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Process Biochemistry 49 (2014) 1196–1204

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Process Biochemistry

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Purification of a thermostable laccase from Leucaena leucocephalausing a copper alginate entrapment approach and the applicationof the laccase in dye decolorization

Nivedita Jaiswal, Veda P. Pandey, Upendra N. Dwivedi ∗

Department of Biochemistry, University of Lucknow, Lucknow 226007, UP, India

a r t i c l e i n f o

Article history:Received 20 December 2013Received in revised form 24 March 2014Accepted 2 April 2014Available online 13 April 2014

Keywords:Celite chromatographyCopper alginateDye decolorizationLaccaseLeucaena leucocephala

a b s t r a c t

Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick two-step procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccasewas found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity oflaccase was found to be highest for catechol and lowest for hydroquinone, however, highest Kcat andKcat/Km were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0and 80 ◦C, respectively. Mn2+, Cd2+, Fe2+, Cu2+ and Na+ activated laccase while Ca2+ treatment increasedlaccase activity up to 3 mM, beyond which it inhibited laccase. Co2+, Hg2+, DTT, SDS and EDTA showedan inhibition of laccase activity. The Leucaena laccase was found to be fairly tolerant to organic solvents;upon exposure for 1 h individually to 50% (v/v) each of ethanol, DMF, DMSO and benzene, more than50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol andchloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized syntheticdyes such as indigocarmine and congo red in the absence of any redox mediator.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Laccases (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) aremulticopper oxidases that catalyze the oxidation of various pheno-lic and non-phenolic aromatic compounds using molecular oxygen[1]. They are widely distributed in nature and have been found inplants, insects, bacteria, fungi and crustaceans. Most laccases havebeen isolated and characterized extensively from fungal sources.However, a considerably lesser number of laccases have beencharacterized from plant sources to date. The laccases reportedfrom plants as well as fungi have been purified using traditionalmethods, such as salt or solvent precipitation and column chro-matography, which not only took longer time but also resulted ina considerably lower yield of the purified enzyme [2–4]. Therefore,the industrial application of laccase demands its bulk production,necessitating the development of efficient, quick and econom-ical purification methods. In this study, we report an alginatebead entrapment method for Leucaena laccase purification thatmay have industrial applications. The alginate bead entrapment

∗ Corresponding author. Tel.: +91 522 2740132; fax: +91 522 2740132.E-mail address: [email protected] (U.N. Dwivedi).

method for the purification of enzymes, directly from crude extract,has recently been used as an attractive, quick and economicalmethod for purifying other enzymes, such as �- and �-amylases,glucoamylase, pectinase, phospholipase and �-galactosidase [5].

Alginate, a copolymer of �-d-mannuronic acid (M) and �-l-guluronic acid (G) residues, has been exploited most commonly forentrapment purposes in the form of calcium alginate beads. How-ever, besides calcium, various other divalent cations have also beenshown to possess an affinity for alginate in the following order:Pb2+ > Cu2+ > Cd2+ > Ba2+ > Sr2+ > Ca2+ > Co2+ > Ni2+ > Zn2+ > Mn2+.Furthermore, the affinity of a metal-alginate bead toward theentrapped enzyme is dependent upon the nature and compositionof the metal as well as its concentration in the bead [6]. Thus, a cal-cium alginate bead will have a higher affinity for a Ca2+-containingenzyme than for another metal-containing enzyme. One suchexample is the purification of �-amylase, a calcium containingmetalloenzyme, using calcium alginate beads [5]. Therefore, thisproperty has been exploited in the present study for purifyinglaccase, a copper containing metalloenzyme, using copper alginatebeads. Though copper alginate beads have been used for theimmobilization of laccase [7–9], it has not yet been used for thepurification of laccase or any other enzyme, to the best of ourknowledge.

http://dx.doi.org/10.1016/j.procbio.2014.04.0021359-5113/© 2014 Elsevier Ltd. All rights reserved.


N. Jaiswal et al. / Process Biochemistry 49 (2014) 1196–1204 1197

Laccases isolated from various sources exhibited diversity withregards to their size and subunit composition. Multimeric, homote-trameric, heterodimeric as well as monomeric forms of laccaseshave been reported, ranging in subunit molecular weight from 36to 175 kDa [2,10–12]. Though the pH optima for plant laccases var-ied from neutral to alkaline range [11–13], while most of the fungaland bacterial laccases exhibited an acidic pH optima [14,15]. Thetemperature optima of laccases have been reported to vary widelyin the range of 25–90 ◦C, depending on the source of the laccase[16–18]. Fungal laccases usually have a lower thermal stabilitythan bacterial laccases [19]. Recently, a laccase from Trametes hir-suta showed an optimum temperature of 85 ◦C [20]. Laccases havebeen inhibited by a number of ions and compounds, such as metalions (e.g., Ca2+, Mg2+, Co2+, Mn2+, Cd2+, Zn2+, Hg2+, etc.), sulfhydrylreagents, EDTA, dithiothreitol, hydroxyglycine, kojic acid, thiourea,detergents, etc. [1].

Laccases have applications in various industrial processes, suchas textile dye bleaching, pulp bleaching, effluent detoxification,bioremediation of contaminating environmental pollutants, enzy-matic conversion of chemical intermediates, and organic synthesis[1]. Therefore, for these industrial applications, laccases that areresistant to extreme conditions of pH, temperature, high salt, andorganic solvents, are highly desirable. Furthermore, laccases havebecome an attractive option (in contrast to peroxidases) for thedecolorization of synthetic dyes from industrial wastes [21], as theydo not require expensive H2O2 as a co-substrate and have broadersubstrate specificity. In the presence of mediators, the substratespecificity of laccases can be further enhanced, leading to the oxi-dation of more complex substrates [22]. Thus, laccases are capableof oxidizing a wide variety of aromatic compounds, such as ortho-,meta- or para- substituted phenols; diamines; aromatic amines andthiols; and inorganic compounds, such as iodine, Mo(CN)8

4−, andFe(CN)6

4− [1,19]. Several microbial laccases have been assessed fortheir potential application in dye decolorization [23,24], however,the reports on dye decolorization by plant laccases is scanty. There-fore, there is a need to search for potential plant laccases with anability to degrade dyes.

In the present paper, we describe a rapid purification protocolfor laccase from the leaves of Leucaena leucocephala, a tree legumewhich is of significance to the fodder and the paper and pulp indus-tries in India. In addition, purified laccase was characterized withregards to the effects of temperature, pH, substrates, various effec-tors, and organic solvents and the ability to oxidize industrial dyes.To the best of our knowledge, this paper is the first report of a pro-tocol for the purification of a laccase and its effectiveness in dyedecolorization.

2. Experimental

2.1. Plant material

Fresh green and young leaves from a 5 years old tree of L. leu-cocephala, which was growing in the garden of the University ofLucknow, Department of Biochemistry, were used as plant material.

2.2. Enzyme assay and protein estimation

Laccase activity was assayed as described by Matijosyte et al.[25]. The reaction mixture contained Tris–HCl buffer (100 mM,pH 7.0), catechol (10 mM) and a suitable enzyme aliquot. After30 min of incubation at 37 ◦C, the increase in absorbance (dueto oxidation of catechol to o-benzoquinone) was measured at390 nm using UV–vis spectrophotometer (Elico SL-177). A parallelcontrol containing all the ingredients of the assay system, exceptthe enzyme, was used as blank and those without substrate

were used as control. Enzyme activity was expressed in terms ofunits. One unit of enzyme activity was defined as the amount ofenzyme required to produce 1 �mol of o-benzoquinone in 1 minunder the specified conditions (ε = 1260 M−1 cm−1). Similarly,enzyme activity using substrates hydroquinone and ABTS, weredetermined by measuring increase in absorbance at 390 nm (ε forp-benzoquinone = 2240 M−1 cm−1), and 420 nm (ε for ABTS+ freeradical = 36,000 M−1 cm−1), respectively.

Protein concentration was estimated by the Bradford dye bind-ing method using bovine serum albumin as the standard [26].

2.3. Extraction from Leucaena leaves

A 30% crude extract was prepared by homogenizing 40 g of Leu-caena leaves in 120 ml of Tris–HCl buffer (100 mM, pH 7.5) using anice-cold blender. Solid PVP (polyvinylpyrrolidone, insoluble; 0.1%(w/v)) and 7 mM �-mercaptoethanol were added at the time ofextraction. The homogenate was centrifuged at 8500 × g for 30 minat 4 ◦C using a Sigma 4K15 centrifuge. The clear supernatant (crudeextract) was subjected to further purification. All of the operationswere performed at 4 ◦C, unless otherwise specified.

2.4. Purification of laccase

2.4.1. Entrapment in copper alginate beadsFor the purification of laccase, a copper alginate-mediated

entrapment (affinity precipitation) method, modified from thatdescribed by Prakash and Jaiswal [5], was used. Crude extract(100 ml) was mixed with 200 ml of sodium alginate (2%, w/v) andkept at 4 ◦C for 30 min with occasional hand swirling. Copper algi-nate beads (containing the entrapped enzyme) were prepared byadding the enzyme–sodium alginate mixture in a drop-wise man-ner to 1 l of a pre-cooled CuSO4 solution (100 mM) with continuousgentle hand swirling. The copper alginate beads formed using thismethod were allowed to harden for 1 h. Afterwards, the beads werewashed twice with Tris–HCl buffer (100 mM, pH 7.5, 200 ml perwash). Entrapped enzymes were eluted from the beads with 100 mlof Tris–HCl buffer (100 mM, pH 7.5) containing 1 M NaCl usingconstant agitation at 100 rpm for 30 min at 30 ◦C. The eluate wascollected by decanting. The eluted enzyme preparation was dia-lyzed overnight against the Tris–HCl buffer (100 mM, pH 7.5) withtwo to three changes. The dialyzed preparation was used for furtherpurification.

2.4.2. Celite adsorption chromatographyTwenty grams of Celite 545 (diatomaceous earth, obtained from

Sigma–Aldrich) was mixed with 250 ml of distilled water, boiled for5 min, and subsequently celite was allowed to settle at room tem-perature. The supernatant was decanted. The celite was washed 3–4times with distilled water to remove the fine particles. The washedcelite was suspended in Tris–HCl buffer (100 mM, pH 7.5) andpacked in a glass column (1.5 cm × 50 cm). The dialyzed enzymepreparation (100 ml) was applied on the top of the celite column.The pass-through fractions (5 ml) containing laccase activity werepooled and concentrated using Centricon with a 50 kDa-molecularmass-cut-off. The concentrated enzyme preparation was stored at4 ◦C for further use.

2.5. Native PAGE and in-gel activity staining

Native polyacrylamide gel-electrophoresis (7.5%; PAGE) wasperformed and visualized using silver staining. In-gel activity stain-ing of the laccase was performed by immersing the gel (after 7.5%native PAGE) in a catechol solution (50 mM) containing Tris–HClbuffer (100 mM, pH 7.5) until brown-colored bands appeared.


1198 N. Jaiswal et al. / Process Biochemistry 49 (2014) 1196–1204

2.6. Native molecular weight determination

The native molecular weight of purified laccase was investi-gated by gel filtration chromatography [27] using a Sephadex G-200column. Blue dextran was used to determine the unoccupied vol-ume (Vo) of the column. Catalase (240 kDa), �-amylase (200 kDa),phosphorylase B (97.4 kDa), bovine serum albumin (67 kDa) andlysozyme (14.3 kDa), were used as standard proteins (1.0 mg/ml)and, were applied onto the column. The amount of protein in thecolumn eluent was estimated using Bradford’s method [26]. Theelution volume (Ve) of each standard protein as well as the purifiedlaccase was measured. The molecular weight of the purified laccasewas calculated from a calibration curve obtained by plotting the logof the molecular weight of the standard proteins against the ratioof the elution volumes of the standard proteins and the unoccupiedvolume of the column (Ve/Vo).

2.7. Subunit molecular weight determination

For subunit molecular weight determination, SDS-PAGE wasconducted in a mini electrophoresis chamber (Bio-Rad) at roomtemperature using a 7.5% resolving and 3% stacking gel inTris–glycine running buffer (pH 8.8) at 100 V for 90 min [28].Standard protein markers containing myosin (205 kDa), phospho-rylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin(43 kDa) and carbonic anhydrase (29 kDa) were used.

2.8. Effect of temperature, pH and substrates

Laccase activity at various temperatures (10–90 ◦C) was investi-gated by incubating the whole assay system at pH 7.0 as describedearlier in Experimental section. The thermostability of the enzymewas determined by incubating the enzyme at 80 ◦C and thenassaying the activity at various time intervals. Similarly, the effectof pH on laccase activity was determined using 100 mM of variousbuffers at different pH values (such as sodium acetate, pH 6.0 and6.5; Tris–HCl, pH 7.0, 7.5, 8.0 and 8.5; sodium borate, pH 9.0 andsodium carbonate, pH 9.5) under standard assay conditions at 37 ◦C.The pH stability of the enzyme was determined by estimating theactivity after incubating the enzyme at different pH values (6.0–9.0)for 24 h. The effect of phenolic (catechol and hydroquinone) andnon-phenolic (ABTS) substrates was investigated at concentrationsranging from 0 to 50 mM at 37 ◦C and pH 7.0. Km and Vmax valueswere obtained by non-linear regression of a plot of enzyme activ-ity vs substrate concentration (hyperbolic Michaelis–Menten plot)using GraphPad Prism software.

2.9. Effect of various metal ions, SDS, DTT, and EDTA

The effect of varying concentrations of different effectors (Na+,Mg2+, Ca2+, Mn2+, Cu2+, Co2+, Fe2+, Cd2+, Hg2+, DTT, SDS, and EDTA)on laccase activity was studied as described above (at 37 ◦C and pH7.0) by performing the activity assay using catechol as the substratein the presence and absence of individual effectors at specifiedconcentrations in the reaction mixture.

2.10. Effect of organic solvents

The stability of Leucaena laccase in the presence of various polar(methanol, ethanol, isopropanol, dimethyl sulfoxide and dimethylfluoride) and non-polar organic solvents (benzene and chloroform)at 20% and 50% concentrations (v/v) was studied by incubating theenzyme in the respective solvents for 1 h at 37 ◦C, and subsequentlymeasuring the activity of enzyme aliquots under the standard assayconditions (at 37 ◦C and pH 7.0).

2.11. Effect on dye decolorization

Synthetic industrial dyes, namely indigocarmine (�max 610 nm)and congo red (�max 500 nm), were used for investigating the effi-cacy of decolorization by purified laccase. Stock solutions (1 mg/ml)of these dyes were prepared in distilled water and diluted to therequired concentration and then used for the decolorization assay.The reaction mixture (3 ml) contained Tris–HCl (100 mM, pH 7.0),a dye solution of a specified concentration and an appropriateamount of enzyme. A reaction mixture without enzyme was alsorun. After incubation for 6 h at 37 ◦C, the change in the absorbancewas measured spectrophotometrically using UV–vis spectropho-tometer (Elico SL-177). The effects of varying enzyme amounts(1.4–22 �g) and dye concentrations (10–250 �g/ml) on dye decol-orization were also investigated.

2.12. Statistical analysis

The experiments were performed in triplicates, and the meanand standard deviation were calculated accordingly.

3. Results and discussion

3.1. Purification of laccase

Laccase was purified to homogeneity from the leaves of Leucaenausing copper alginate bead entrapment followed by celite chro-matography (Table 1). The homogeneity of the purified enzyme wasestablished using native PAGE, where a single band was obtained(Fig. 1A). The purified laccase was found to be catalytically activeas established through in-gel activity staining (Fig. 1B). The laccasewas purified to 110.6-fold with an overall recovery of 51.0% anda specific activity of 58.5 units/mg. Thus, the copper alginate beadentrapment method (affinity precipitation) has been exploited suc-cessfully for the first time to purify laccase from a plant source.

To date, laccases isolated from various sources, such as plants,bacteria and fungi, have been purified using traditional multi-step

Fig. 1. (A) Native-PAGE analysis and silver staining of L. leucocephala laccase dur-ing purification. Lane 1: crude extract, Lane 2: eluate obtained after salt elutionof the entrapped enzyme from the copper alginate beads, Lane 3: purified laccaseobtained after celite chromatography. (B) In-gel activity staining of purified lac-case. The gel after Native-PAGE was immersed in 50 mM catechol solution until thebrown-colored band appeared.



Table 1Summary of laccase purification from Leucaena leucocephala leaves.

Steps Volume (ml) Total activity(U)

Total protein(mg)

Specific activity(U/mg protein)

Yield (%) Foldpurification

Crude extract 100 31.5 59.6 0.53 100 –Affinity precipitation 100 18.5 2.7 6.8 58.7 12.8Celite chromatography

(concentrated usingcentricon)

10 16.1 0.3 58.5 51.0 110.6

procedures, such as ammonium sulfate or organic solvent precip-itation and column chromatography techniques, which result inlow enzymatic yields. For example, a plant laccase isolated from axerophyte species, Opuntia vulgaris, was purified by acetone pre-cipitation, Sephadex A-50 anion exchange chromatography, andSephadex G-100 gel filtration chromatography with a 6.6% yield,21.6-fold purification, and 120.9 × 10−4 (IU/mg) specific activ-ity [11]. A fungal laccase isolated from Trichoderma harzianumnwas purified 151.7-fold with a yield of 0.39% and specific activ-ity of 130.5 units/mg through acetone precipitation, ultrafiltration,Sephadex G-100 column chromatography and Concanavalin-Aaffinity chromatography [14]. Similarly, a Scytalidium thermophilumlaccase was also reported to be purified to 7.9-fold using varioussteps, such as acetone precipitation, gel filtration on Biogel S200,anion exchange chromatography on Mono-Q and Resource QTM

and gel filtration on Superdex 200, with an overall recovery of 30%and 139.4 units/mg specific activity [4]. A fungal laccase was iso-lated from T. hirsuta was purified to homogeneity by ammoniumsulfate precipitation, DEAE-sepharose anion exchange chromatog-raphy and Sephacryl S-200 gel exclusion chromatography with ayield of 31%, 180-fold purification and 360 units/mg specific activ-ity [20]. These traditional methods of enzyme purification did notperform satisfactorily in our hands, which led to the developmentof the copper alginate entrapment (affinity precipitation) methodfor the purification of laccase. This purification protocol proved tobe economic, using inexpensive and easily obtainable materials,making it suitable for large scale commercial production of laccases.

3.2. Native and subunit molecular weight determination oflaccases

The native molecular weight of the purified laccase, wasfound to be ∼220 kDa (Fig. 2) by gel filtration chromatography.SDS-PAGE analysis of the purified laccase revealed two subunits,one of 100 kDa and other of 120 kDa, suggesting a heterodimericstructure for Leucaena laccase (Fig. 3). The majority of laccasesreported from plant sources are monomeric, having a subunit

Fig. 2. Calibration plot for the determination of the native molecular weight ofpurified laccase using gel filtration chromatography. The standard protein mark-ers (1.0 mg/ml) used were: catalase (240 kDa), �-amylase (200 kDa), phosphorylaseB (97.4 kDa), bovine serum albumin (67 kDa) and lysozyme (14.3 kDa).

molecular weight in the range of 60–100 kDa [16,29]. Recently,multimeric laccase isoforms of O. vulgaris (OV137 and OV90) havebeen reported, exhibiting a subunit molecular mass of 43 kDa andnative molecular weight of 137 kDa [11]. There are also reportsof laccases from fungal and bacterial sources being homodimeric[30–32], heterodimeric [10] and homotrimeric [4]. Dimantidis andcoworkers [33] have reported a multimeric laccase from a soil bac-terium Azospirillum lipoferum that consisting of one catalytic chainof 16.3 kDa and one or two regulatory/structural heavy chains of81.5 kDa.

3.3. Effect of temperature, pH, and substrates on laccase activity

3.3.1. Effect of temperatureThe effect of temperature on purified laccase was investigated,

and the results are presented in Fig. 4A. The data revealed a rapidincrease in laccase activity from 50 to 80 ◦C followed by a decline inlaccase activity of approximately 26% at 90 ◦C. Similar reports of lac-cases that are active at higher temperature have been obtained fromplant (O. vulgaris (OV137, 80 ◦C and OV90, 70 ◦C, [11]) and Cereuspterogonus (90 ◦C, [12])), bacterial (Thermus thermophilus (80 ◦C)[34]) and fungal (basidiomycete strain PM1 (80 ◦C, [35]), Marasmiusquercophilus (80 ◦C, [36]) and T. hirsuta (85 ◦C, [20])) sources. TheQ10 value of Leucaena laccase was found to be >1 between 10 and70 ◦C suggesting that the reaction rate is temperature-dependent.The energy of activation (Ea) as determined from the slope of theArrhenius plot was found to be 6.9 kJ mol−1 (Fig. 4A).

Fig. 3. SDS-PAGE and silver staining of purified laccase. Lane 1: purified laccase,Lane 2: molecular weight markers. Standard protein markers containing myosin(205 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin(43 kDa) and carbonic anhydrase (29 kDa) were used.



Fig. 4. (A) Effect of temperature on the activity of L. leucocephala laccase. The enzyme was incubated at different temperature (10–90 ◦C) and activity was measured understandard assay conditions. The inset shows the Arrhenius plot. (B) Thermal stability of L. leucocephala laccase at 80 ◦C. Percent relative activity represents the enzyme activityrelative to the control (0 min), which was set at 100%.

Time-dependent thermostability of Leucaena laccase was alsoinvestigated at 80 ◦C (Fig. 4B). The data revealed that Leucaena lac-case was activated (by approximately 45%) upon pre-incubation at80 ◦C for up to 50 min, after which there was a decline in activation,suggesting that the enzyme was fairly thermostable. Similar acti-vation upon pre-incubation of laccases at higher temperatures hasbeen reported from a number of fungal sources [35,37]. The hyper-activation of the Leucaena laccase observed in the present studymight be attributed to interactions between the active site copperions as well as ionic and hydrogen bonding interactions, leading tostabilization of more active conformation, as suggested by Hildenet al. [37].

3.3.2. Effect of pHThe effect of pH on the purified Leucaena laccase revealed pH

optima of 7.0 with a steep decline in both acidic as well as basicpH ranges (Fig. 5A). Laccase stability was also investigated and itrevealed that the enzyme retained more than 80% of its activity atpH values ranging from 6.0 to 9.0 within 24 h at 37 ◦C (Fig. 5B). Sim-ilar to our observations, pH optima of approximately 7.0 have beenreported for laccase from R. vernicifera and R. succedanea using cat-echol as a substrate [13]. The pH optimum of the Leucaena laccase inthe neutral range shows the potential of the enzyme for its applica-tion in the biobleaching industry, as these processes require neutralto alkaline conditions [38]. Recently, laccase isoforms isolated fromtwo xerophytic plant species, C. pterogonus and O. vulgaris, have

been found to show an optimum pH of 10 [17]. However, bacte-rial and fungal laccases are reported to exhibit acidic pH optima,e.g., A. lipoferum (6.0, [33]), Streptomyces cyaneus and Trametes ver-sicolor (3–5, [39]), Pleurotus ostreatus (3.5, [40]). The Melanocarpesalbomyces laccase exhibited an atypical pH optimum of 7.0 for phe-nolic substrates [41]. Some alkaline laccases have been reportedfrom a fungus, Myrothecium verrucaria (9.0, [42]), and bacteria,Streptomyces coelicolor (9.4, [43]) and Thermobifida fusca (8.0, [44]).

3.3.3. Effect of substrates on laccase activityThe effects of various polyphenolic (catechol and hydroquinone)

and non-phenolic (ABTS) substrates on the purified enzyme wereinvestigated and various kinetic parameters were calculated whichis presented in Table 2. Km and Vmax values were found to be 1.24,1.92, and 6.86 mM and 0.063, 0.088, and 0.351 �M min−1 ml−1,for catechol, ABTS and hydroquinone, respectively. Kcat of theenzyme for these substrates were found to be in the order: hydro-quinone (14.03 min−1) > ABTS (3.53 min−1) > catechol (2.52 min−1).However, the order of catalytic efficiency of the purified lac-caase for the substrates (as presented by Kcat/Km) was foundto be in the order: hydroquinone (2.05 mM−1 min−1) > catechol(2.03 mM−1 min−1) > ABTS (1.83 mM−1 min−1). As evident from theKm values, catechol exhibited the highest affinity, while hydro-quinone exhibited the least affinity for laccase. Km of catechol forlaccase from various plant sources has been found to vary widely;for example, Km values of 3.13, 15 and 45 mM have been reported

Fig. 5. (A) Effect of pH on the activity of L. leucocephala laccase. The activity was assayed at different pH (6.0–9.0) under standard assay conditions. (B) pH stability of L.leucocephala laccase. The enzyme was incubated for 24 h at different pH (6.0–9.0) and activity assayed using a suitable pre-incubated enzyme aliquot under standard assayconditions. Percent relative activity represents enzyme activity calculated by setting the activity, at optimum pH, as 100%.



Table 2Kinetic properties of L. leucocephala laccase with various phenolic and non-phenolic substrates.

Substrates Km (mM) Vmax (�M min−1 ml−1) Kcat (min−1) Kcat/Km (mM−1 min−1)

Catechol 1.24 0.063 2.52 2.03ABTS 1.92 0.088 3.53 1.83Hydroquinone 6.86 0.351 14.03 2.05

for laccases from Amorphophallus campanulatus, Rhus succedaneaand R. vernicifera, respectively [13,45]. Thus, the Leucaena laccaseexhibited a lower Km value compared with laccases isolated fromother plants. Furthermore, based on the ability of the Leucaenalaccase to efficiently oxidize both o- and p-diphenol substrates(catechol and hydroquinone, respectively), the purified enzyme isvalidated as a laccase, as suggested by Ferrar and Walker [46]. TheKcat value of L. leucocephala laccase for ABTS (3.53 min−1) was lowerthan those found for several other laccases, such as those of T. hir-suta (197 s−1 [20]), Pleurotus pulmonarius (1520 s−1 [31]), and P.ostreatus (244.32 s−1 [40]).

A comparison of the physico-chemical properties, namely Km forvarious substrates, pH, temperature optima, and molecular weight,of L. leucocephala laccase, with those of laccases reported from var-ious plant, bacterial, and fungal sources are summarized in Table 3.

3.4. Effect of various metal ions, SDS, DTT, and EDTA

The effects of different effectors, such as metal ions, detergents(SDS), reducing agents (DTT), and chelating agents (EDTA) on Leu-caena laccase activity were investigated and are shown in Table 4.

Mn2+, Cd2+, and Na+ activated laccase in a concentration-dependentmanner (0.1–10 mM). However, Mg2+ did not have any effect, whileCa2+ exhibited activation of laccase up to 3 mM, beyond whichit exhibited an inhibition of laccase activity. Fe2+ and Cu2+ acti-vated laccase in a concentration-dependent manner up to 1 mM. Aconcentration-dependent inhibition of laccase activity with Hg2+

and Co2+ up to 1 mM was observed. The effect of metals, suchas Fe2+, Cu2+, Hg2+ and Co2+, on laccase activity at concentra-tions higher than 1 mM could not be determined because of theinterference of high concentrations of these metal salts with colordevelopment. Similar to our results, the addition of Mn2+, Cd2+, Cu2+

and Fe2+ have been reported to increase the activity of laccases fromO. vulgaris [17], P. ostreatus [19], and Streptomyces psammoticus [3].The involvement of four copper ions, distributed at the three dif-ferent copper centers via type-1 (T1) or blue copper center, type-2(T2) or normal copper and type-3 (T3) or coupled binuclear coppercenters, exhibiting characteristic UV/vis and electron paramagneticresonance (EPR) spectra, have been suggested during the catalysisof laccase [1]. Thus, the increase in activity with divalent metal ionsmight be due to their competition with Cu2+ in the electron trans-port system, leading to a positive cooperative relationship between

Table 3A comparison of physico-chemical properties of purified L. leucocephala laccase with other reported plant, bacterial and fungal laccases.

Organism Substrates with Km Optimumtemp. (◦C)

OptimumpH

Mol. wt.(kDa)

Ref.

PlantsLeucaena leucocephala Catechol, 1.24 mM

ABTS, 1.92 mMHydroquinone, 6.86 mM

80 7.0 220 Present study

Opuntia vulgaris(OV137) 2,6-DMP, 2.2 mM 80 10.0 137 [11](OV90) 2,6-DMP, 2.2 mM 70 10.0 90Cereus pterogonus(CP137) 2,6-DMP, 2.1 mM 90 10.0 137 [12](CP90) 2,6-DMP, 2.1 mM 90 10.0 90(CP43) 2,6-DMP, 2.1 mM 60 10.0 43Rhus vernicifera Catechol, 45 mM 40 7.0 – [13]Rhus succedanea Catechol, 15 mM 50 7.0 – [13]Morus alba 4-Methylcatechol, 6 mM 45 7.0 62–64 [16]

BacteriaThermus thermophilus – 92 – 53 [34]Azospirillum lipoferum Syringaldazine, 34.65 �M – 6.0 179.3 [33]Streptomyces psammoticus Pyrogallol, 0.25 mM

ABTS, 0.39 mM45 8.5 43 [3]

Streptomyces ipomoea ABTS, 0.40 mM2,6-DMP, 4.27 mM

60 5.0 79 [24]

Thermobifida fusca – 60 8.0 73.3 [44]

FungiTrametes hirsuta ABTS, 0.07 mM

DMP, 0.2 mM85 2.4–2.5 90 [20]

Trichoderma harzianum ABTS, 180 �MGuaiacol, 60 �M

85 4.5 79 [14]

Ganoderma lucidum ABTS, 77 �MGuaiacol, 217 �M

50 4.5 62 [18]

Pleurotus pulmonarius ABTS, 210 �MGuaicacol, 550 �M

45 4.0–5.5 46 [31]

Marasmius quercophilus ABTS, 50 mMSyringaldazine, 7.7 mM

80 6.2 65 [36]

Pleurotus ostreatus ABTS, 46.51 mMDMP, 400 mMGuaiacol, 100 mMo-Dianisidine, 23.52 mM

50 4.5 68.4 [40]



Table 4Effect of various effectors on purified laccase activity. Percent relative activity represents enzyme activity relative to control (without any effector) which was taken ashundred percent. The values presented as the mean ± SD of triplicate tests.

Effectors % Relative activity

Concentration (mM)

0 0.1 0.5 1 3 5 10

Mn2+ 100 110 ± 1.7 149 ± 1.5 195 ± 2.0 376 ± 2.2 403 ± 2.0 431 ± 2.5Fe2+ 100 128 ± 1.6 178 ± 1.4 186 ± 1.7 * * *

Cd2+ 100 105 ± 1.5 114 ± 1.5 128 ± 1.8 159 ± 2.4 189 ± 2.2 189 ± 2.2Na+ 100 116 ± 1.3 129 ± 1.7 142 ± 1.7 148 ± 2.0 154 ± 1.5 159 ± 1.6Cu2+ 100 112 ± 1.3 123 ± 1.2 130 ± 1.4 * * *

Ca2+ 100 105 ± 1.8 112 ± 1.3 126 ± 1.6 139 ± 1.8 124 ± 2.0 104 ± 2.1Mg2+ 100 100 100 100 100 100 100Hg2+ 100 85 ± 1.7 66 ± 1.6 50 ± 1.8 * * *

Co2+ 100 70 ± 1.4 52 ± 1.4 37 ± 1.3 * * *

DTT 100 170 ± 1.8 147 ± 1.4 72 ± 2.1 33 ± 1.5 9 ± 1.5 0SDS 100 84 ± 1.5 71 ± 1.8 65 ± 1.7 53 ± 1.6 42 ± 2.3 11 ± 2.0EDTA 100 91 ± 1.8 38 ± 1.5 5 ± 1.8 0 0 0

* salts of Fe2+, Cu2+, Hg2+ and Co2+ at concentrations beyond 1 mM interfered with the color development during enzyme activity assay and hence the effect could not bemeasured.

the enzyme and substrate, as suggested by Chao et al. [47]. Theactivation of laccase by Cu2+ may be due to the filling of type-2 cop-per binding sites with Cu2+ ions [14]. The inhibition by the metalions Hg2+ and Co2+ have also been reported [14,48], and the reasonbehind which might be due to the amino acid residue modifications,conformational changes or copper chelation [49].

DTT activated laccase at lower concentrations, while it inhibitedthe enzyme at concentrations above 0.1 mM in a concentration-dependent manner. SDS inhibited laccase activity at all of theconcentrations tested in a concentration-dependent manner. EDTAwas found to strongly inhibit laccase in such a way that at concen-trations above 1 mM, the enzyme was completely inhibited. It islikely that at lower concentration of DTT, only a limited number ofS S bonds is split (may be the only one which supports the enzymeactive structure), providing the enzyme with some optimal flexi-bility and activity. However, at higher concentrations of DTT theunique three-dimensional conformation of the enzyme might haveaffected, thus resulting in an inhibition in enzyme activity [50]. Theinhibition by SDS and EDTA was found almost similar to the lac-cases isolated from peach [29], S. cyaneus [51], and S. psammoticus[3]. The disulfide reducing agent, DTT, and the anionic detergent,SDS, might have also caused a conformational change in the pro-tein, resulting in the inhibition of the enzyme activity. The stronginhibition of the enzyme by EDTA showed that the purified Leu-caena laccase was highly sensitive to copper chelation, resulting inthe conformational change of the protein and the loss of enzymeactivity. It has been reported that the type-2 Cu2+ can be reversiblyremoved from the protein with the chelating reagent EDTA, suchthat the copper-depleted protein is enzymatically inactive, indi-cating that the type-2 Cu2+ of laccase has a functional role in theprotein [52].

3.5. Effect of organic solvents on the stability of laccase

The effects of various polar (methanol, ethanol, isopropanol,DMF, DMSO) and non-polar (benzene and chloroform) organicsolvents on the stability of Leucaena laccase have been investigatedand are presented in Fig. 6. The Leucaena laccase was found to bequite stable in presence of 20% (v/v) of all the organic solventstested. The enzyme was found to retain more than 80% activity inthe presence of all of the organic solvents except isopropanol, inwhich it retained approximately 60% activity. In the presence ofhigher concentrations (50% (v/v)) of ethanol, DMF, DMSO and ben-zene, laccase retained more than 50% activity, while the enzymeretained 42, 37 and 37% activity in the presence of methanol,

Fig. 6. Organic solvent stability of L. leucocephala laccase. The enzyme was incu-bated for 1 h with 20% and 50% (v/v) of organic solvents at 37 ◦C and subsequentlythe activity was assayed using a suitable aliquot of pre-incubated enzyme understandard assay conditions. Percent residual activity represents the enzyme activityrelative to the control (without any organic solvent), which was taken as 100%.

isopropanol and chloroform, respectively. In the literature, thereare few reports of organic solvent-tolerant laccases from plant,Rhus vernicifera [53]; white-rot fungus, Ganoderma fornicatum[54]; and bacteria, Bacillus licheniformis [47] and T. fusca [44].Laccases tolerant to organic solvents appear to be quite attractivefor their industrial applications, such as in the bioremediationof industrial waters contaminated with organic solvents, and inorganic synthesis and chiral resolution.

3.6. Effect of laccase on dye decolorization

The ability of Leucaena laccase to oxidize industrial dyes wasinvestigated to demonstrate its industrial applicability. The effectof enzyme concentration on the dye decolorization revealedthat with increasing concentration of laccase, decolorization ofindigocarmine (10 �g/ml) and congo red (10 �g/ml) increasedprogressively and the complete decolorization of both the dyeswere achieved within 6 h at 11 and 22 �g of enzyme, respectively(Fig. 7A). The rate of decolorization (oxidation) of the two dyes bythe Leucaena laccase was also investigated at various dye concen-trations (10–250 �g/ml) and the data are shown in Fig. 7B. Thoughthe rate of dye decolorization was found to increase with dye con-centration but it was not directly proportional to the concentration



Fig. 7. (A) Effect of L. leucocephala laccase concentration on the decolorization of indigocarmine (10 �g/ml) and congo red (10 �g/ml) dyes. The reaction mixture (3 ml)containing dyes (30 �g) in Tris–HCl (100 mM, pH 7.5) buffer was incubated for 6 h at 37 ◦C and the change in color was measured at 610 nm (for indigocarmine) and 500 nm(for congo red). (B) Rate of decolorization of indigocarmine and congo red dyes by L. leucocephala laccase. The change in color was measured upon varying the dye concentration(10–250 �g/ml) in the reaction mixture keeping the enzyme amount 11 �g and 22 �g for indigocarmine and congo red, respectively.

of the dye. Thus, in case of indigocarmine, the rate of dye decol-orization at a dye concentration of 10 �g/ml was 5.4 �M/min/mgwhile at 250 �g/ml dye concentration, the rate of decolorizationwas 33.8 �M/min/mg. Similarly, in case of congo red, the rateof dye decolorization at a dye concentration of 10 �g/ml was1.81 �M/min/mg while at 250 �g/ml dye concentration, the rateof decolorization was 11.3 �M/min/mg. The rate of decolorization(oxidation) of indigocarmine was about three times faster thanthat of congo red at all dye concentrations. The variation in thedecolorization efficiency of the two dyes might be attributed tothe structural variation of the dyes, as suggested by Nyanhongoand coworkers [55]. Laccases from fungi, such as Panus rudis [23]and T. versicolor [15], have been reported to decolorize the indigo-carmine dye in the presence of ABTS. The purified Leucaena laccasehad an advantage of decolorizing the tested dyes (indigocarmineand congo red) without any additional redox mediators, such asABTS. Thus, the Leucaena laccase possesses an additional advantagewith regards to its application in textile industries.

4. Conclusion

A quick purification protocol using copper alginate bead entrap-ment with celite chromatography has been successfully utilized topurify laccase to homogeneity. The purified laccase was found tobe a heterodimeric protein showing a pH optimum in the neutralrange and thermostability up to 80 ◦C. The enzyme was found tobe active with both phenolic and non-phenolic substrates. Laccasewas potentially activated by Mn2+, Cd2+, Fe2+, Cu2+ and Na+ andinhibited by Co2+, Hg2+, DTT, SDS and EDTA, in a concentration-dependent manner. The enzyme was tolerant toward a number ofpolar and non-polar organic solvents. The remarkable decoloriza-tion ability of the enzyme suggests that it may have great potentialin the decolorization of effluents of dyes and in textile industries.

Acknowledgements

Financial support from UGC, New Delhi, India, in the form ofa Dr. D. S. Kothari Post doctoral Fellowship to N.J. is gratefullyacknowledged. Department of Higher Education, Government ofUttar Pradesh, India under the Centre of Excellence in Bioinformat-ics, Department of Biotechnology, Government of India under theBIF Scheme, New Delhi and Department of Science and Technology,New Delhi, under Promotion of University Research and ScientificExcellence (DST-PURSE) program are also gratefully acknowledgedfor providing infrastructure facilities.

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