Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization

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Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of 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

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Article history: Received 20 December 2013 Received in revised form 24 March 2014 Accepted 2 April 2014 Available online xxx Keywords: Celite chromatography Copper alginate Dye decolorization Laccase Leucaena leucocephala

a b s t r a c t Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick twostep procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccase was found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity of laccase was found to be highest for catechol and lowest for hydroquinone, however, highest Kcat and Kcat /Km were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0 and 80 ◦ C, respectively. Mn2+ , Cd2+ , Fe2+ , Cu2+ and Na+ activated laccase while Ca2+ treatment increased laccase activity up to 3 mM, beyond which it inhibited laccase. Co2+ , Hg2+ , DTT, SDS and EDTA showed an 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 than 50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol and chloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized synthetic dyes 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) are multicopper oxidases that catalyze the oxidation of various phenolic and non-phenolic aromatic compounds using molecular oxygen [1]. They are widely distributed in nature and have been found in plants, insects, bacteria, fungi and crustaceans. Most laccases have been isolated and characterized extensively from fungal sources. However, a considerably lesser number of laccases have been characterized from plant sources to date. The laccases reported from plants as well as fungi have been purified using traditional methods, such as salt or solvent precipitation and column chromatography, which not only took longer time but also resulted in a 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 economical purification methods. In this study, we report an alginate bead entrapment method for Leucaena laccase purification that may 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 economical method for purifying other enzymes, such as ␣- and ␤-amylases, glucoamylase, pectinase, phospholipase and ␣-galactosidase [5]. Alginate, a copolymer of ␤-d-mannuronic acid (M) and ␣-lguluronic acid (G) residues, has been exploited most commonly for entrapment purposes in the form of calcium alginate beads. However, besides calcium, various other divalent cations have also been shown 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 the entrapped enzyme is dependent upon the nature and composition of the metal as well as its concentration in the bead [6]. Thus, a calcium alginate bead will have a higher affinity for a Ca2+ -containing enzyme than for another metal-containing enzyme. One such example is the purification of ␣-amylase, a calcium containing metalloenzyme, using calcium alginate beads [5]. Therefore, this property has been exploited in the present study for purifying laccase, a copper containing metalloenzyme, using copper alginate beads. Though copper alginate beads have been used for the immobilization of laccase [7–9], it has not yet been used for the purification of laccase or any other enzyme, to the best of our knowledge.

http://dx.doi.org/10.1016/j.procbio.2014.04.002 1359-5113/© 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Jaiswal N, et al. Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.002

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Laccases isolated from various sources exhibited diversity with regards to their size and subunit composition. Multimeric, homotetrameric, heterodimeric as well as monomeric forms of laccases have been reported, ranging in subunit molecular weight from 36 to 175 kDa [2,10–12]. Though the pH optima for plant laccases varied from neutral to alkaline range [11–13], while most of the fungal and bacterial laccases exhibited an acidic pH optima [14,15]. The temperature optima of laccases have been reported to vary widely in the range of 25–90 ◦ C, depending on the source of the laccase [16–18]. Fungal laccases usually have a lower thermal stability than bacterial laccases [19]. Recently, a laccase from Trametes hirsuta showed an optimum temperature of 85 ◦ C [20]. Laccases have been inhibited by a number of ions and compounds, such as metal ions (e.g., Ca2+ , Mg2+ , Co2+ , Mn2+ , Cd2+ , Zn2+ , Hg2+ , etc.), sulfhydryl reagents, EDTA, dithiothreitol, hydroxyglycine, kojic acid, thiourea, detergents, etc. [1]. Laccases have applications in various industrial processes, such as textile dye bleaching, pulp bleaching, effluent detoxification, bioremediation of contaminating environmental pollutants, enzymatic conversion of chemical intermediates, and organic synthesis [1]. Therefore, for these industrial applications, laccases that are resistant to extreme conditions of pH, temperature, high salt, and organic solvents, are highly desirable. Furthermore, laccases have become an attractive option (in contrast to peroxidases) for the decolorization of synthetic dyes from industrial wastes [21], as they do not require expensive H2 O2 as a co-substrate and have broader substrate specificity. In the presence of mediators, the substrate specificity of laccases can be further enhanced, leading to the oxidation of more complex substrates [22]. Thus, laccases are capable of oxidizing a wide variety of aromatic compounds, such as ortho-, meta- or para- substituted phenols; diamines; aromatic amines and thiols; and inorganic compounds, such as iodine, Mo(CN)8 4− , and Fe(CN)6 4− [1,19]. Several microbial laccases have been assessed for their potential application in dye decolorization [23,24], however, the reports on dye decolorization by plant laccases is scanty. Therefore, there is a need to search for potential plant laccases with an ability to degrade dyes. In the present paper, we describe a rapid purification protocol for laccase from the leaves of Leucaena leucocephala, a tree legume which is of significance to the fodder and the paper and pulp industries in India. In addition, purified laccase was characterized with regards to the effects of temperature, pH, substrates, various effectors, and organic solvents and the ability to oxidize industrial dyes. To the best of our knowledge, this paper is the first report of a protocol for the purification of a laccase and its effectiveness in dye decolorization. 2. Experimental 2.1. Plant material Fresh green and young leaves from a 5 years old tree of L. leucocephala, which was growing in the garden of the University of Lucknow, 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. After 30 min of incubation at 37 ◦ C, the increase in absorbance (due to oxidation of catechol to o-benzoquinone) was measured at 390 nm using UV–vis spectrophotometer (Elico SL-177). A parallel control containing all the ingredients of the assay system, except the enzyme, was used as blank and those without substrate

were used as control. Enzyme activity was expressed in terms of units. One unit of enzyme activity was defined as the amount of enzyme required to produce 1 ␮mol of o-benzoquinone in 1 min under the specified conditions (ε = 1260 M−1 cm−1 ). Similarly, enzyme activity using substrates hydroquinone and ABTS, were determined by measuring increase in absorbance at 390 nm (ε for p-benzoquinone = 2240 M−1 cm−1 ), and 420 nm (ε for ABTS+ free radical = 36,000 M−1 cm−1 ), respectively. Protein concentration was estimated by the Bradford dye binding 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 Leucaena leaves in 120 ml of Tris–HCl buffer (100 mM, pH 7.5) using an ice-cold blender. Solid PVP (polyvinylpyrrolidone, insoluble; 0.1% (w/v)) and 7 mM ␤-mercaptoethanol were added at the time of extraction. The homogenate was centrifuged at 8500 × g for 30 min at 4 ◦ C using a Sigma 4K15 centrifuge. The clear supernatant (crude extract) was subjected to further purification. All of the operations were performed at 4 ◦ C, unless otherwise specified. 2.4. Purification of laccase 2.4.1. Entrapment in copper alginate beads For the purification of laccase, a copper alginate-mediated entrapment (affinity precipitation) method, modified from that described by Prakash and Jaiswal [5], was used. Crude extract (100 ml) was mixed with 200 ml of sodium alginate (2%, w/v) and kept at 4 ◦ C for 30 min with occasional hand swirling. Copper alginate beads (containing the entrapped enzyme) were prepared by adding the enzyme–sodium alginate mixture in a drop-wise manner to 1 l of a pre-cooled CuSO4 solution (100 mM) with continuous gentle hand swirling. The copper alginate beads formed using this method were allowed to harden for 1 h. Afterwards, the beads were washed twice with Tris–HCl buffer (100 mM, pH 7.5, 200 ml per wash). Entrapped enzymes were eluted from the beads with 100 ml of Tris–HCl buffer (100 mM, pH 7.5) containing 1 M NaCl using constant agitation at 100 rpm for 30 min at 30 ◦ C. The eluate was collected by decanting. The eluted enzyme preparation was dialyzed overnight against the Tris–HCl buffer (100 mM, pH 7.5) with two to three changes. The dialyzed preparation was used for further purification. 2.4.2. Celite adsorption chromatography Twenty grams of Celite 545 (diatomaceous earth, obtained from Sigma–Aldrich) was mixed with 250 ml of distilled water, boiled for 5 min, and subsequently celite was allowed to settle at room temperature. The supernatant was decanted. The celite was washed 3–4 times with distilled water to remove the fine particles. The washed celite was suspended in Tris–HCl buffer (100 mM, pH 7.5) and packed in a glass column (1.5 cm × 50 cm). The dialyzed enzyme preparation (100 ml) was applied on the top of the celite column. The pass-through fractions (5 ml) containing laccase activity were pooled and concentrated using Centricon with a 50 kDa-molecular mass-cut-off. The concentrated enzyme preparation was stored at 4 ◦ C for further use. 2.5. Native PAGE and in-gel activity staining Native polyacrylamide gel-electrophoresis (7.5%; PAGE) was performed and visualized using silver staining. In-gel activity staining of the laccase was performed by immersing the gel (after 7.5% native PAGE) in a catechol solution (50 mM) containing Tris–HCl buffer (100 mM, pH 7.5) until brown-colored bands appeared.

Please cite this article in press as: Jaiswal N, et al. Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.002

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2.6. Native molecular weight determination

2.11. Effect on dye decolorization

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

Synthetic industrial dyes, namely indigocarmine (max 610 nm) and congo red (max 500 nm), were used for investigating the efficacy of decolorization by purified laccase. Stock solutions (1 mg/ml) of these dyes were prepared in distilled water and diluted to the required 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 appropriate amount of enzyme. A reaction mixture without enzyme was also run. After incubation for 6 h at 37 ◦ C, the change in the absorbance was measured spectrophotometrically using UV–vis spectrophotometer (Elico SL-177). The effects of varying enzyme amounts (1.4–22 ␮g) and dye concentrations (10–250 ␮g/ml) on dye decolorization were also investigated. 2.12. Statistical analysis

2.7. Subunit molecular weight determination For subunit molecular weight determination, SDS-PAGE was conducted in a mini electrophoresis chamber (Bio-Rad) at room temperature using a 7.5% resolving and 3% stacking gel in Tris–glycine running buffer (pH 8.8) at 100 V for 90 min [28]. 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. 2.8. Effect of temperature, pH and substrates Laccase activity at various temperatures (10–90 ◦ C) was investigated by incubating the whole assay system at pH 7.0 as described earlier in Experimental section. The thermostability of the enzyme was determined by incubating the enzyme at 80 ◦ C and then assaying the activity at various time intervals. Similarly, the effect of pH on laccase activity was determined using 100 mM of various buffers at different pH values (such as sodium acetate, pH 6.0 and 6.5; Tris–HCl, pH 7.0, 7.5, 8.0 and 8.5; sodium borate, pH 9.0 and sodium carbonate, pH 9.5) under standard assay conditions at 37 ◦ C. The pH stability of the enzyme was determined by estimating the activity after incubating the enzyme at different pH values (6.0–9.0) for 24 h. The effect of phenolic (catechol and hydroquinone) and non-phenolic (ABTS) substrates was investigated at concentrations ranging from 0 to 50 mM at 37 ◦ C and pH 7.0. Km and Vmax values were obtained by non-linear regression of a plot of enzyme activity vs substrate concentration (hyperbolic Michaelis–Menten plot) using GraphPad Prism software.

The experiments were performed in triplicates, and the mean and standard deviation were calculated accordingly. 3. Results and discussion 3.1. Purification of laccase Laccase was purified to homogeneity from the leaves of Leucaena using copper alginate bead entrapment followed by celite chromatography (Table 1). The homogeneity of the purified enzyme was established using native PAGE, where a single band was obtained (Fig. 1A). The purified laccase was found to be catalytically active as established through in-gel activity staining (Fig. 1B). The laccase was purified to 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. Thus, the copper alginate bead entrapment method (affinity precipitation) has been exploited successfully 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

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 pH 7.0) by performing the activity assay using catechol as the substrate in the presence and absence of individual effectors at specified concentrations 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 dimethyl fluoride) and non-polar organic solvents (benzene and chloroform) at 20% and 50% concentrations (v/v) was studied by incubating the enzyme in the respective solvents for 1 h at 37 ◦ C, and subsequently measuring the activity of enzyme aliquots under the standard assay conditions (at 37 ◦ C and pH 7.0).

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

Please cite this article in press as: Jaiswal N, et al. Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.002

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Table 1 Summary of laccase purification from Leucaena leucocephala leaves. Steps

Volume (ml)

Total activity (U)

Total protein (mg)

Specific activity (U/mg protein)

Yield (%)

Fold purification

Crude extract Affinity precipitation Celite chromatography (concentrated using centricon)

100 100 10

31.5 18.5 16.1

59.6 2.7 0.3

0.53 6.8 58.5

100 58.7 51.0

– 12.8 110.6

procedures, such as ammonium sulfate or organic solvent precipitation and column chromatography techniques, which result in low enzymatic yields. For example, a plant laccase isolated from a xerophyte species, Opuntia vulgaris, was purified by acetone precipitation, Sephadex A-50 anion exchange chromatography, and Sephadex G-100 gel filtration chromatography with a 6.6% yield, 21.6-fold purification, and 120.9 × 10−4 (IU/mg) specific activity [11]. A fungal laccase isolated from Trichoderma harzianumn was purified 151.7-fold with a yield of 0.39% and specific activity of 130.5 units/mg through acetone precipitation, ultrafiltration, Sephadex G-100 column chromatography and Concanavalin-A affinity chromatography [14]. Similarly, a Scytalidium thermophilum laccase was also reported to be purified to 7.9-fold using various steps, 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 isolated from T. hirsuta was purified to homogeneity by ammonium sulfate precipitation, DEAE-sepharose anion exchange chromatography and Sephacryl S-200 gel exclusion chromatography with a yield of 31%, 180-fold purification and 360 units/mg specific activity [20]. These traditional methods of enzyme purification did not perform satisfactorily in our hands, which led to the development of the copper alginate entrapment (affinity precipitation) method for the purification of laccase. This purification protocol proved to be economic, using inexpensive and easily obtainable materials, making it suitable for large scale commercial production of laccases.

molecular weight in the range of 60–100 kDa [16,29]. Recently, multimeric laccase isoforms of O. vulgaris (OV137 and OV90) have been reported, exhibiting a subunit molecular mass of 43 kDa and native molecular weight of 137 kDa [11]. There are also reports of laccases from fungal and bacterial sources being homodimeric [30–32], heterodimeric [10] and homotrimeric [4]. Dimantidis and coworkers [33] have reported a multimeric laccase from a soil bacterium Azospirillum lipoferum that consisting of one catalytic chain of 16.3 kDa and one or two regulatory/structural heavy chains of 81.5 kDa. 3.3. Effect of temperature, pH, and substrates on laccase activity 3.3.1. Effect of temperature The effect of temperature on purified laccase was investigated, and the results are presented in Fig. 4A. The data revealed a rapid increase in laccase activity from 50 to 80 ◦ C followed by a decline in laccase activity of approximately 26% at 90 ◦ C. Similar reports of laccases that are active at higher temperature have been obtained from plant (O. vulgaris (OV137, 80 ◦ C and OV90, 70 ◦ C, [11]) and Cereus pterogonus (90 ◦ C, [12])), bacterial (Thermus thermophilus (80 ◦ C) [34]) and fungal (basidiomycete strain PM1 (80 ◦ C, [35]), Marasmius quercophilus (80 ◦ C, [36]) and T. hirsuta (85 ◦ C, [20])) sources. The Q10 value of Leucaena laccase was found to be >1 between 10 and 70 ◦ C suggesting that the reaction rate is temperature-dependent. The energy of activation (Ea ) as determined from the slope of the Arrhenius plot was found to be 6.9 kJ mol−1 (Fig. 4A).

3.2. Native and subunit molecular weight determination of laccases The native molecular weight of the purified laccase, was found 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 heterodimeric structure for Leucaena laccase (Fig. 3). The majority of laccases reported from plant sources are monomeric, having a subunit

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

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.

Please cite this article in press as: Jaiswal N, et al. Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.002

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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 under standard assay conditions. The inset shows the Arrhenius plot. (B) Thermal stability of L. leucocephala laccase at 80 ◦ C. Percent relative activity represents the enzyme activity relative to the control (0 min), which was set at 100%.

Time-dependent thermostability of Leucaena laccase was also investigated at 80 ◦ C (Fig. 4B). The data revealed that Leucaena laccase was activated (by approximately 45%) upon pre-incubation at 80 ◦ C for up to 50 min, after which there was a decline in activation, suggesting that the enzyme was fairly thermostable. Similar activation upon pre-incubation of laccases at higher temperatures has been reported from a number of fungal sources [35,37]. The hyperactivation of the Leucaena laccase observed in the present study might be attributed to interactions between the active site copper ions as well as ionic and hydrogen bonding interactions, leading to stabilization of more active conformation, as suggested by Hilden et al. [37]. 3.3.2. Effect of pH The effect of pH on the purified Leucaena laccase revealed pH optima of 7.0 with a steep decline in both acidic as well as basic pH ranges (Fig. 5A). Laccase stability was also investigated and it revealed that the enzyme retained more than 80% of its activity at pH values ranging from 6.0 to 9.0 within 24 h at 37 ◦ C (Fig. 5B). Similar to our observations, pH optima of approximately 7.0 have been reported for laccase from R. vernicifera and R. succedanea using catechol as a substrate [13]. The pH optimum of the Leucaena laccase in the neutral range shows the potential of the enzyme for its application in the biobleaching industry, as these processes require neutral to alkaline conditions [38]. Recently, laccase isoforms isolated from two xerophytic plant species, C. pterogonus and O. vulgaris, have

been found to show an optimum pH of 10 [17]. However, bacterial and fungal laccases are reported to exhibit acidic pH optima, e.g., A. lipoferum (6.0, [33]), Streptomyces cyaneus and Trametes versicolor (3–5, [39]), Pleurotus ostreatus (3.5, [40]). The Melanocarpes albomyces laccase exhibited an atypical pH optimum of 7.0 for phenolic substrates [41]. Some alkaline laccases have been reported from 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 activity The effects of various polyphenolic (catechol and hydroquinone) and non-phenolic (ABTS) substrates on the purified enzyme were investigated and various kinetic parameters were calculated which is 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 the enzyme for these substrates were found to be in the order: hydroquinone (14.03 min−1 ) > ABTS (3.53 min−1 ) > catechol (2.52 min−1 ). However, the order of catalytic efficiency of the purified laccaase for the substrates (as presented by Kcat /Km ) was found to 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 the Km values, catechol exhibited the highest affinity, while hydroquinone exhibited the least affinity for laccase. Km of catechol for laccase 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 assay conditions. Percent relative activity represents enzyme activity calculated by setting the activity, at optimum pH, as 100%.

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Table 2 Kinetic 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 ABTS Hydroquinone

1.24 1.92 6.86

0.063 0.088 0.351

2.52 3.53 14.03

2.03 1.83 2.05

for laccases from Amorphophallus campanulatus, Rhus succedanea and R. vernicifera, respectively [13,45]. Thus, the Leucaena laccase exhibited a lower Km value compared with laccases isolated from other plants. Furthermore, based on the ability of the Leucaena laccase to efficiently oxidize both o- and p-diphenol substrates (catechol and hydroquinone, respectively), the purified enzyme is validated as a laccase, as suggested by Ferrar and Walker [46]. The Kcat value of L. leucocephala laccase for ABTS (3.53 min−1 ) was lower than those found for several other laccases, such as those of T. hirsuta (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 for various substrates, pH, temperature optima, and molecular weight, of L. leucocephala laccase, with those of laccases reported from various 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 Leucaena laccase activity were investigated and are shown in Table 4.

Mn2+ , Cd2+ , and Na+ activated laccase in a concentration-dependent manner (0.1–10 mM). However, Mg2+ did not have any effect, while Ca2+ exhibited activation of laccase up to 3 mM, beyond which it exhibited an inhibition of laccase activity. Fe2+ and Cu2+ activated laccase in a concentration-dependent manner up to 1 mM. A concentration-dependent inhibition of laccase activity with Hg2+ and Co2+ up to 1 mM was observed. The effect of metals, such as Fe2+ , Cu2+ , Hg2+ and Co2+ , on laccase activity at concentrations higher than 1 mM could not be determined because of the interference of high concentrations of these metal salts with color development. Similar to our results, the addition of Mn2+ , Cd2+ , Cu2+ and Fe2+ have been reported to increase the activity of laccases from O. vulgaris [17], P. ostreatus [19], and Streptomyces psammoticus [3]. The involvement of four copper ions, distributed at the three different copper centers via type-1 (T1) or blue copper center, type-2 (T2) or normal copper and type-3 (T3) or coupled binuclear copper centers, exhibiting characteristic UV/vis and electron paramagnetic resonance (EPR) spectra, have been suggested during the catalysis of laccase [1]. Thus, the increase in activity with divalent metal ions might be due to their competition with Cu2+ in the electron transport system, leading to a positive cooperative relationship between

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

Opuntia vulgaris (OV137) (OV90) Cereus pterogonus (CP137) (CP90) (CP43) Rhus vernicifera Rhus succedanea Morus alba Bacteria Thermus thermophilus Azospirillum lipoferum Streptomyces psammoticus Streptomyces ipomoea Thermobifida fusca Fungi Trametes hirsuta Trichoderma harzianum Ganoderma lucidum Pleurotus pulmonarius Marasmius quercophilus Pleurotus ostreatus

Substrates with Km

Optimum temp. (◦ C)

Optimum pH

Mol. wt. (kDa)

Catechol, 1.24 mM ABTS, 1.92 mM Hydroquinone, 6.86 mM

80

7.0

220

Present study

2,6-DMP, 2.2 mM 2,6-DMP, 2.2 mM

80 70

10.0 10.0

137 90

[11]

2,6-DMP, 2.1 mM 2,6-DMP, 2.1 mM 2,6-DMP, 2.1 mM Catechol, 45 mM Catechol, 15 mM 4-Methylcatechol, 6 mM

90 90 60 40 50 45

10.0 10.0 10.0 7.0 7.0 7.0

137 90 43 – – 62–64

[12]

– Syringaldazine, 34.65 ␮M Pyrogallol, 0.25 mM ABTS, 0.39 mM ABTS, 0.40 mM 2,6-DMP, 4.27 mM –

92 – 45

– 6.0 8.5

53 179.3 43

[34] [33] [3]

60

5.0

79

[24]

60

8.0

73.3

[44]

ABTS, 0.07 mM DMP, 0.2 mM ABTS, 180 ␮M Guaiacol, 60 ␮M ABTS, 77 ␮M Guaiacol, 217 ␮M ABTS, 210 ␮M Guaicacol, 550 ␮M ABTS, 50 mM Syringaldazine, 7.7 mM ABTS, 46.51 mM DMP, 400 mM Guaiacol, 100 mM o-Dianisidine, 23.52 mM

85

2.4–2.5

90

[20]

85

4.5

79

[14]

50

4.5

62

[18]

45

4.0–5.5

46

[31]

80

6.2

65

[36]

50

4.5

68.4

[40]

Ref.

[13] [13] [16]

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

Effectors

Concentration (mM)

Mn2+ Fe2+ Cd2+ Na+ Cu2+ Ca2+ Mg2+ Hg2+ Co2+ DTT SDS EDTA

0

0.1

100 100 100 100 100 100 100 100 100 100 100 100

110 128 105 116 112 105 85 70 170 84 91

0.5 ± 1.7 ± 1.6 ± 1.5 ± 1.3 ± 1.3 ± 1.8 100 ± 1.7 ± 1.4 ± 1.8 ± 1.5 ± 1.8

149 178 114 129 123 112 66 52 147 71 38

1 ± 1.5 ± 1.4 ± 1.5 ± 1.7 ± 1.2 ± 1.3 100 ± 1.6 ± 1.4 ± 1.4 ± 1.8 ± 1.5

195 186 128 142 130 126 50 37 72 65 5

± 2.0 ± 1.7 ± 1.8 ± 1.7 ± 1.4 ± 1.6 100 ± 1.8 ± 1.3 ± 2.1 ± 1.7 ± 1.8

3

5

10

376 ± 2.2

403 ± 2.0

431 ± 2.5

*

159 ± 2.4 148 ± 2.0

*

189 ± 2.2 154 ± 1.5

*

139 ± 1.8 100

*

124 ± 2.0 100

*

*

*

*

33 ± 1.5 53 ± 1.6 0

9 ± 1.5 42 ± 2.3 0

*

189 ± 2.2 159 ± 1.6 *

104 ± 2.1 100 * *

0 11 ± 2.0 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 be measured.

the enzyme and substrate, as suggested by Chao et al. [47]. The activation of laccase by Cu2+ may be due to the filling of type-2 copper binding sites with Cu2+ ions [14]. The inhibition by the metal ions Hg2+ and Co2+ have also been reported [14,48], and the reason behind which might be due to the amino acid residue modifications, conformational changes or copper chelation [49]. DTT activated laccase at lower concentrations, while it inhibited the enzyme at concentrations above 0.1 mM in a concentrationdependent manner. SDS inhibited laccase activity at all of the concentrations tested in a concentration-dependent manner. EDTA was found to strongly inhibit laccase in such a way that at concentrations above 1 mM, the enzyme was completely inhibited. It is likely that at lower concentration of DTT, only a limited number of S S bonds is split (may be the only one which supports the enzyme active structure), providing the enzyme with some optimal flexibility and activity. However, at higher concentrations of DTT the unique three-dimensional conformation of the enzyme might have affected, thus resulting in an inhibition in enzyme activity [50]. The inhibition by SDS and EDTA was found almost similar to the laccases 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 protein, resulting in the inhibition of the enzyme activity. The strong inhibition of the enzyme by EDTA showed that the purified Leucaena laccase was highly sensitive to copper chelation, resulting in the conformational change of the protein and the loss of enzyme activity. It has been reported that the type-2 Cu2+ can be reversibly removed from the protein with the chelating reagent EDTA, such that the copper-depleted protein is enzymatically inactive, indicating that the type-2 Cu2+ of laccase has a functional role in the protein [52].

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

isopropanol and chloroform, respectively. In the literature, there are 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 attractive for their industrial applications, such as in the bioremediation of industrial waters contaminated with organic solvents, and in organic synthesis and chiral resolution. 3.6. Effect of laccase on dye decolorization

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) organic solvents on the stability of Leucaena laccase have been investigated and are presented in Fig. 6. The Leucaena laccase was found to be quite stable in presence of 20% (v/v) of all the organic solvents tested. The enzyme was found to retain more than 80% activity in the presence of all of the organic solvents except isopropanol, in which it retained approximately 60% activity. In the presence of higher concentrations (50% (v/v)) of ethanol, DMF, DMSO and benzene, laccase retained more than 50% activity, while the enzyme retained 42, 37 and 37% activity in the presence of methanol,

The ability of Leucaena laccase to oxidize industrial dyes was investigated to demonstrate its industrial applicability. The effect of enzyme concentration on the dye decolorization revealed that with increasing concentration of laccase, decolorization of indigocarmine (10 ␮g/ml) and congo red (10 ␮g/ml) increased progressively and the complete decolorization of both the dyes were achieved within 6 h at 11 and 22 ␮g of enzyme, respectively (Fig. 7A). The rate of decolorization (oxidation) of the two dyes by the Leucaena laccase was also investigated at various dye concentrations (10–250 ␮g/ml) and the data are shown in Fig. 7B. Though the rate of dye decolorization was found to increase with dye concentration but it was not directly proportional to the concentration

Please cite this article in press as: Jaiswal N, et al. Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.002

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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 decolorization at a dye concentration of 10 ␮g/ml was 5.4 ␮M/min/mg while at 250 ␮g/ml dye concentration, the rate of decolorization was 33.8 ␮M/min/mg. Similarly, in case of congo red, the rate of dye decolorization at a dye concentration of 10 ␮g/ml was 1.81 ␮M/min/mg while at 250 ␮g/ml dye concentration, the rate of decolorization was 11.3 ␮M/min/mg. The rate of decolorization (oxidation) of indigocarmine was about three times faster than that of congo red at all dye concentrations. The variation in the decolorization efficiency of the two dyes might be attributed to the structural variation of the dyes, as suggested by Nyanhongo and coworkers [55]. Laccases from fungi, such as Panus rudis [23] and T. versicolor [15], have been reported to decolorize the indigocarmine dye in the presence of ABTS. The purified Leucaena laccase had an advantage of decolorizing the tested dyes (indigocarmine and congo red) without any additional redox mediators, such as ABTS. Thus, the Leucaena laccase possesses an additional advantage with regards to its application in textile industries. 4. Conclusion A quick purification protocol using copper alginate bead entrapment with celite chromatography has been successfully utilized to purify laccase to homogeneity. The purified laccase was found to be a heterodimeric protein showing a pH optimum in the neutral range and thermostability up to 80 ◦ C. The enzyme was found to be active with both phenolic and non-phenolic substrates. Laccase was potentially activated by Mn2+ , Cd2+ , Fe2+ , Cu2+ and Na+ and inhibited by Co2+ , Hg2+ , DTT, SDS and EDTA, in a concentrationdependent manner. The enzyme was tolerant toward a number of polar and non-polar organic solvents. The remarkable decolorization ability of the enzyme suggests that it may have great potential in the decolorization of effluents of dyes and in textile industries. Acknowledgements Financial support from UGC, New Delhi, India, in the form of a Dr. D. S. Kothari Post doctoral Fellowship to N.J. is gratefully acknowledged. Department of Higher Education, Government of Uttar Pradesh, India under the Centre of Excellence in Bioinformatics, Department of Biotechnology, Government of India under the BIF Scheme, New Delhi and Department of Science and Technology, New Delhi, under Promotion of University Research and Scientific Excellence (DST-PURSE) program are also gratefully acknowledged for providing infrastructure facilities.

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Please cite this article in press as: Jaiswal N, et al. Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization. Process Biochem (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.002