleaching of the lignin in kraftpulp (Lopez et al. 2002).

One promising strategy is the use of microbesincluding white-rot fungal and bacterial strains thatpossess the ability to decolorize synthetic dyes(Ferreira et al. 2000). Microbial decolorization anddegradation are an environmentally friendly and cost-competitive alternative to physico-chemical decom-position processes for the treatment of industrialeffluents (Verma and Madamwar 2003). There is aconsiderable number of recent reports on decoloriza-tion and degradation of individual synthetic dyes bywhite-rot fungi (Asgher et al. 2006; Hamedaani et al.2007; Tavaker et al. 2006).

The biodegradation ability of the white-rot fungi isassumed to be associated with the production oflignolytic enzymes such as lignin peroxidase andlaccase (Couto and Herrera 2006; Ghodake et al.2008). Laccase (EC 1.10.3.2, benzenediol/oxygenoxidoreductase) is an multicopper oxidase enzymesecreted by the most of the lignin degrading white-rotbasidiomycetes, and it has been reported as anessential enzyme for lignin degradation in fungiwithout peroxidases (Kahraman and Gurdal 2002). Itcatalyzes the oxidation of a broad range of organic

and inorganic substrates, including diphenols, poly-phenols, diamines, aromatic amines, and ascorbate viaa one-electron transfer mechanism but its substraterange has been extended to non-phenolic compoundsin the presence of low molecular mass compoundsacting as mediators (Eggert et al. 1998; Thurston1994). Most of the studies have been carried out withlaccases from eukaryotes, principally with enzymessecreted by basidiomycetes being their distribution inprokaryotes more recently reported (Claus 2003).

Previously, a white-rot fungus F022 was success-fully isolated from a tropical rain forest. The fungusshowed ligninolytic activity, and the potential todegrade synthetic dyes. In this study, therefore weinvestigated the ability of strain F022 to performbiological decolorization of azo, triphenylmethane,anthraquinone. Moreover, because there is no detailedpublished report on the effect of the time-point of dyeaddition on decolorization by fungi, the present studywas undertaken to explore the effect of dye addition.We also investigated the importance of laccases of thestrain F022 for dye decolorization including optimumcondition of pH, temperature and laccase activity fordecolorization.

2 Materials and Methods

2.1 Dye and Chemicals

2,2-Azino-di-(3-ethylbenzthiazoline sulfonic acid)(ABTS) was purchased from Tokyo Chemical Indus-try (Tokyo, Japan). Azo dyes (Reactive Black 5[RB5]), triphenylmethane dyes (Brilliant Green) aswell as anthraquinonic dye (Remazol Brilliant Blue R[RBBR]) were of analytical grade. The structure of alldyes is shown in Table 1. All other chemicals werepurchased from Wako Pure Chemical Industry(Osaka, Japan) at the highest purity available.

2.2 Microorganism and Culture Maintenance

White-rot F02 was maintained on malt extracts agar(ME) containing (in g/l): glucose (20), malt extracts(20), polypeptone (2), and agar (20). The growthmedium for production of laccase and decolorizationof dyes was prepared in ME with glucose as a carbonsource. An inoculum of white-rot F022 for liquidculture was prepared as follows: three agar plugs

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(5 mm in diameter) punched from the periphery of a7-day agar plate were cultivated in a 250-ml flaskcontaining 100 ml culture solution, and the flask wasincubated for 7–8 days at 25°C in a shaking incubator(120 rpm).

2.3 Morphologic and Molecular Characterization

Micro- and macroscopic observations were performedon Petri dishes with PDA and malt extract agar(MEA), respectively. The optimum temperature andpH of isolates were also determined for both theisolates. Molecular identification of F022 was con-ducted on the basis of variation of 18S rRNA gene.Extracted DNA genome was amplified using univer-sal primers, NS1 and NS8. Component polymerasechain reaction (PCR) included buffer PCR, MgCl2,primers, enzyme Taq polymerase, dNTPs Mix, andDNA template. PCR was performed using thefollowing procedure: 1 cycle at 94°C for 3 min;25 cycles at 94°C for 30 s, 50°C for 30 s, and 72°Cfor 2 min; and ended with 1 cycle at 72°C for10 min. Amplification products were cloned intopGEM-T Easy (Promega) before sending them to1st BASE Laboratory Sdn. Bhd. Malaysia forsequencing. The resulting DNA sequence was read

and edited by BIOEDIT. The sequence was furthercompared with other 18S rRNA gene sequencesobtained from the NCBI GenBank database. Phylo-genetic analysis was conducted based upon neighborjoining method.

2.4 Dry Mycelium Weight of Strain F022

Dry mycelium weight (DMW) of the fungal mass wasobtained by removing the culture solution, which isdone by filtering the contents of each flask throughpre-weighed Whatman no. 1 filter paper. The paperholding the filtered fungal mass then was left to dry toa constant weight at 100°C. DMW was expressed interms of g/l biomass.

2.5 Production, Preparation and Analysis of Laccase

The time course of laccase production by F022 wasstudied in 100 ml nutrient broth at 30°C at staticcondition. Laccase activity (as discussed in thefollowing section) was measured in a crude cellextract of F022 cells which were grown at differenttime intervals. For higher laccase production, 10%inoculum of F022 was inoculated in 3 l nutrientmedium and incubated at 30°C for 12 h. Cells were

Table 1 Structures and maximum visible wavelengths of dyes

Dye max (nm) Structure

Reactive Black 5 (Azo)

Remazol Brilliant Blue R (Anthraquionone)

Brilliant Green (Triphenylmethane)

598

495

625

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collected by centrifugation at 8,000×g for 15 min andsuspended (150 g/l) in a 50 mM sodium phosphatebuffer (pH 7.0) containing 5 g/l lysozyme. Cells werefurther incubated at 37°C for 45 min in a water bathand then disrupted by sonication. This cell-freeextract was solubilized in cholic acid on a magneticstirrer at 4°C for 30 min. The cell lysate obtained wascentrifuged twice at 15,000×g at 4°C for 30 min, andthe clear supernatant is used immediately or storedat −20°C until its use to purify laccase.

The supernatant containing laccase activity 0.04 Uwas heated at 60°C for 10 min and centrifuged at8,000×g for 20 min. The clear supernatant obtainedafter centrifugation was loaded onto a DEAE cellulosefast flow column (15×120 mm). The column waswashed with the same buffer by twice the columnvolume and the enzyme was eluted with a lineargradient of 0–1.0 M NaCl. Fractions containing laccaseactivity were pooled and dialyzed against a 1-mMsodium phosphate buffer (pH 6.0). The dialyzedsample was concentrated (1–2 ml) by ultrafiltrationand loaded on a Bio-gel column (10×500 mm)equilibrated with a 50 mM sodium phosphate buffer(pH 6.0). Protein elution was carried with the samebuffer at 6 ml/h flow rate. Fractions containing laccaseactivity were pooled and stored at −20°C until use.

Laccase activity was determined at 30°C bymeasuring the increase of optical density at 420 nmin a reaction mixture of 2 ml containing 0.66 mMABTS in a 0.1 M acetate buffer (pH 4.9) and 100 μlenzyme (Hatvani and Mecs 2001). One unit ofenzyme activity was defined as a change in absor-bance U mg protein−1 min−1. The protein concentra-tion of each fraction was monitored by absorbance at280 nm or Lowry methods with bovine serumalbumin as a standard (Lowry et al. 1951).

2.6 Decolorization by the Fungus F022

The decolorization of azo, triphenylmethane andanthraquinonic dyes was also evaluated using MEliquid medium. The amounts of Reactive Black 5,Brilliant Green and RBBR in the culture were 100–500 mg/l, respectively. Dyes were added eitherinitially or after 5 days of cultivation. At regularintervals, samples (1 ml) were withdrawn from theflasks and centrifuged at 8,000×g. The supernatantswere analyzed by measuring the decrease in absor-bance at the absorbance maxima (λmax) of each

dye using a UV–visible spectrophotometer. Dyeremoval was determined according to the followingformulation:

Decolorization %ð Þ ¼ 1� C

C0

� �� 100

where C0 is the absorbance of the dye beforedecolorization and C is the absorption of the dye afterdecolorization at each sampling time (Sayan 2006).Cultures containing only fungi but no dye were used ascontrol groups.

2.7 Treatment of Dyes by Purified Laccase

Decolorization of all dyes by purified laccase wasperformed using 3-ml disposable cuvettes with 2 mlfinal reaction volume. The reaction mixture wascomposed of 100 mM acetate buffer pH 4, 200 mg/l dyesand 0.5 U/ml laccase. Decolorization was monitoredin 24-h intervals by scanning the spectrum between400 and 800 nm using a UV–Vis spectrophotometer.All experiments were performed in triplicate, andincubated with shaking (120 rpm) for an appropriatetime. Controls were performed using heat inactivatedenzymes after incubation at 100°C for 10 min.Calculation of decolorization percentage was done asdescribed as above.

3 Results and Discussion

3.1 Isolation and Identification of Fungi

Awhite-rot fungus, isolated from the decayed wood ina tropical rain forest, was designated F022. This strainF022, when grown on ME agar, had a white spore (7–9×6–7 μm), smooth, more or less elliptical, inamy-loid with a prominent apiculus, and has a clampconnection between hyphae. F022 has a sturdy,fibrous stem, with a pendent, thin, white to pale greyring and convex to umbonate, tawny to ochre capwith sparse and fibrous scale. The gills are attached orbeginning to run down the stem; nearly distant;whitish, sometimes bruising or discoloring darker(Laessoe 2002; Pace 1998). Based on these macro-scopic morphological characteristics and the phyloge-netic position of the sample, F022 is classified asbelonging to the genus Armillaria (Fig. 1).

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3.2 Fungal Growth in Liquid Media Containing Dye

Dye toxicity for Armillaria sp. F022 growth wascalculated by growing fungi in liquid media contain-ing different dyes, and then we compared the DMWto a control group growing in liquid media withoutdyes. As shown in Fig. 2, dyes were added at differenttimes during culture in order to evaluate dye effectson fungal growth. When dyes were added at 0 h ofculture, the maximum DMW values in the mediumcontaining Brilliant Green were 1/6 of that achievedby the control group. For other dyes, the DMW wassimilar with control. When dyes were added after 24 hof cultivation, similar DMW values were obtained inexperimental and control media of Armillaria sp.

F022. These results indicated that Brilliant Green hastoxic effects on Armillaria sp. F022.

To determine the maximum dye concentration toler-ated by the cell in a culture, experiments with differentinitial dye concentrations, ranging from 100 to 500mg/l,were performed. Even with the dye concentration ashigh as 500 mg/l, more than 60% of color removal wasobtained at 96 h incubation time except BG. A typicalMonod-type profile was observed as the specificdecolorization rate (rdye) initially increased with theconcentration of RB5, RBBR and Brilliant Green(Fig. 3). Figure 3 also indicates that the toxic toleranceof dye for the cell beads was excellent at least up to aconcentration of 500 mg/l. These results suggest thatcell in this work was highly promising for practical

Fig. 1 Phylogenetic tree ofArmillaria species

0

1

2

3

Black 5Control Reactive RBBR Brilliant Green

Dyes

DM

W (

g/L

)

0h 48h

Fig. 2 Dry myceliumweights (DMW) ofArmillaria sp. F022 in MEliquid medium containingthree synthetic dyes(incubation time: 96 h).The dyes were added intothe culture at 0 and 48 hof cultivation

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applications involving the biodegradation of syntheticdyes at high concentrations (Carliell et al. 1995).

3.3 Treatment of Dyes by Fungal Culture

To investigate the ability of Armillaria sp. F022 todecolorize dye solutions, several of the dyes describedabove were selected and added to fungal cultures. Theculture supernatant was analyzed at 24-h intervals totest total dye absorbance. Armillaria sp. F022 wascapable of decolorizing all dyes tested. With theexception of Brilliant Green, more than 65% of theazo dye RB5 and anthraquinonic dye RBBR weredegraded by Armillaria sp. F022 in 96 h. In contrast,only 30% of triphenylmethane dye Brilliant Green wasremoved in 96 h. When incubated with Armillaria sp.F022, RB5 and RBBR were degraded more than twotimes faster compared to Brilliant Green (Fig. 4).

Previous result showed that anthraquinone dyes weredecolorized by Trametes versicolor laccases, and thatthese dyes can even function as mediator in thedecolorization of azo and indigoid dyes (Wong andYu 1999). In general, the overall complexity of thestructure alone is not an indicator of the difficulty ofdecoloration of a particular dye. An early step in azodye decoloration is the breaking of the azo bond, theease of which has been found to depend on the identity,number, and position of functional groups in thearomatic region and the resulting interactions with theazo bond. Further degradation of the azo dyes involvesaromatic cleavage, which has also been found to bedependent on the identity of the ring substituents withthe presence of phenolic, amino, acetamido, 2-methoxyphenol, or other easily biodegradable func-tional groups resulting in a greater extent of degrada-tion (Paszczynski et al. 1992; Spadaro et al. 1992).

0

20

40

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120

0 100 200 300 400 500 600

Initial dye concentration (mg/L)S

pes

ific

dec

olo

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tio

n

rate

(m

g d

ye/g

cel

l)

RB5 RBBR Brilliant GreenFig. 3 Spesific decolorizationrate of three synthetic dyesusing cell in the culture ofArmillaria sp. F022

0

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0 24 48 72 96

Incubation time (h)

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Reactive Black 5 RBBR Brilliant Green

Fig. 4 Decolorization ofsynthetic dyes by the cultureof Armillaria sp. F022

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A previous study also showed that a biosorptionmechanism might also play an important role in thedecolorization of synthetic dyes by living fungi inaddition to biodegradation (Fu and Viraraghavan2001). This result also similar to a previous study, inwhich addition of Myrothecium verrucaria into a dyesolution showed that more than 50% of the dye wasremoved by absorption in the beginning of cultivation(Mou et al. 1991). Other researchers studied the colorabsorption by T. versicolor mycelia and reported thatthe adsorption accounted for only 5–10% of the totalcolor removal (Benito et al. 1997). Results of thatstudy were similar to our study results, where amaximum of 6.0% of the added dye was found tobind to the mycelia after 96 h of incubation. In thisstudy, therefore, absorption of dyes to mycelia wasnot a significant contributor to dye losses.

3.4 Treatment of Dyes by Purified Laccase

Purified Armillaria sp. F022 laccase (0.5 U/ml) wasable to degrade all of the dyes tested. However, onlytwo dyes, RB5 and RBBR, were completely degradedby laccase of Armillaria sp. F022 in the culture. RB5and RBBR were good substrates for laccase, and weredegraded more than 80% in 96 h (Figs. 5, 6 and 7).From the triphenylmethane dye tested, Brilliant Greenwas not the optimum substrate for laccase, becausethe decolorization rate was less than 40% in 96 h(Fig. 7). From the three classes of dyes tested, RBBR(anthraquinone dye) was the best substrate forArmillaria sp. F022 laccase, as the purified enzyme(0.5 U/ml) was able to decolorize more than 70% ofthis dye in 48 h over a range of concentrationsbetween 100 and 200 mg/l at 25°C, pH 4 (Fig. 6a).The rate of decolorization decreased when dyeconcentration was raised to 500 mg/l, and only 54%was degraded after the end of incubation. Therecolorization efficiency of RBBR increased with theincreasing laccase activity, with a 100% decoloriza-tion of the supplied RBBR (300 mg/l) obtained within

A

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0.5U/mL 1U/mL 1.5U/mL 2U/mL

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3 4 5 6 7

4 15 25 40 70

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tion

(%)

Fig. 5 Effects of dye concentration (a), laccase activity (b), pH(d) and temperature (e) on decolorization of azo dye ReactiveBlack 5 by laccase of Armillaria sp. F022. Incubationcondition: a dye concentration in mg/l, laccase (0.5 U/ml),pH 4, 25°C; b laccase activity in U/ml, dye concentration(300 mg/l), pH 4, 25°C; c dye concentration (300 mg/l), laccase(0.5 U/ml), 25°C; d dye concentration (300 mg/l), laccase(0.5 U/ml), pH 4

�

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72 h incubation at 25°C with 1.5–2 U/ml of laccase(Fig. 6b). The effect of pH on decolorization ofRBBR (300 mg/l) is shown in Fig. 6c. The optimalpH value for dye decolorization was determined to bepH 4, at which pH the decolorization yield exceeded89% after incubation at 25°C for 96 h. Temperaturevariation had a little effect on decolorization of RBBRbelow 70°C, and a maximum decolorization percent-age was observed at 25–40°C after 96 h (Fig. 6d).RB5 was also a good substrate for laccase ofArmillaria sp. F022. The enzyme was able to degradeRB5 more than 80% in 96 h while the concentrationof the dye was increased up to 300 mg/l. At the highconcentration (500 mg/l), the dye was degraded by42% (Fig. 5a). Increasing the enzyme activity to1.5 U/ml was able to degrade the dye totally in 96 h(Fig. 5b). The optimum pH and temperature was thesame as RBBR: at pH 4 and 40°C (Fig. 5c and d).

The effect of dye concentration on decolorizationrate has been rarely reported. A general tendencyobserved is that high dye concentration will causeslower decolorization rate. Each dye molecule con-tains a chromophore such as azo and anthraquinone,and its color disappears only after the chromophorestructure is destroyed. Destroying the chromophorestructure of one dye molecule may require numerousattacks of the enzymes. High dye concentrationimplies less average attacks of enzyme to each dyemolecule, and hence slower color removal rate(Young and Yu 1997). The laccase was stable at 40–50°C but lost 90% of its activity at more than 60°C(Zouari-Mechichi et al. 2006). These results alsoindicate that the rate of laccase-catalyzed decoloriza-tion of the dyes increased with the increase intemperature up to a certain degree above which thedye decolorization decreased or did not take place atall because the rate of enzyme inactivation becamefaster than the enzymatic catalysis rate at hightemperatures (Nyanghong et al. 2002). It is clear thatlaccase with possible contributions by unknownenzymes played a role in the decolorization process

A

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400 mg/L 500 mg/L

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%)

Fig. 6 Effects of dye concentration (a), laccase activity (b), pH(d) and temperature (e) on decolorization of anthraquinone dyeRBBR by the laccase of Armillaria sp. F022. Incubationcondition: a dye concentration in mg/l, laccase (0.5 U/ml),pH 4, 25°C; b laccase activity in U/ml, dye concentration(300 mg/l), pH 4, 25°C; c dye concentration (300 mg/l), laccase(0.5 U/ml), 25°C; d dye concentration (300 mg/l), laccase(0.5 U/ml), pH 4

�

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carried out by Armillaria sp. F022 cultures and thatbiosorption contributed a negligible part to thedecolorization by cultures.

Next, we determined the kinetic parameter, byperforming an assay in 100 mM citrate phosphatebuffer (pH 4) with 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) at room temperature. Typically,ABTS was used as a mediator in the determination oflaccase activity. Next, we determined the steady-statekinetics of Armillaria sp. F022 spores displayinglaccase with ABTS as the substrate at 37°C(Table 2). Kinetic parameters were calculated usingthe Michaelis–Menten equation. The apparent Km

and Vmax values of the displayed enzyme for ABTSwere 325±76 μM and 95±11 μmol/min of mg perspore, respectively.

4 Conclusion

Armillaria sp. F022, a newly isolated white-rotfungus, is capable of decolorizing a variety ofsynthetic dyes, including azo, anthraquinone, andtriphenylmethane dyes, with RBBR and ReactiveBlack 5 dyes being favored. The time-point of dyesupplementation either greatly affected fungal growthor significantly influenced the efficiency of decolor-ization by Armillaria sp. F022. All dyes tested weredecolorized by the purified enzyme. Among the dyesused, RBBR was the best substrate. In contrast,Brilliant Green was not a good substrate for laccase,with only 75% degraded in maximum laccaseconcentration (2 U/ml). Therefore, we suggest that

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%)

Fig. 7 Effects of dye concentration (a), laccase activity (b), pH(d) and temperature (e) on decolorization of triphenylmethanedye Brilliant Green by the laccase of Armillaria sp. F022.Incubation condition: a dye concentration in mg/l, laccase(0.5 U/ml), pH 4, 25°C; b laccase activity in U/ml, dyeconcentration (300 mg/l), pH 4, 25°C; c dye concentration(300 mg/l), laccase (0.5 U/ml), 25°C; d dye concentration(300 mg/l), laccase (0.5 U/ml), pH 4

�

Table 2 Kinetic parameters of Armillaria sp. F022 usingABTS as substrate

Enzyme Km (μM) Vmax

(U [mg/spores])Vmax/Km

(l mg spore−1 min−1)

Laccase onspore

325±76 95±11 0.29

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laccase ofArmillaria sp. F022 is an excellent biologicalapproach in the treatment of colored effluent fromsynthetic dye.

Acknowledgement A part of this research was financiallysupported by Research University Grant from the UniversitiTeknologi Malaysia (Vote No. 00J31), which is gratefullyacknowledged.

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