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Enzyme and Microbial Technology 41 (2007) 794–799

A pH-stable laccase from alkali-tolerant �-proteobacterium JB:Purification, characterization and indigo carmine degradation

Gursharan Singh a, Neena Capalash b, Rashmi Goel a, Prince Sharma a,∗a Department of Microbiology, Panjab University, Chandigarh 160014, India

b Department of Biotechnology, Panjab University, Chandigarh 160014, India

Received 24 January 2007; received in revised form 17 June 2007; accepted 4 July 2007

bstract

�-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified toomogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel elec-rophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu2+-centre.t worked optimally at 55 ◦C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4–10 evenfter 60 days at 4 ◦C. Km and Vmax values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, l-methyl DOPA and guaiacol substratesere determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively

sing guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 ◦C) to anthranilic acid via isatin as determined spectropho-ometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoiccid (91.6%) but not HOBT.

2007 Elsevier Inc. All rights reserved.

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eywords: Laccase; pH-stable; �-Proteobacterium; Indigo; Isatin; Degradation

. Introduction

Laccase (benzenediol oxygen oxidoreductase, EC 1.10.3.2),member of blue multicopper oxidase family, catalyses the

xidation of diphenols and related substances using molecu-ar oxygen as electron acceptor. Laccases act non-specificallyn a broad range of substrates including diphenols, polyphe-ols, different substituted phenols, diamines, aromatic amines,enzenethiols and other xenobiotic compounds [1]. The mostxtensively studied applications of laccases include pulp delig-ification, textile dye bleaching, effluent detoxification, additiono detergents and biopolymer modification [2]. Since their dis-overy more than one century ago in the Japanese tree Rhusernicifera, laccases have been found to be widely distributedmong plants and fungi. There is an increasing evidence now

or the widespread occurrence of laccase and laccase-like activ-ty in bacteria [3], but only a few bacterial laccases have beenurified and characterized to date. The first prokaryotic laccase,

∗ Corresponding author. Tel.: +91 172 2534147; fax: +91 172 2545425.E-mail address: princess@pu.ac.in (P. Sharma).

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141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2007.07.001

eported in soil bacterium Azospirillum lipoferum [4], was a mul-imeric enzyme exhibiting three bands with molecular masses of8.9, 97.8 and 179.3 kDa in SDS-PAGE corresponding to a cat-lytic subunit and one or two larger chains [5]. A heterologouslyxpressed laccase from melanogenic bacterium, Marinomonasediterranea, showed both the tyrosinase and laccase activities

6]. The best-studied bacterial laccase to date is the cotA generoduct (CotA), which is a component of the spore coat of Bacil-us subtilis [7]. The CotA protein which was overexpressed inscherichia coli, had a molecular mass of 65 kDa and exhibitedhigher thermal stability with a half life at 80 ◦C of about 2 h andptimum temperature at 75 ◦C [8]. Some laccase-like enzymesave also been characterized, e.g. EpoA from Streptomycesriseus is a homotrimer stably maintained during migrationn SDS-PAGE [9]. Laccase-like activity was also shown ineptothrix discophora [10] and marine �-proteobacterium SD-1 [11]. Two Mn2+-oxidizing factors (180 and 250 kDa) fromseudomonas strain GB-1 [12] were purified and found to

e associated with laccase-like activity. We have previouslyeported laccase activity from a new non-melanogenic alkali-olerant �-proteobacterium JB [13] whose production increased

arkedly in the presence of copper, dyes and xenobiotics [14].

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G. Singh et al. / Enzyme and Mic

his work reports purification and characterization of this lac-ase and its ability to degrade indigo carmine via isatin pathwayoth in presence and absence of redox mediators at alkalineH. The study indicates the usefulness of bacterial laccase inhe textile finishing industry which has been using acidic fungalaccases and encourages further studies on use in the treatmentf dye effluents.

. Materials and methods

.1. Microorganisms and growth conditions

�-Proteobacterium JB, isolated previously in our laboratory [13], was main-ained as a suspension in 20% glycerol at −70 ◦C and was routinely cultured on

162 medium (g/l): CaSO4·2H2O, 0.4; MgCl2·6H2O, 2.0; nitrilotriacetic acid,.0; 0.01 M ferric citrate solution, 5 ml; micronutrient solution (g/L: H2SO4,.5 ml; MnSO4H2O, 2.28; ZnSO47·H2O, 0.5; H3BO3, 0.5; CuSO4·5H2O, 0.025;a2MO4·2H2O, 0.025; COCl2·6H2O, 0.045), 10 ml; yeast extract, 2; trypton, 2

15]. One millilitre of inoculum (overnight culture) was used to inoculate 100 mlf M162 medium, incubated at 37 ◦C and 150 rpm for 24 h. Culture supernatantas obtained by centrifugation at 10,000 × g, 4 ◦C for 10 min and used as crude

xtracellular enzyme. Cells obtained were washed twice and resuspended inml of 0.1 M phosphate buffer (pH 6.5). The cells were disrupted by ultrasoni-ation using a Braun Labsonic (four to five bursts of 1 min each at 100% power).ell extract, obtained as supernatant after centrifugation at 13,000 × g, 4 ◦C for0 min, was used as crude intracellular enzyme. Trametes hirsuta was obtainedrom Dr. G.M. Gubitz (Graz University of Technology, Austria), and maintainedn PDA slants at 4 ◦C. Laccase from this organism, was prepared and purifieds described [16].

.2. Enzyme assays and protein determination

Bacterial laccase activity was determined using 2 mM guaiacol as substrate,t 55 ◦C in 0.1 M phosphate buffer (pH 6.5). The change in absorbanceue to oxidation of guaiacol in the reaction mixture was monitored at65 nm (ε = 48,000 M−1 cm−1) for 10 min of incubation. Laccase assayor T. hirsuta was performed at pH 5.0 (0.1 M acetate buffer) and 30 ◦Csing 2 mM guaiacol as substrate. Enzyme units were expressed in nkatnmol of substrate converted/s/ml of enzyme). Spectrophotometric measure-ents of substrate oxidation by the purified enzymes were obtained at the

ptimal pH and temperature using 4 ml reaction mixtures containing testubstrates, viz. syringaldazine (ε = 50,000 M−1 cm−1, λmax = 525 nm), catecholε = 26,000 M−1 cm−1, λmax = 450 nm), pyrogallol (ε = 35,000 M−1 cm−1,

max = 450 nm), p-phenylenediamine (ε = 14,685 M−1 cm−1, λmax = 450 nm),uaiacol (ε = 48,000 M−1 cm−1 λmax = 465 nm) and l-methyl DOPAε = 32,000 M−1 cm−1, λmax = 475 nm). Molar absorption coefficients (ε)ere determined by measuring absorption of substrates after their completexidation by bacterial laccase. Protein contents were estimated by Lowry’ sethod [17] and the specific activities were expressed as nkat/mg of protein.ll experiments were carried out in triplicates at least. The results presented

re mean values. Standard deviation was 1.3–5.3% ± all are average values.

.3. Purification and characterization of laccase

Ammonium sulphate saturation was standardized by using different cuts0–40, 40–60 and 60–80%) of ammonium sulphate. Proteins were precipitatedrom the supernatant by addition of ammonium sulphate and centrifugation ofalted-out proteins at 4 ◦C, 10,000 × g for 20 min. The precipitate was resus-ended in 0.1 M phosphate buffer (pH 6.5) and dialysed at 4 ◦C against theame buffer. The enzyme (16.5 ml) was applied to an anion-exchange DEAE-

epharose (Amersham Pharmacia Biotech, Freiburg, Germany) column (bedolume, 26.7 ml), equilibrated with 50 mM Tris–HCl buffer (pH 8.0). The pro-eins were eluted by KCl gradient (0–2 M in equilibrating buffer) at a flow rate ofml min−1 and 4 ml fraction size. Fractions with laccase activity were pooled,esalted and concentrated by ultrafiltration, using10 kDa cut off membrane

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Technology 41 (2007) 794–799 795

Amicon, Millipore, USA). The concentrated proteins (400 �l) were subjectedo 10% preparative non-denaturing polyacrylamide gel electrophoresis (Miniel electrophresis Bio-Rad, Germany) at 100 V for 2 h and laccase was elutedrom the gel slice by immersing in elution buffer (50 mM Tris–HCl, pH 8.0nd 10% glycerol) overnight at 4 ◦C. Protein purification and molecular weightere analysed by silver-stained, native and SDS-PAGE [18]. Molecular weightf native protein was also estimated by elution through Sephadex-G-200 usingtandard proteins (BSA, 66 kDa; phosphorylase b, 97 kDa; aldolase, 152 kDa).accase was zymographed by incubating the native gel in 2 mM guaiacol pre-ared in 0.1 M phosphate buffer (pH 6.5) at 55 ◦C for 10 min. Michaelis–Mentonarameters (Km and Vmax) for purified laccase (10 �g ml−1) were determinedt optimal temperature (55 ◦C) and pH for different substrates. Initial velocityas measured in glass cuvettes with 1 cm path lengths. Reactions were initiatedy adding laccase. Initial rates were calculated from the linear portion of therogress curve [18]. Inhibition constants (Ki) and type of inhibtion were anal-sed by preincubation of inhibitors (0–2 mM) with purified laccase (10 �g ml−1)or 10 min at 55 ◦C before adding the substrate. Thereafter reaction mixture wasept for another 10 min.

The UV–vis (200–700 nm) absorption spectrum of the purified laccase from-proteobacterium JB was determined at wavelengths between 700 and 200 nmt room temperature in 0.1 M phosphate buffer (pH 6.5) using a UV/visualpectrophotometer (Perkin–Elmer, Model-�35).

.4. Application of purified laccase to degrade indigo carmine

Purified laccase from �-proteobacterium JB was evaluated for indigoarmine degradation by UV/visual scanning at 200–700 nm. Indigo carminet 10 �M concentration was degraded using 34 U of bacterial or fungal laccasest optimal temperatures of 55 and 30 ◦C and pH 6–10.6 and 5.0, respectively.he dye and its degradation products were subjected to HPLC analysis on aARIAN HPLC system (VISITA 5560) consisting of a system controller andmodel VARIAN 9300 auto injector connected to JASCO UV 975 intelli-

ent UV–vis detector set at 608 nm (Full scale deflection, 0–1 V). Separationf sample was achieved on RP18 techsphere octadecyl silane (ODS) column4.0 mm × 250 mm). The operating conditions used for analysis were: injectionolume, 1 ml; mobile phase, acetonitrile–water gradient; flow rate, 1 ml/min.ffect of various mediators (at 0.5 mM) and their combinations was also seenn the degradation of indigo carmine.

. Results and discussion

.1. Growth and enzyme production

The bacterium attained maximum growth after 8 h of incu-ation (Fig. 1). The extracellular enzyme was detectable after2–14 h of the onset of growth. The intracellular activity was half10 nkat) in case of buffered (50 mM) medium when comparedith the intracellular activity produced in unbuffered medium

fter 16 h of growth. pH of medium showed significant role inaccase production by �-proteobacterium JB. The organism pro-uced maximum laccase only when pH shifted from 7 to ∼8 innbuffered medium. This shift in pH of medium may be dueo release of alkali compounds by organism itself. Since maxi-

um laccase yield was reported at pH 10 in our previous work13], alkaline conditions seem to favour enzyme production.ig. 1 shows that there was delayed growth and less enzymeroduction in buffered (0.05 M (pH 7.0) buffer) medium. Whenedium was buffered again with 0.1 M of phosphate buffer there

as no shift of pH and no laccase production and much delayedrowth of bacterium. All reported bacterial laccases are intra-ellular [4,6] except for the laccase in spores of B. subtilis [7].-Proteobacterium JB too produced only intracellular laccase.

796 G. Singh et al. / Enzyme and Microbial Technology 41 (2007) 794–799

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ig. 1. Profiles of growth and laccase production by �-proteobacterium JB innbuffered and buffered (0.05 and 0.1 M, phosphate buffer pH 7.0) medium.

his intracellular laccase became extracellular on autolysis ofells after ∼16 h of incubation as shown in Fig. 1. Purified intra-ellular and extracellular laccase from �-proteobacterium JB hadame molecular weight (∼120 kDa) and bands of laccase weret the same position in SDS-PAGE analysis (data not shown).here was no isozyme formation (as revealed by zymogramictudy of laccase) as this organism was protease negative. Theseesults show that �-proteobacterium JB produced only one typef laccase.

.2. Purification of laccase

The laccase activity in the culture supernatant (from lysed

ells) obtained after 72 h of growth was purified (Table 1).mmonium sulphate saturated (40–60% saturation showedaximum laccase activity (>60%)) and dialysed protein was

pplied to DEAE-sepharose anion exchange column. The lac-

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urification step Volume (ml) Activity (nkat) Total protein (m

rude extract 1050 8264 126NH4)2SO4 precipitation (60%) 16.5 6690 33EAE sepharose 16 2722 8reparative PAGE 13.2 747 0.528

a Guaiacol was used as a substrate in the whole process of purification.

ig. 2. Silver-stained SDS-PAGE. Lane B, purified laccase; lane C, molecularize markers; lane A, guaiacol-stained zymograph of laccase.

ase fraction was eluted at 0.8–1.2 M KCl concentration showingtrong binding. Thereafter, several preparatory native PAGE gelsere run. Each gel was loaded with 400 �l of enzyme and the

accase was eluted. Enzyme kinetics with purified laccase wereossible due to high specific activity of homogeneous laccase.he purified laccase (Fig. 2) obtained exhibited specific activityf 1414.7 nkat mg−1 of protein and the purification factor was1.5-fold, which corresponded to a final yield of 9%.

.3. Characterization of the purified laccase

The purified laccase produced one band on SDS-PAGE gelith a molecular mass of 120 kDa (Fig. 2). Elution throughephadex-G-200 also showed the molecular weight ≈125 kDa,howing that laccase protein is monomeric and is differentrom molecular weights reported for intracellular laccases of

. subtilis (65 kDa) [8], S. griseus (100 kDa) [9] and S. cyaneus

75 kDa) [19]. Multimeric laccase has also been reported in A.ipoferum (48.9, 97.8, 179.3 kDa) [5]. The low molecular weightxtracellular fungal laccases have been reported for Volvariella

g) Specific activity (nkat mg−1 protein) Yield (%) Purification (fold)

65.5 100 1202 81 3340 33 5.2

1414.7 9 21.5

G. Singh et al. / Enzyme and Microbial

Table 2Kinetic constants of purified laccase at 55 ◦C, and pH optima of varioussubstrates

Substrates Optimum pH Km (mM) Vmax (mM min−1 mg−1)

Syringaldazine 6.0 0.01 909Catechol 6.0 0.055 100Pyrogallol 6.5 0.04 71.43p-Phenylenediamine 7.2 0.28 256.4Guaiacol 6.5 0.58 111.1l

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bha1 or blue copper atom. Type 2 copper exhibits weak visibleabsorbance and Type 3 copper has two copper centers and isresponsible for a shoulder at around 330 nm. The laccase from�-proteobacterium JB showed no peak at 330 nm.

Table 3Ki for the inhibitors of purified laccase at 55 ◦C and pH 6.5

Inhibitor Ki (mM)

Sodium azide 0.18

-Methyl DOPA 7.0 0.04 111.1

olvacea (58 kDa) [20], Marasmius quercophilus (63 kDa) [21]ompared to the intracellular bacterial laccases which have higholecular weight.The optimum temperature for the purified laccase activity

as 55 ◦C. Half life (t1/2) of laccase was 120 and 30 min at 55nd 60 ◦C, respectively at pH 6.5. Optimum pH for different sub-trates are shown in Table 2. Laccase was stable (>80%) in theH range 4–9 and >70% at pH 3 and 10.6 even after 48 h of incu-ation at 37 ◦C, at respective pH. The enzyme was 100, 60 and9% stable at pH 9.0 (Tris–HCl, 0.1 M), 10.6 (glycine–NaOH,.1 M) and 4.0 (citrate, 0.1 M), respectively after 60 days at 4 ◦C.ris–HCl was the best buffer for activity and stability. Stabilityf the enzyme can be because the organism, did not producerotease.

Activity was enhanced in the presence of chloridealts of 1 mM Na+(110.26%), Co2+(105.60%), Ca2+(108.0%),

g2+(107.50%) and Mn2+(111.85%) but decreased in theresence of Zn2+(87.80%), Cd2+(23.30%) and Ag+(19.78%).uSO4 at 10–100 mM increased the activity to 150% andad no effect from 1 to 10 mM. NaCl (10 mM–1 M) had noffect on activity indicating the potential of the enzyme toe used in industry particularly in kraft pulp biobleachinghere excess amount of chlorine is used for increasing therightness of the pulp. Laccase from T. hirsuta retained 50%f its activity at 50 mM NaCl [22] and IC50 range between.4 and 600 mM Cl− for fungal laccases has been reported23].

The kinetic constants of the purified laccase were determinednder the optimal assay conditions. The Km and Vmax for dif-erent substrates are shown in Table 2. Syringaldazine seemso be the best substrate for this laccase, as it showed maximummax and least Km. Syringaldazine is a non-autooxidizable com-ound which does not give a reaction with tyrosinase, lignineroxidase or with hydrogen peroxidase alone and has beenonsidered to be uniquely a laccase substrate [24]. Laccaserom Chaetomium thermophilum has been reported to exhibitKm of 0.034 mM and Vmax of 4.1 �M O2 min−1 [25] and that

rom Magnaporthe grisea, a Km of 0.118 mM, using syringal-azine [26]. For Cot A laccase from B. subtilis, syringaldazinead a Km of 26 ± 2 �M and Vmax of 4 ± 1 �mol min−1 mg−1

f protein. Guaiacol was one of the poor substrates of fungalaccases with Km values ranging from 0.4 mM for C. ther-

ophilum and 0.917 mM for Lentinula edodes laccase [27].he affinity of laccase from �-proteobacterium JB, for otherubstrates tested, i.e. l-methyl DOPA, catechol, pyrogallol and

SSC

Technology 41 (2007) 794–799 797

-phenylenediamine fell in decreasing order, and in betweenhat for syringaldazine and guaiacol. Fungal laccases too showimilar patteren but with the affinity varying from one specieso the other [25–27]. Sodium azide, sodium diethlydithiocar-amate, sodium thioglycollate and cysteine-hydrochloride allnhibited laccase non-competitively indicating that the substratend inhibitor molecules bind simultaneously to the enzymeolecule at different binding sites, decreasing the turnover rather

han diminishing the proportion of enzyme molecules that areound to the substrate. Hundred percentage inhibition of lac-ase activity was observed in the presence of 1.0 mM sodiumzide, 3.5 mM sodium diethlydithiocarbamate or sodium thio-lycollate and 1.0 mM cysteine hydrochloride. Ki values arehown in Table 3. Detailed studies to determine the patternf inhibition have not been carried out previously for laccases,specially those of bacterial origin. Inhibition of laccases fromood rotting fungi (family Coprinaceae) has been determined

o be competitive in case of sodium azide and noncompeti-ive for DEDTC (N,N-diethyldithiocarbamic acid) and EGTA28]. Laccase from A. lipoferum was affected by metal chela-ors such as EDTA and sodium azide and more specificallyy copper chelators such as diethyldithiocarbamate [4]. Sim-lar results were obtained with �-proteobacterium JB laccasehich suggests that copper is necessary for its enzyme activ-

ty. Difference in kinetic and inhibition constants of purifiedaccase from �-proteobacterium JB can be due to the degreef specificity and affinity of enzyme for various substrates andnhibitors. Laccases A and B from Trametes sp. AH28-2 showedemarkably different kinetic constants for various laccase sub-trates [29]. Xu et al. [30] reported that catalytic properties ofaccase from the fungus Myceliophthora thermophilica coulde appreciably changed by site directed mutagenesis. In a tripleutant, catalytic ability of enzyme as well as inhibiton werearkedly altered. Properties of this laccase could be changed,

ither by changes in their amino acid sequences or in com-osition of the carbohydrate moiety. The use of compoundsike dithiotheritrol, diethyldithiocarbamic acid and sodium azides inhibitors of phenoloxidases lies in their effect seen onther metallo-enzymes rather than their being laccase specific25,31].

The UV–vis spectrum of the purified laccase was typical oflue oxidases with a peak at 590 nm. Laccase from B. subtilisad four copper binding sites [7] and produced spectra with anbsorption maximum at 605 nm which corresponded to a Type

odium diethyldithiocarbamate 0.163odium thioglycolate 3.15ysteine-hydrochloride 0.0205

798 G. Singh et al. / Enzyme and Microbial Technology 41 (2007) 794–799

F e as measured spectroscopically. Spectra were recorded at 0 h and then every 4 h fora e) and upward (increase) arrows showing change in absorbance with time.

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ig. 3. Time course of indigo carmine degradation by �-proteobacterium laccastotal time of 24 h. Inset shows peak formation at 235 nm. Downward (decreas

.4. Application of purified laccase to degrade indigoarmine

Bacterial laccase degraded indigo carmine optimally at tem-erature 55 ◦C and pH 9 in 0.1 M Tris–HCl buffer. Purifiedungal laccases from T. hirsuta (pH 5) [21] and Sclerotiumolfsii (pH 5.5) [16] worked best on indigo carmine in acidicH. The Km and Vmax for indigo carmine were 0.0096nd 96.4 mM min−1 mg−1, respectively. The UV/visual scan200–700 nm) showed a clear decline of peak at 608 nm (λmaxf indigo carmine) in case of reaction containing the enzymeFig. 3) implying that indigo carmine was degraded by thenzyme as boiled enzyme showed no such peak decline. Also, aew peak was seen at 235 nm (Fig. 3, inset), which is the λmax forsatin-5 sulfonic acid, an intermediate formed in indigo carmineegradation [22]. The dye and its degradation products werenalysed by HPLC at 608 nm. Different patterns were obtainedor standard solution of indigo carmine, fungal enzyme and bac-erial enzyme-treated indigo carmine. Untreated indigo carmine,nthranilic acid and isatin-5 sulfonic acid gave single peaks withetention times of 1.20, 1.28 and 1.41 s. Peaks with retentionimes corresponding to both isatin (1.4 s) and anthranilic acid1.28 s) were seen in samples treated with bacterial and fungalnzyme. The amount of anthranilic acid formed in case of fun-al enzyme was more (98.3%) as compared to bacterial (56.4%)accase owing to higher rate of reaction of fungal laccases.he present study compares the degradation pathway of indigoarmine by laccase from �-proteobacterium JB with T. hirsutaaccase, as degradation pathway for the latter is well defined [16].omparison of degraded products was also important because noacterial laccase has been reported for such degradation. Medi-tors like syringaldehyde, vanillin, p-hydroxybenzoic acid andOBT were used alone and in combinations to enhance theegradation. Syringaldehyde (571%), p-hydroxybenzoic acid91.6%) and vanillin (156%) increased the degradation. HOBT

ad no effect. The combination of syringaldehyde with vanillin,-hydroxybenzoic acid or HOBT was no better than syringalde-yde alone (Fig. 4). Syringaldehyde was probably the preferredediator when used in combination. It was seen that bacterial

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ig. 4. Effect of mediators (0.5 mM) and their combinations on the decolouri-ation of indigo carmine at pH 9.0, 55 ◦C.

accase degraded indigo carmine the same way as fungal laccase.lectron donating methyl and methoxy substituents seemed tonhance laccase activity, while electron withdrawing chloro,uoro and nitro substituents are known to inhibit oxidation ofzophenols and other substituted phenols and phenol analogs byungal laccases [32]. On the other hand, extent to which redoxediators enhanced laccase-catalysed reaction was dependent

n the nature of dye [32]. In this study, potential of several media-ors has been evaluated, with the aim of identifying a cheaper and

ore efficient mediator for indigo carmine degradation by bacte-ial laccase. In 2001, Zytex Pvt. Ltd. (Mumbai), India developedformulation based on ‘LMS’ capable of degradation of indigoarmine in a very specific way [33].

. Conclusion

A new bacterial laccase has been purified and character-zed. Evidence for laccase activity was obtained from substratepecificity data and kinetic, biochemical and spectral character-stics combined with the ability of enzyme to degrade indigo

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armine in presence and absence of mediators. Lignin-derivedhenolic compound, syringaldehyde represented ecofriendlylternative to synthetic (including NOH type) mediators foraccase-catalysed degradation of indigo carmine.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.enzmictec.2007.07.001.

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