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Biochemical Engineering Journal 70 (2013) 106– 114

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Biochemical Engineering Journal

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Regular article

Characterization of optimized production, purification and application of laccasefrom Ganoderma lucidum

Tamilvendan Manavalana, Arulmani Manavalanb,c, Kalaichelvan P. Thangavelua,∗, Klaus Heesed,∗∗

a Centre for Advanced Studies in Botany, University of Madras, Chennai 600 025, Tamil Nadu, Indiab School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singaporec Institute of Advanced Studies, Nanyang Technological University, 60 Nanyang View, Singapore 639673, Singapored Department of Biomedical Engineering, College of Engineering, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 133-791, Republic of Korea

a r t i c l e i n f o

Article history:Received 24 July 2012Received in revised form 12 October 2012Accepted 18 October 2012Available online xxx

Keywords:Ganoderma lucidumLaccaseEthanolTamarind shellAcid Fast Red AMethyl Violet 2BRemazol Yellow G

a b s t r a c t

We show for the first-time Ganoderma lucidum laccase enzyme production using medium containing 3%(v/v) ethanol, which enhanced the enzyme production up to 14.1 folds. A more than 400-folds increasecould be achieved if grown in the presence of the novel lignocellulosic biomass tamarind shell plusethanol (3%, v/v), CuSO4 (0.4 mM) and gallic acid (1 mM). A 38.3 kDa laccase enzyme was purified fromthe initial protein preparation with an overall yield of 32% using Sephadex G-100 and DEAE-cellulose col-umn chromatography. The enzyme was identified through MALDI-TOF/TOF tandem mass spectrometry(MS/MS) as G. lucidum laccase-3. This enzyme exerted its optimal activity at a pH of 5 and a tempera-ture of 55 ◦C with ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as an ideal substrate. Thecatalytic efficiencies (kcat/Km) determined for ABTS and guaiacol were 11.5 × 105 and 3.9 × 105 s−1 M−1,respectively. The G. lucidum laccase decolorized various textile dyes and industrial textile dye effluent upto 90% and 97%, respectively.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2), anoxidase belonging to the group of multi-copper proteins of lowspecificity acting on both o- and p-quinols and often acting alsoon aminophenols and phenylenediamine, is produced mainly bywhite-rot fungi to degrade lignin compounds in order to natu-rally recycle these bio-polymers [1,2]. The distribution of laccaseis widespread among plants [3], fungi [4] and bacteria [5]. Theseenzymes are involved in various physiological functions. In plants,they seem to be involved in lignin synthesis [6], whereas in fungi,they are involved in lignin degradation, pigmentation and patho-genesis [7]. Extensive studies made on fungal laccases have proventheir potentiality [1], which could be tapped for paper-pulp bleach-ing [8,9], synthetic dye decolorization [10], bioremediation [10],biosensors [11] and immunoassays [12].

Worldwide, over 10,000 different dyes and pigments are usedin dyeing and printing industries. The total world colorant pro-duction is estimated to be 800,000 tons per annum and at least

∗ Corresponding author. Tel.: +91 44 22202754; fax: +91 44 22352498.∗∗ Corresponding author. Tel.: +82 2 2220 0397; fax: +82 2 2296 5943.

E-mail addresses: [email protected] (K.P. Thangavelu),[email protected], [email protected] (K. Heese).

10% of the used dyestuffs are discharged as environmental waste[13]. Most of the dyes are very stable because of their high bio-logical and chemical oxygen demand (BOD/COD), pH, presence ofmetal and resistance to light, temperature and microbial attacks,thus making them persistent against most naturally occurringbiodegradable metabolisms [14]. Various reports have mentionedthe direct and indirect toxic effects of those dyes and their metalsthat can lead to the formation of tumors, cancers and allergiesbesides growth inhibition of bacteria, protozoan, algae and plants[15]. The major concern of textile dye effluents is their toxicity andvarious attempts to reduce the toxicity of such effluents by means ofbiological systems has been reported earlier [16,17]. Recently sev-eral reports concluded that microbial systems not only decolorizeand degrade but also detoxify dyes, thus providing an essentialenvironmentally advantage over other chemical decompositionprocedures [16,18,19], though there are some reports also indi-cating that after microbial-mediated dye degradation eventuallysome metabolites could be obtained that are even more toxic thanthe original parent dye compounds [20]. White rot fungi are themost efficient ligninolytic organisms capable of degrading varioustypes of dyes such as azo, heterocyclic, reactive and polymeric dyes[10,21]. Besides, the great industrial interest in laccases is based ontheir potential to catalyze the oxidation of both phenolic and non-phenolic compounds [22] and thus can mineralize a wide range ofsynthetic dyes [23].

1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.bej.2012.10.007


T. Manavalan et al. / Biochemical Engineering Journal 70 (2013) 106– 114 107

However, a major limitation for the extensive industrial appli-cations of laccases is their high cost. Therefore, a good strategyto increase the productivity of the laccase fermentation processwould be the optimization of the fermentation medium and alsoenhancing the laccase activity by using inducers. Moreover, manyaromatic compounds have been widely used to stimulate the pro-duction of laccase. For instance, pyrogallol and ferulic acid werereported to be the most effective inducers for the production oflaccase by Ganoderma lucidum [24] and Pleurotus sajor-caju [23].Gallic acid is another example of inducer, which enhances the pro-duction of laccase from Botrytis cinerea [25]. Unfortunately, mostof these aromatic compounds are either very harmful to humansor quite expensive, precluding their use from industrial applica-tions. Lee et al. [26], Songulashvili et al. [27], and Meza et al. [28]reported that alcohols, e.g. ethanol, could be more suitable andeconomically advantageous to stimulate the laccase production.Several hypotheses on the role of alcohols in laccase productionhave been formulated: (i) increase in membrane permeability andpromotion of protein secretion, (ii) inhibition of melanin formationand consequent increase in phenolic monomers, and (iii) activationof oxidative stress, which could be indirectly responsible for the lac-case induction [26]. However, the real influence of various alcoholson the level of expression of the various laccase genes remained tobe elucidated.

The main objective of the present study was thus to isolate andcharacterize laccase from G. lucidum, a medicinal white-rot basid-iomycete, grown under optimized production conditions – relevantfor economic production of laccase at an industrial level – by mod-ulating the fermentation medium using various alcohols, aromaticinducers, different concentrations of copper sulfate and various lig-nocellulosic biomasses. Accordingly, in the current investigationwe document for the first time that ethanol (3%, v/v) alone is themost effective alcohol-stimulator for G. lucidum laccase productionin the absence of any other potent inducer: it was increased upto 14.1 folds – the highest increase shown so far for any alcoholused. In addition, we compared ethanol with various other ((non-)aromatic) inducers, including gallic acid and copper sulfate, and incombination with various lignocellulosic biomasses, and describeherein their ability to stimulate laccase production. Further, weaimed to purify G. lucidum laccase from submerged culture in orderto characterize the purified enzyme in terms of its activity, stabil-ity and potential toward the decolorization and detoxification ofdifferent textile dyes and effluents.

2. Materials and methods

2.1. Materials

Sephadex G-100, DEAE-cellulose, guaiacol, ABTS(2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), 2,6-dimethoxyphenol, 4-methylcatechol, ferulic acid, hydroquinone,tyrosine, xylidine, veratryl alcohol, vanillin and gallic acid werepurchased from Sigma–Aldrich (St. Louis, MO, USA). Proteinmolecular weight marker was purchased from Bangalore GeneiIndia Pvt. Ltd. (Bangalore, Karnataka, India) and all other chemicalswere analytical grade obtained from M/s. HiMedia LaboratoriesPvt. Ltd. (LBS Marg, Mumbai, India).

2.2. Fungal strain and culture conditions

G. lucidum was isolated from a tamarind trunk wood nearby theIndian Institute of Technology (Chennai, Tamil Nadu, India) andidentified as G. lucidum by amplifying and sequencing the ITS (Inter-nal Transcribed Spacer) DNA using ITS1 and ITS4 primers accordingto previous reports [29] (GenBank accession number FJ982798)

by polymerase chain reaction (PCR) and sequencing as describedpreviously [30]. Stock cultures of G. lucidum (strain ‘TVK1’) weremaintained on the potato dextrose agar (PDA) slant at 4 ◦C in thedark. The mycelium from the slant was transferred to PDA platesand incubated at 30 ◦C for 7 days. Mycelial discs from the peripheralregion of actively growing culture were used as inoculum.

The production medium containing 10 g starch, 2.5 g yeastextract, 1.0 g KH2PO4, 0.5 g MgSO4, 0.05 g Na2HPO4, 0.01 g CaCl2,0.01 g FeSO4, 0.001 g thiamine hydrochloride and 1 ml trace ele-ment solution per liter was used for the laccase enzyme production(basal medium). The stock solution of trace elements contained100 mg B4O7Na2, 10 mg MnSO4, 10 mg (NH4)6Mo7O24, 10 mgZnSO4 and 100 mg CuSO4 in 1 l distilled water (pH 5.5). Laccase pro-duction from G. lucidum was carried out in a lab scale bioreactor,which had a provision for 5 l working volume (Bioreactor, ScigenicsIndia Pvt. Ltd., Chennai, Tamil Nadu, India). Three liter (3000 ml)of the production medium were added to the reactor and steril-ized for 20 min at 121 ◦C at 15 psi. The pre-cultures were preparedin 250-ml Erlenmeyer flasks containing nutrient medium withstarch (10 g l−1) in static conditions at 30 ◦C. Seven-days-old pre-cultures were homogenized before transferring into the mediumin the bioreactor (15 g l−1 wet mycelium (G. lucidum)). The agitatorand flow rate of filter-sterilized air were set at 75 rpm and 3 l h−1,respectively. The internal temperature of the bioreactor was main-tained at 30 ◦C. The basal medium was eventually supplementedwith inducers, such as CuSO4 (e.g. 0.4 mM), gallic acid (e.g. 1 mM)and ethanol (e.g. 3% (v/v)) as described in the text, to enhance thelaccase production.

2.3. G. lucidum laccase’s enzyme activity and protein content

G. lucidum-derived laccase activity was determined by theoxidation of 2,2-azino-bis(3-ethylbenzylthiozoline-6-sulfonate)(ABTS, 1 mM) as described previously [8]. One activity unit (U) isdefined as the amount of laccase transforming 1 �mol/min ABTS toits cation radical (ε436 nm = 29.3 mM−1 cm−1) in the sodium acetatebuffer (100 mM, pH 5) at 30 ◦C estimated by measuring the OD at436 nm using a Beckman Coulter DU 50 spectrophotometer. Theassay mixture contained 100 �l of G. lucidum culture filtrate orpurified laccase enzyme, 100 �l of ABTS substrate (final concentra-tion 1 mM), and 800 �l of sodium acetate buffer (100 mM, pH 5.0).The protein concentration in the culture supernatant was deter-mined following the dye-binding method as described previouslyby Bradford [31] with bovine serum albumin (BSA) as the referencestandard.

2.4. Effect of various alcohols and different concentration ofethanol on G. lucidum laccase production

To study the influence of various alcohols on G. lucidum laccaseproduction, the production medium was modified using variousalcohols – namely methanol, ethanol, 2-methyl-1-propanol, 1-propanol and 2-propanol (iso-propanol) at 1% (v/v). Additionally,the dose-dependent effect of ethanol on the laccase production wasanalyzed by applying various concentrations (0, 1, 2, 3, 4 and 5%,v/v).

2.5. Effect of different inducers and copper sulfate on G. lucidumlaccase production

The G. lucidum laccase production medium was furtheramended using various inducers such as gallic acid, veratryl alco-hol, xylidine, ferulic acid and vanillin – all set at a pre-definedconcentration of 1 mM as described previously [32] to induce theproduction of laccase. Various concentrations of CuSO4 (0.1, 0.2, 0.3,


108 T. Manavalan et al. / Biochemical Engineering Journal 70 (2013) 106– 114

0.4, 0.5 and 0.6 mM) were applied with the production medium totest the enhancing effect on G. lucidum laccase synthesis.

2.6. Combinatorial effect of different inducers and lignocellulosicbiomasses on G. lucidum laccase production

Ethanol (3%, v/v), CuSO4 (0.4 mM) and gallic acid (1 mM) wereapplied in different combinations to study their additive effect on G.lucidum laccase production as described in the text. Various grindedlignocellulosic biomasses (such as: kodo millet (Paspalum scrobicu-latum, a member of the family Poaceae, commonly also called kodamillet or kodra) bran, orange peel, pomelo peel, rice bran, tamarindshell and wheat bran, all at 1%, w/v) were tested with regard totheir effect on laccase production in the presence of ethanol (3%,v/v), CuSO4 (0.4 mM) and gallic acid (1 mM).

2.7. Protein isolation and laccase purification

Fifteen-days-old G. lucidum culture was used for laccase purifi-cation. The G. lucidum culture supernatant was initially filteredthrough cheese cloth to remove mycelial debris. The filtrate wascentrifuged (8000 × g for 30 min at 4 ◦C) and concentrated by salt-ing out with 80% (w/v) ammonium sulfate. The protein precipitatewas collected by centrifugation (8000 × g for 30 min at 4 ◦C), dis-solved in the sodium acetate buffer (100 mM, pH 5.0) and dialyzedagainst 10 mM sodium acetate buffer (pH 5.0). The dialyzed proteinwas then concentrated by lyophilization (Freeze Dryer, FD-10M,Labfreez Instruments Co., Ltd., Beijing, PR China) and used in fur-ther purification steps. The precipitated and dialyzed protein wasloaded onto a Sephadex G-100 column (Sigma) (Fraction collec-tor: LKB Bromma 7000 Ultrovac; LKB, Bromma, Sweden). Proteinswere eluted with the same buffer and 3 ml fractions were col-lected. The protein content of each fraction was determined bymeasuring the absorbance at 280 nm (Beckman Coulter DU 50 spec-trophotometer (Brea, CA, USA)). Laccase-positive fractions weredetermined as described previously [8], pooled, concentrated, dia-lyzed against sodium acetate buffer (10 mM, pH 5.0), and used forfurther purification on a DEAE-cellulose column (Sigma) (Fractioncollector: LKB Bromma) that was pre-equilibrated with sodiumacetate buffer (10 mM, pH 5.0). Unbound proteins were washedout with sodium acetate buffer (10 mM, pH 5.0) and the boundproteins were eluted in the same buffer with a linear gradient ofNaCl (0–0.5 M). Three ml of fractions were collected and laccase-rich fractions were pooled, concentrated, dialyzed against sodiumacetate buffer (10 mM, pH 5.0), and stored at −20 ◦C until furtheruse.

2.8. Characterization of purified G. lucidum laccase

SDS-PAGE was carried out following the protocol of Laemmli[33] with 5% (w/v) stacking gel and 10% (w/v) resolving gel at 20 mAin Genei’s Mini-electrophoresis unit (Bangalore Genei India Pvt.Ltd.) as described previously [34]. Protein bands were stained withsilver nitrate and the molecular mass of the purified laccase wasdetermined by calculating the relative mobility based on a proteinmolecular weight marker that runs alongside. Zymography for G.lucidum laccase activity was performed on native-PAGE using 1 mMguaiacol as substrate in 100 mM sodium acetate buffer at pH 5.0[21].

Optimum pH for the purified laccase activity was determinedby incubating the enzyme with various buffers over a wide rangeof pH values, such as citrate–phosphate buffer (100 mM 2.5–4.5),sodium acetate buffer (100 mM, pH range 4.5–5.5), phosphatebuffer (100 mM, pH range 5.5–7.5), and Tris–HCl buffer (100 mM,pH range 7.5–9.0). Ten �g of purified protein were incubated withthe above buffers for 5 min, then the substrate (ABTS, 1 mM) was

added to the mixture and the enzyme activity was assayed at 30 ◦Cby measuring the OD at 436 nm using a Beckman Coulter DU 50spectrophotometer as described previously [8].

The optimal working temperature for the purified G. lucidumlaccase activity was determined by incubating the purified laccasefor 1 h at various temperatures (20–100 ◦C; 10 ◦C increment) afterwhich the remaining enzyme activity was estimated by incubationin the sodium acetate buffer (100 mM, pH 5) and applying ABTS(1 mM) as substrate. The enzyme activity was assayed by measuringthe OD at 436 nm using a Beckman Coulter DU 50 spectrophotome-ter as described previously [8].

The kinetic constants of G. lucidum laccase were determinedby varying the concentrations of ABTS (5–500 �M) and guaiacol(20–1000 �M) in sodium acetate buffer (100 mM, pH 5.0) contain-ing 0.1 ml of laccase (0.11 �M) in a total volume of 1 ml and theenzyme activity was assayed by measuring the OD at 436 nm usinga Beckman Coulter DU 50 spectrophotometer as described previ-ously [8]. A Lineweaver–Burk plot was used to calculate the kineticconstants [35,36]. The Michaelis–Menten equation was used todetermine the Michaelis constant (Km) and the maximum veloc-ity of the enzyme (Vmax). Linear regression was used to analyze Km

and Vmax estimation by the values obtained in the assay; kcat wascalculated as Vmax/enzyme concentration (0.11 �M).

2.9. Identification of G. lucidum laccase by MALDI-TOF/TOFMS/MS

The identification of G. lucidum laccase by MALDI-TOF/TOFMS/MS was performed by cutting the laccase band obtained bySDS-PAGE analysis. The gel sample was digested with trypsin, andthe resulting peptides were spotted on a MALDI target plate. Thepeptides were then analyzed by MALDI-TOF/TOF (4800 MALDITOF/TOFTM Analyzer; Applied Biosystems, Foster City, CA, USA) andthe peptide fingerprint was compared with the National Center forBiotechnology Information (NCBInr) protein database as describedpreviously [37].

2.10. Dye decolorization activity of purified laccase and industrialtextile dye effluent treatment by G. lucidum laccase

To determine the optimum quantity of the purified G. lucidum-derived laccase for decolorization, the enzyme decolorization assaywas performed in a 1 ml reaction mixture, consisting of the purifiedenzyme (3–30 U ml−1) solution (sodium acetate buffer (100 mM,pH 5.0)) supplemented with the various dyes at a final concen-tration of 50 �M for MV (Methyl Violet 2B; Sigma), RY (RemazolYellow G; HiMedia Laboratories) and AFR (Acid Fast Red A; HiMediaLaboratories), respectively, at 30 ◦C for 24 h. All dye decoloriz-ing experiments were carried out in triplicates. Control sampleswere run alongside using a heat-inactivated laccase. The effect ofvarious dye concentrations on the laccase enzyme activity waschecked using 10–100 �M of each dye. Dye decolorization wasmeasured in an UV–Visible scanning spectrophotometer (BeckmanCoulter DU 50) for each dye and expressed in terms of percent-age.

The industrial textile dye effluent was collected from Rama-krishna dying industry (Tirupur, India) which is mostly using thereactive and acid dyes. The characteristics of textile dye effluentsused were as follows: pH 9.8; dark blue in color; BOD 1400 mg l−1;COD 4500 mg l−1. The production medium contained the industrialtextile dye effluent instead of water and the pH of the effluentwas adjusted to 5.5, autoclaved, inoculated with 0.5% of G. luciduminoculum and a control was maintained without G. lucidum inocula-tion. To determine the industrial textile dye effluent decolorization,BOD and COD levels, an aliquot of 3 ml was withdrawn at differenttime intervals (every 3 days and up to 21 days) from the test flask



and centrifuged at 8000 × g for 30 min at 4 ◦C. Clear supernatantwas used to determine the industrial textile dye effluent decoloriza-tion in a Beckman Coulter DU 50 spectrophotometer [21,38–40]. Alldecolorization experiments were performed in the three sets andthe decolorization activity was expressed in terms of the percent-age decolorization as follows:

Decolorization (%) = Initial absorbance − Final absorbanceInitial absorbance

× 100.

2.11. Phytotoxicity

The phytotoxicity study was carried out (at room temperature)using Oryza sativa (Monocotyledon) and Vigna radiata (Dicotyle-don) (10 seeds of each) by adding separately a 10 ml filtrate sampleof untreated (control) and G. lucidum-treated textile dyes (AFR, MVand RY) as well as untreated (control) and G. lucidum-treated indus-trial textile dye effluent samples (collected from Tirupur textileindustry). An additional control set for seed germination was car-ried out using water only at the same time. Germination (%) and thelength of shoot (plumule) and root (radicle) were recorded after 5days [17,38].

2.12. Statistical analysis

The data obtained in this investigation are illustrated asmean ± SD. Differences between the groups were established usingan unpaired Student’s t-test while within-group comparisons wereperformed using the paired Student’s t-test.

3. Results

3.1. Effect of various alcohols and different concentrations ofethanol on G. lucidum laccase production

An experiment was designed to investigate time-dependentlythe effect of different alcohols (at 1%, v/v) on the G. lucidum lac-case production. After 15 days of incubation, the laccase productionwas as high as 1.18 U ml−1 with ethanol, which was 6.5 timeshigher than the control. Addition of methanol to the mediumyielded 3.2 times higher laccase activity only. However, with 2-propanol, cell growth was severely inhibited, and thus laccaseproduction was negligible (Fig. 1A). Further, the laccase produc-tion could be enhanced by varying the concentration of ethanol.After 15 days incubation, the laccase production reached a max-imum of 2.53 U ml−1 when the medium was supplemented with3% (v/v) ethanol. The yield obtained was 14.1 times higher com-pared with ethanol-free conditions. At an ethanol concentration of5% (v/v), conversely, cell growth was severely inhibited, and the lac-case production was less significantly increased (1.88 U ml−1 only)(Fig. 1B).

3.2. Effect of different inducers and copper sulfate (CuSO4) on G.lucidum laccase production

Investigating the effect of various individual enhancing compo-nents revealed that gallic acid (1 mM) was the best inducer of G.lucidum laccase production (3.79 U ml−1) followed by ethanol (at3%, v/v) (2.53 U ml−1) and xylidine (1 mM) (2.36 U ml−1) (Fig. 2A).

Copper sulfate (CuSO4) has also proven to be a promisinginducer for laccase production [27]. Laccase production was sig-nificantly increased (1.5 U ml−1) when the culture medium wasamended with 0.4 mM CuSO4, but less increased at 0.3 mM(1.2 U ml−1) and 0.5 mM (1.02 U ml−1), respectively (Fig. 2B).

Fig. 1. Effect of alcohol on G. lucidum laccase production. (A) Time-dependent effectof various alcohols (1%, v/v). (B) Dose-dependent effect of ethanol (concentrationEtOH in %, v/v), cultivation at 30 ◦C for 15 days. The data in (A) and (B) are the valuesobtained in triplicate assays and are shown as mean ± SD (*P < 0.05, compared withcontrols).

Fig. 2. Effect of various inducers on G. lucidum laccase production. (A) Effect of vari-ous inducers (ethanol (3%, v/v) and other inducers (all 1 mM)). (B) Effect of differentcopper sulfate concentrations. In (A) and (B) cells were cultivated at 30 ◦C for 15days. Values shown in (A) and (B) (mean ± SD) represent data obtained in triplicatefrom two independent experiments (*P < 0.05, compared with controls).



Fig. 3. Combinatorial effect of various inducers on G. lucidum laccase production.(A) Combinatorial effect of different inducers. (B) Effect of various lignocellulosesubstrates (1%, w/v). The control media was composed of basal medium withoutlignocellulose substrate. In (A) and (B) cells were cultivated at 30 ◦C for 15 days.Values shown in (A) and (B) (mean ± SD) represent data obtained in triplicate fromtwo independent experiments (*P < 0.05, compared with controls).

3.3. Combinatorial effect of different inducers and lignocellulosicbiomasses on G. lucidum laccase production

Ethanol (3%, v/v), CuSO4 (0.4 mM) and gallic acid (1 mM) wereapplied in different combinations and highest laccase productionwas observed for the combination of ethanol (3%, v/v) plus CuSO4(0.4 mM) and gallic acid (1 mM) (Fig. 3A). However, the combina-tion of the above three components was not much more significantthan the combination of ethanol (3%, v/v) and CuSO4 (0.4 mM) orethanol (3%, v/v) and gallic acid (1 mM). Moreover, ethanol is muchcheaper and environmentally more safer than gallic acid (Fig. 3A).

Various lignocellulosic substrates (1%, w/v) were applied tocharacterize their effect on the laccase production with and with-out the combination of ethanol (3%, v/v), CuSO4 (0.4 mM) and gallicacid (1 mM). The control media was composed of basal mediumwithout any lignocellulosic biomass. Interestingly, we show herefor the first time that tamarind shell with ethanol (3%, v/v), CuSO4(0.4 mM) and gallic acid (1 mM)-containing medium was the bestgrowth condition for the G. lucidum laccase production, yieldingabout 74.84 U ml−1 after 15 days. This was 416 times higher thanthe control (Fig. 3B).

Fig. 4. Identification of purified G. lucidum laccase-3. (A) Molecular mass determi-nation of purified laccase on SDS-PAGE. Lanes: 1: crude protein extract, 2: purifiedlaccase after DEAE-cellulose chromatography, 3: partially purified laccase afterSephadex G-100 size exclusion chromatography, 4: standard marker proteins. (B)Zymography of laccase activity was performed using 1 mM guaiacol as substrate.Lanes: 1: purified laccase, 2: crude laccase, 3: partially purified laccase. (C) MALDI-TOF/TOF MS/MS analysis revealed the de novo peptide sequence of purified G.lucidum laccase (as indicated in red letters) to be identical with laccase 3 (ABK59821)and highly homolog with other published G. lucidum laccases (e.g. AAG17009).

3.4. Purification and characterization of extracellular laccasefrom G. lucidum

The summary of the laccase purification steps, obtained from G.lucidum, is presented in Table 1.

The laccase enzyme was purified 5.57 folds from its initial cul-ture broth with a final yield of 32%. The specific activity of thepurified enzyme was 145 U mg−1 of protein as estimated applyingthe ABTS substrate (1 mM) as described in Materials and methods.The crude protein showed four laccase isoforms by zymography ona native-PAGE while the purified laccase-3 (Glac 3) protein had asingle band on SDS-PAGE and native-PAGE with a molecular massof 38.3 kDa (Fig. 4A and B).

In a small-scale pilot study, we tested the 4 different laccasesand found that the 38.3 kDa-related laccase (Glac 3) had the highestactivity and thermostability (data not shown), thus we decided toisolate, purify and characterize the 38.3 kDa laccase.

Testing various buffers with different pH values, the optimumpH for achieving maximum activity of the purified laccase from G.lucidum was identified as 5.0 while the enzyme was stable withina relative wide range of pH 4–7.5.

Testing the temperature-dependent activity and thermal stabil-ity, we found that the purified laccase from G. lucidum showed anoptimum activity in the sodium acetate buffer (100 mM, pH 5) at

Table 1Summary of the purification steps of extracellular laccase from G. lucidum.

Purification step Volume (ml) Total protein (mg) Total activity (U) Specific activity (U mg−1) Purification fold Percent recovery

Crude culture filtrate 3000 521 13,500 26 1 100Ammonium sulfate

fractionation100 315 10,740 34 1.3 79

Sephadex G-100 20 110 7570 68 2.6 56DEAE-cellulose 16 30 4356 145 5.57 32



55 ◦C while an increase in temperature above 55 ◦C reduced theenzyme activity drastically. The thermal stability of the purifiedlaccase in sodium acetate buffer (100 mM, pH 5) from G. lucidumwas maximal at 30 ◦C, started to drop above 40 ◦C and decreasedabruptly beyond 60 ◦C. The half-life of the laccase at 60 ◦C was lessthan 60 min.

Substrate oxidizing activity of the purified laccase was deter-mined using different aromatic substrates, at a final concentrationof 1 mM. The substrate oxidation rate was followed by measuringthe absorbance change with the molar extinction coefficient (ε)obtained from the literature [41,42]. Like other laccases, our puri-fied G. lucidum laccase oxidizes a wide range of substrates includingphenolic and aromatic amine substrates.

Our purified laccase of G. lucidum preferred ABTS as theideal substrate similar to laccases derived from other fungi[6,43,44]. Substrate-specific oxidization of this enzyme (repre-sented as relative activity) was observed in the following order:ABTS (100%) > guaiacol (91%) > 2,6-dimethoxyphenol (83%) > 4-methylcatechol (68%) > ferulic acid (45%) > hydroquinone (27%),and no activity was detected with tyrosine (0%).

The kinetic constants of purified G. lucidum laccase weredetermined for the two substrates ABTS and guaiacol. Thecatalytic efficiency (specificity constant, kcat/Km) was slightlyhigher for ABTS (11.5 × 105 s−1 M−1) compared with guaiacol(3.9 × 105 s−1 M−1). The Km values for ABTS and guaiacol were47 �M and 94 �M, respectively. The catalytic constant (turnovernumber, kcat) of the laccase was calculated to be 54 and 37 for ABTSand guaiacol, respectively.

3.5. MALDI-TOF/TOF MS/MS analysis identifies G.lucidum-derived laccase

The purified 38.3 kDa laccase was identified by MALDI-TOF/TOFMS/MS while the peptide fingerprint was compared with the NCBIprotein database. The tryptic fragments of G. lucidum laccase werematched with already existing tryptic fragments of laccases from G.lucidum with 100% identity and confirmed as laccase-3 of G. lucidum(Fig. 4C) [45,46]. Hence, this result confirmed that the 38.3 kDa G.lucidum laccase belongs to the laccase family.

3.6. Dye decolorization by purified G. lucidum laccase andindustrial textile dye effluents treatment by G. lucidum laccase

Purified laccase from G. lucidum, used at various dilutions(3–30 U ml−1), showed effective dye decolorization for AFR, RY andMV dyes. While increasing the enzyme concentration the rate ofdye decolorization also increased accordingly. More than 90% ofdecolorization activity was observed during 24 h incubation for allthe dyes tested (Fig. 5A). A time course of the decolorization ratewas also monitored using an enzyme concentration of 20 U ml−1 forall dyes at a concentration of 50 �M (Fig. 5B). The extent of decol-orization was higher for AFR (92%), followed by RY (83%) and MV(78%).

The decolorization rate was increased with increasing the incu-bation time. Fig. 5C shows the decolorization of all dyes relativeto the initial dye concentration. A complete decolorization (100%)was observed at lower dye concentrations (10–30 �M) whichdecreased markedly when the dye concentrations increased from40 to 100 �M with 20 U ml−1 laccase enzyme.

Our study clearly depicts the ability of decolorization of indus-trial textile dye effluents by G. lucidum. The industrial textile dyeeffluent was decolorized up to 97% within 21 days (Table 2). Underthese conditions, the extracellular laccase activity (28 U ml−1)increased as the decolorization rate also increased. The reductionin the BOD and COD was also analyzed before and after the treat-ment of the industrial textile dye effluent by G. lucidum. Reduction

Fig. 5. Effect of the G. lucidum laccase enzyme on dye decolorization. (A) Differ-ent purified laccase enzyme concentrations tested for dye decolorization. (B) Dye(50 �M) decolorization by purified laccase (20 U ml−1). (C) Effect of dye concentra-tions on the decolorization ability of purified laccase (20 U ml−l). Values shown in(A)–(C) (mean ± SD) represent data obtained in triplicate from two independentexperiments.

of BOD and COD after treatment with G. lucidum reached a maxi-mum of 75% and 70%, respectively. Thus our investigation stronglydemonstrates the ability of G. lucidum to decolorize the industrialtextile dye effluent without any further dilution component.

Table 2Decolorization of industrial textile dye effluent by G. lucidum and changes in BOD,COD, decolorization and laccase production.

Time (day) Decolorization (%) Laccase (U ml−1) BOD (%) COD (%)

3 20 1.2 5 36 45 8.5 9 79 60 15 23 18

12 78 24 48 3915 86 28 62 5518 91 22 68 6421 97 18 75 70

Values represent the means of values from three independent experiments, witha maximal sample mean deviation of ±5%. BOD, biological oxygen demand; COD,chemical oxygen demand.



Table 3Phytotoxicity study of untreated and G. lucidum-treated textile dye and effluent. The relative sensitivities toward the textile dyes and effluent against the germination ofseeds of Oryza sativa and Vigna radiata.

Plant name Dyes Shoot length (cm) Root length (cm)

Control Untreated Treated Control Untreated Treated

Oryza sativa Acid Fast Red A (30 �M) 3.5 0.7 3.1 5 1 4.3Methyl Violet 2B (30 �M) 3.5 0.4 2.6 5 0.6 3.8Remazol Yellow G (30 �M) 3.5 0.6 2.8 5 0.8 4Textile dye effluent (100%) 3.5 0.3 2.2 5 0.4 2.9

Vigna radiata Acid Fast Red A (30 �M) 5.6 1.3 4.9 7 1.8 6.5Methyl Violet 2B (30 �M) 5.6 0.6 3.6 7 1 5.4Remazol Yellow G (30 �M) 5.6 1.1 4.5 7 1.4 5.8Textile dye effluent (100%) 5.6 0.5 3.3 7 0.7 4.4

Values represent the means of germinated seeds from three independent experiments, with a maximal sample mean deviation of ±5%.

3.7. Phytotoxicity

The results of the phototoxicity tests revealed that the G.lucidum-treated textile dyes (AFR, MV and RY) and the G. lucidum-treated industrial textile dye effluent show better seed germination(root and shoot length) compared with untreated (G. lucidumfree) controls both for O. sativa (Monocotyledon) and V. radiata(Dicotyledon) but it was adversely inhibited in untreated dyes (G.lucidum-free controls) (Table 3). Our detoxification tests clearlyindicate not only decolorization but also detoxification of varioustextile dyes and industrial textile dye effluent by G. lucidum.

4. Discussion

A laccase from G. lucidum was produced under the noveloptimized media conditions containing 3% (v/v) ethanol, which sig-nificantly enhanced the enzyme production up to 14.1 folds, whichis significantly higher than the laccase production from Pycnoporuscinnabarinus which was only enhanced up to 7.1 and 5.3 times withethanol and methanol at a concentration of 20 g l−1 [28], thoughit was less compared with laccase production using ethanol/starchfree residue (EPSFR)-supplemented media coming from wheat bran[27].

Though a recent report showed that ferulic acid was sup-posed to be the best inductor for laccase production by G.lucidum (0.14 U ml−1) [24], our G. lucidum-derived laccase activ-ity (2.53 U ml−1) at 3% (v/v) ethanol was significantly better thanthe usage of xylidine, ferulic acid, vanillin or veratryl alcohol alone.Thus, since ethanol is much cheaper and environmentally moreacceptable than gallic acid, the use of ethanol as a stimulator forlaccase production is highly recommendable.

While we found that 0.4 mM copper sulfate was the optimalconcentration to induce G. lucidum laccase production (1.5 U ml−1

in the basal medium), Fonseca et al. [47] reported that the additionof 0.5 mM copper sulfate considerably stimulated laccase produc-tion in Ganoderma applanatum (1.85 U ml−1). Songulashvili et al.also showed that 1–3 mM Cu2+ can increase the laccase produc-tion up to 2-fold [27]. However, our data show that ethanol (3%,v/v) alone seems to be good enough for industrial G. lucidum lac-case production (2.53 U ml−1) because the addition of CuSO4 and/orferulic acid increased the yield only slightly.

In addition, we are describing here for the first time that thecombinatorial application of tamarind shell (1%, w/v), ethanol (3%,v/v), CuSO4 (0.4 mM) with gallic acid (1 mM) was the most potentG. lucidum laccase production (74.84 U ml−1)-inducing medium,which is 6.32 times higher than the G. lucidum laccase productionusing pomelo peel as a lignocellulose substrate [35]. This innova-tive potential industrial application, in terms of the best-possiblemix of value-for-money performance ratio, flexibility, and quality,needs to be elucidated further.

Our purified laccase-3 from G. lucidum showed a molecularweight of 38.3 kDa. Similarly, some white-rot fungi have beenreported with molecular weights from 38 up to 150 kDa [48] asalso shown by our zymography analysis. D’Souza et al. [49] reportedlaccase enzymes with a molecular mass of 40 and 66 kDa from G.lucidum. On the other hand, Ganoderma sp. WR-1 shows a laccasewith a molecular weight of 60 kDa [2]. Similarly, Ganoderma sp.KU-Alk4 (G. philippii) produced multiple forms of laccase in thepresence of veratryl alcohol [43]. Ko et al. [45] detected three lac-case isozymes in the mycelia of G. lucidum. The recent report byDing et al. could identify only one isoform at about 68 kDa (proba-bly Glac 2) [35]. The variation in the production of various laccaseisoforms between different genera, species and strains might arisefrom different ecological origins of these mushrooms.

Our purified laccase-3 from G. lucidum exhibits stability over abroad range of pH (4–7.5) and temperature (30–60 ◦C) with optimalworking conditions at pH 5.0 and 55 ◦C.

Several fungal laccases show maximum activity at a pH rangingfrom 4.0 to 6.0, thus confirming our results [12,50,51]. In contrast,others found that the ideal pH observed for laccases from G. lucidumand G. fornicatum was unusual low at pH = 2.6 and 2.5 with ABTSused as substrate [44,52]. The more acidic activity may eventuallyalso depend on the different isoforms as the 68 kDa isoform (Glac 2)showed an optimal pH of 3 [35]. Interestingly, Ko et al. [45] discussabout 3 laccases with a molecular weight in a range of 65–68 kDaand an optimal pH value around 3.

Our purified laccase from G. lucidum showed an optimum activ-ity at 55 ◦C, which is much higher than that for laccase from G.lucidum published by others [45] but similar to a very recent reportby Ding et al. [35]. Similar to our data obtained, Huang et al. [52],Sun et al. [44] and Ding et al. [35] reported that the optimum tem-perature for laccase activity was 55 ◦C. The loss of enzyme activity athigher temperature may be due to the release of copper ions fromthe enzyme laccase as suggested by Palonen et al. [6]. However,higher temperature laccase activity has also been reported from G.lucidum (70 ◦C; Wang and Ng [53]).

The G. lucidum laccase’s substrate-specific oxidizationorder (ABTS > guaiacol > 2,6-dimethoxyphenol > 4-methyl cate-chol > ferulic acid > hydroquinone, and no activity was detectedwith tyrosine) suggests that the substituted methoxy groups playan important role [42,43].

Our G. lucidum laccase’s Km values for ABTS and guaiacol (47 �Mand 94 �M) can be considered to be within a normal range becauseprevious investigations described a very wide range of Km valuesfor laccases from different fungi. For instance, while Huang et al.[52] reported Km values of 103.9 �M and 1263 �M for ABTS andguaiacol from G. fornicatum, Sun et al. [44] reported Km values of0.9665 mM and 1.1122 mM for ABTS and guaiacol from G. lucidum.The Km value reported by Ko et al. for one of their 68 kDa laccases(“GaLc 3”) is described as 3.7 �M for ABTS [45]. The recent report



about the 68 kDa laccase (Glac 2) describe Km and kcat values of0.114 M and 74.63 s−1, respectively, and the specificity constant(kcat/Km) as 654.65 s−1 M−1 [35], which are similar to our valuesobtained.

Due to the variations described for the various G. lucidum-derived laccases (in terms of molecular weight, optimal pH andtemperature as well as kcat/Km values), it is highly recommendablethat in future studies scientists isolate and sequence (and makingthem available in public data bases) their laccases in order to beable to make an appropriate comparison and to see if the differ-ences are based on the gene or protein sequence only or eventuallydue to post-translational modifications – e.g. caused by different (i)environmental isolation loci, (ii) growth and fermentation as wellas (iii) purification conditions.

Enzyme-based decolorization is an efficient method to treatindustrial effluent [4]. The variation of decolorization is possi-bly due to the structural variation of the many different dyes[54]. Laccase-mediated dye decolorization has been described withcrude and purified forms from many fungi; however, most ofthe laccases require redox mediators [4,10]. It has been reportedthat extracellular enzymes produced by G. lucidum under suspen-sion culture completely decolorized several phenolic azo dyes, andmost of the dye decolorization activity was mainly due to laccase[10,55,56]. The results obtained in our study reveals that the dyedecolorization ability of the purified laccase against the dyes MV,RY and AFR are more than 80% with 40 �M dye concentration. Thisactivity is significantly higher than that reported for the laccasepurified from Thelephora sp. [57], which showed only 12.5% decol-orization. Besides, our described decolorization conditions are freefrom any redox mediators.

The result obtained in decolorization of industrial textile dyeeffluent (97%) was slightly better than the previously reported oneusing Ganoderma sp. En3 [38]. G. lucidum could decolorize anddetoxify the textile dyes (AFR, MV and RY) and industrial textiledye effluent effectively.

5. Conclusion

G. lucidum is an efficient laccase producer reaching 2.53 U/mlwith 3% (v/v) of ethanol even in the absence of any further inducer.The lignocellulosic biomass tamarind shell is a potent G. lucidumlaccase stimulator opening new innovative avenues for potentialindustrial applications. G. lucidum-derived laccase exhibits highactivity and stability over a broad range of pH and temperature.The dye decolorization and detoxification ability of G. lucidum lac-case both in textile dyes and industrial textile dye effluent, withoutany further supplemental redox mediator, suggests that the herepresented growth and fermentation conditions might become use-ful for the economic production of laccase at industrial levels tofacilitate the benefit of using immobilized enzymes for bioremedi-ation.

Acknowledgments

The authors thank the University Grants Commission for pro-viding the scholarship to T.M. through in Science for MeritoriousStudents and Dr. R. Rengasamy, CAS in Botany, University ofMadras, for providing the adequate laboratory facility to carry outresearch.

This study was supported by the Institute of Advanced Studies,Nanyang Technological University and by the Hanyang University.

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Characterization of optimized production, purification and application of laccase from Ganoderma...

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