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Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography
Accepted Manuscript Title: Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography Author: Nivedita Jaiswal Veda P. Pandey Upendra N. Dwivedi PII: DOI: Reference:
S0141-8130(14)00576-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.08.032 BIOMAC 4556
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
5-5-2014 11-8-2014 12-8-2014
Please cite this article as: N. Jaiswal, V.P. Pandey, U.N. Dwivedi, Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.08.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography
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Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi*
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Department of Biochemistry, University of Lucknow, Lucknow-226007, U.P., India
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*Corresponding author. Tel.: +91 522 2740132; Fax: +91 522 2740132.
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E-mail address: [email protected] (U.N. Dwivedi).
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Abstract A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70oC) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold
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purification of laccase with a specific activity of 41.3 units mg-1 and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ~260 kDa. The purified
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enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate
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(ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable upto 70oC such that it retained ~80% activity upon 30 min incubation at 70oC. The
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Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol-1. The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (Kcat/Km) in the
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order of 7.25 mM-1min-1>0.67 mM-1min-1>0.27 mM-1min-1 for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn2+, Cd2+, Ca2+,
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Na+, Fe2+, Co2+ and Cu2+ while weakly inhibited by Hg2+. The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising
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candidate enzyme for industrial exploitation.
Keywords: Alkaline laccase, Papaya, Thermostability.
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1. Introduction Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a copper-containing
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enzyme that catalyzes the oxidation of a wide variety of organic and inorganic compounds by coupling it to the reduction of oxygen to water. It is widely distributed in bacteria, fungi, plants,
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and insects [1]. Extensive studies have been made on bacterial and fungal laccases as compared
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to plant laccases. Though laccase was first discovered in exudates of the Japanese lacquer tree, Rhus vernicifera [2], only a few plant laccases have been purified and characterized till date.
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Structurally, laccases can be either monomeric, dimeric or multimeric containing four copper atoms per monomer and varied molecular weight [3-6]. Plant laccases have been reported to
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show higher molecular mass than fungal laccases which may be due to a higher extent of glycosylation than fungal ones [7]. The glycosylation has been reported play a role in copper
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retention, thermal stability and activity of the enzyme [8]. The temperature, pH optima and substrate specificity of laccases have been found to vary depending on the source [9-13]. Laccase
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is a metalloenzyme requiring four copper atoms for its catalytic activity [14]. Other metal ions such as Na+, Ca2+, Mg2+, Mn2+, Fe2+, Co2+, Hg2+, etc. have been reported to either activate or inhibit the laccases depending upon the source [1, 15]. Laccase has been a focus of attention as since it can be used for diverse biotechnological applications such as dye decolorization, biopulping, biobleaching, degradation of xenobiotics, food processing, biopolymer modification, ethanol production, development of biosensors, drug synthesis and organic synthesis, etc [1, 6]. Most of the laccases isolated so far either exhibit lower yield of enzyme activity or are found sensitive to extreme conditions of temperature, pH, metal ions, etc. which result in loss of catalytic activity limiting their large-scale commercial and industrial applications. Thus, thermostability is one of the general demands of enzymes which 3 Page 3 of 32
not only offers the enzymes to work at high process temperatures with higher reaction rates but also reduces the risk of microbial contamination [16]. Thermostable laccases is of demand in biobleaching of pulp and treatment of colored industrial effluents [17, 18]. Such thermostable
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laccases have generally been reported from thermophilic bacteria and fungi [16]). However, reports on plant laccases as a source of thermostable enzymes are very scanty.
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Besides, other obstacles which impede the practical application of laccases in
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biotechnological industries is the lowered laccase activity in neutral to alkaline pH as well as in the presence of various metal ions which is generally found in wastewaters discharged from
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textile industries [19, 20]. Thus, use of laccases active at alkaline pH is another ‘green technology alternative’ over chemical treatment as many industrial applications such as the
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textile industry (for denim bleaching), cosmetic industry (for hair coloring), and waste water
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treatment, etc. occur at neutral to alkaline pH [21, 22]. Thus, there is a need to explore newer
process applications.
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sources of laccases with improved properties to facilitate more bio-catalytic and industrial
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The present study therefore reports purification of a thermostable, alkaline and metaltolerant laccase from the leaves of an easily available and economically important medicinal plant, papaya (Carica papaya) employing heat treatment followed by Con-A affinity chromatography.
2. Experimental
2.1 Chemicals and Plant material Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich, USA and were of certified reagent grade. Fresh green and young papaya leaves growing in the garden of Department of Biochemistry, University of Lucknow, were used as plant material.
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2.2 Enzyme assay and protein estimation Laccase activity was determined by incubating the reaction mixture containing a suitable enzyme
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aliquot and 10 mM catechol (substrate) in 100 mM Tris-HCl buffer (pH 7.5) for 30 min at 37 oC [23]. The increase in absorbance due to oxidation of catechol to o-benzoquinone was measured
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at 390 nm using UV-vis spectrophotometer (Elico SL-177). A parallel control containing all the
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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
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enzyme activity was defined as the amount of enzyme required to produce 1 μmol of obenzoquinone (ε =1260 M-1cm-1) in 1 min under the specified conditions. Similarly, enzyme
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activity using substrates hydroquinone and ABTS, were determined by measuring increase in absorbance at 390 nm (ε for p-benzoquinone = 2240 M-1cm-1) and 420 nm (ε for ABTS+• =36000
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M-1cm-1), respectively.
Protein concentration was estimated by the Bradford dye binding method using bovine
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serum albumin as the standard [24].
2.3 Enzyme isolation from papaya leaves A 30% crude extract was prepared by homogenizing 20 g papaya leaves in 60 ml Tris-HCl buffer (100 mM, pH 7.5) using an ice-cold blender. Solid PVP (polyvinylpyrrolidone, insoluble; 0.1% (w/v)) and 7 mM β-mercaptoethanol were added at the time of extraction. The homogenate was centrifuged at 8500 x g for 30 min at 4oC using a Sigma 4K15 centrifuge. The clear supernatant that was obtained (crude extract) was subjected to purification. All the operations were done at 4oC, unless and otherwise specified. 2.4 Purification of laccase The following two quick steps were undertaken to purify the crude enzyme preparation. 5 Page 5 of 32
2.4.1 Heat treatment The crude extract (50 ml) was heated at 70oC for 30 min in a water bath and was immediately chilled in crushed ice followed by centrifugation at 8500 x g for 15 min at 4oC using Sigma
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4K15 centrifuge. The supernatant was collected and subjected to further purification step. 2.4.2 Concanavalin-A affinity chromatography
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The heat treated enzyme (48 ml) was loaded onto a Concanavalin-A CL Agarose column (5 cm x
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2.5 cm) (Genei, Bangalore, India) pre-equilibrated with 100 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl, 0.1 mM CaCl2 and 0.1 mM MnCl2. The bound proteins were eluted from
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the column by a linear (50-300 mM) sucrose gradient in the same buffer. The fractions containing high laccase activity were pooled and stored at 4oC for further use.
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2.5 Native PAGE and in-gel acivity staining
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Native polyacrylamide gel electrophoresis (PAGE; 7.5%) was performed in order to check the
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homogeneity of the preparation using resolving gel (7.5%), stacking gel (3%) and Tris-glycine running buffer (pH 8.8) as described by Davis [25]. The enzyme samples were loaded into the
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wells of gel and electrophoresis was carried out at 4oC at constant power of 100 V for 90 min. Ingel activity staining of the laccase was done by immersing the gel (after 10% native PAGE) in a catechol solution (50 mM) containing Tris-HCl buffer (100 mM, pH 7.5) until brown colored band appeared.
2.6 Native and subunit molecular weight determination Native molecular weight of the purified laccase was determined by gel filtration chromatography [26] using Sephadex G-200 column chromatography. The void/unoccupied volume (Vo) of the sephadex column was determined using blue dextran. Catalase (240 kDa), β-amylase (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (67 kDa) and lysozyme (14.3 kDa) were
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used as standard proteins (1.0 mg/ml) and were applied onto the column and the amount of protein in column eluent was estimated by Bradford’s method [24]. The elution volume (Ve) of each standard protein as well as purified laccase was measured. The molecular weight of the
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purified laccase was calculated from a calibration curve obtained by plotting the log molecular weight of the standard proteins against the ratio of the elution volumes of the standard proteins
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and the void/unoccupied volume of the column (Ve/Vo).
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The subunit molecular mass was determined using SDS PAGE carried using a 7.5% resolving gel, 3% stacking gel and Tris-glycine running buffer (pH 8.8) run at a constant power
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of 100 V for 90 min according to the method of Laemmli [27]. Proteins were visualized by silver staining. Standard protein markers containing myosin (205 kDa), phosphorylase b (97.4 kDa),
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bovine serum albumin (66 kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa) were
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2.7 Effect of temperature
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used.
Laccase activity at various temperatures (10-90oC) was investigated under standard assay
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conditions as described earlier in Experimental section. The thermostability of the papaya laccase was determined by incubating the enzyme at different temperatures (50, 60, 70 and 80oC) and assaying the activity at various time intervals. 2.8 Effect of pH
The effect of pH on the enzyme activity was studied over a pH range of 6.5-9.0 with the phenolic substrate, catechol and 5.0-9.0 with the non-phenolic substrate, ABTS. The different buffers (100 mM) used were sodium acetate, pH 6.5; Tris-HCl, pH 7.0, 7.5, 8.0 and 8.5; and sodium borate, pH 9.0. The pH stability of the papaya laccase was determined by estimating the activity after
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incubation of the enzyme at different pH values (6.5-9.0) for 24 h under standard assay conditions. 2.9 Kinetic constants of papaya laccase
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Kinetic constants of papaya laccase for the phenolic and non-phenolic substrates, namely catechol, ABTS and hydroquinone (0-50 mM) were investigated under standard assay
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conditions, respectively. Km and Vmax values were obtained by non-linear regression of a plot of
Prism software.
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2.10 Effect of various effectors on laccase activity
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enzyme activity vs substrate concentration (hyperbolic Michaelis-Menten plot) using GraphPad
The effect of varying concentration (0-10 mM) of several effectors, Na+, Mg2+, Ca2+, Mn2+, Cu2+,
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Co2+, Fe2+, Cd2+, Hg2+, DTT, SDS and EDTA on laccase activity was determined by performing
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the activity assay under standard assay conditions in the absence (control) as well as presence of
3. Results and Discussion
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3.1 Purification of laccase
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the individual effectors in the reaction mixture.
For purification of laccase, crude homogenate of papaya leaves were initially subjected to heat treatment at 70oC for 30 min which led to removal of more than 96% proteins with a recovery of ~80% of the laccase activity corresponding to ~22 fold purification of laccase. This partially purified laccase preparation was further subjected to Con-A affinity chromatography which led to ~1377 fold purification of laccase with overall recovery of ~62% and a specific activity of ~41 units/mg (Table 1). Table 1 The homogeneity of the purified laccase was established by running a native PAGE where a
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single band was obtained (Fig. 1A). The purified laccase was found to be catalytically active as established through in-gel activity staining (Fig. 1B). Laccase from Trichoderma harzianum was purified to 151.7 fold by Con-A affinity chromatography with only 0.39% yield and 130.5 units/
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Fig. 1
mg specific activity [9]. Similarly, laccase from Chaetomium thermophilum was purified to 10-
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fold using the same Con-A affinity chromatography with 40% yield and 37 units/mg specific
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activity [28]. However, the two-step purification method employed in our study resulted in purified papaya laccase with good yield, thus serving for various industrial applications of
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laccases.
3.2 Native and subunit molecular weight determination of papaya laccase
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The native molecular weight of the purified papaya laccase was found to be ~260 kDa by gel
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Fig. 2
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filtration chromatography (Fig. 2).
For the determination of subunit structure of purified laccase, SDS-PAGE was run where four
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bands were visualized on silver staining (Fig. 3A). For determination of size of these bands, a calibration graph between relative mobility and log molecular weight was plotted (Fig. 3B). Based on this calibration plot, the size of these bands was found to correspond to 52, 45, 38 and 25 kDa, suggesting a hexameric structure (consisting of two subunits each of 52 and 45 while one each of 38 and 25 kDa) for the native laccase. Fig. 3 Most of the plant laccases reported so far was found to be monomeric having subunit molecular within the range of 60-100 kDa [11, 29]. Recently, a heterodimeric laccase from a tree legume, Leucaena leucocephala consisting of two subunits, one of 100 kDa and other of 120
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kDa, have been reported [6]. Multimeric laccases having a native molecular weight of 137 kDa each have been reported from two xerophytic plant species, Cereus pterogonus (CP137) and Opuntia vulgaris (OV137) [12]. Reports of fungal and bacterial laccases in homodimeric,
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heterodimeric and homotrimeric forms have also been made in literature [30-34]. 3.3 Effect of temperature on papaya laccase activity
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The effect of temperature on purified laccase was investigated and it was found that laccase
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activity increased upto 70oC and afterwards decreased rapidly (Fig. 4A). The optimum Fig. 4
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temperature for laccases from plants like Leucaena leucocephala, Cereus pterogonus and Opuntia vulgaris have been reported to be 80, 90, and 70°C, respectively [4-6]. Some
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thermostable bacterial and fungal laccases have also been reported [9, 35, 36]. The Q10 value of
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papaya laccase was found to be >1 upto 70oC similar to L. leucocephala and Cerrena unicolor
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laccase suggesting that the reaction rate is temperature-dependent [6, 37]. The energy of activation (Ea) as determined from the slope of the Arrhenius plot was found to be 7.7 kJmol-1
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which was also almost similar to those found from L. leucocephala (6.9 kJmol-1 [6]) and Cerrena unicolor (6.9 kJmol-1 [37]).
Time-dependent thermostability of papaya laccase was also investigated by incubating the enzyme at temperatures 50, 60, 70 and 80oC for different time intervals and it was found that the enzyme lost 25, 18, 47 and 100% activity within 1h of incubation at 50, 60 and 70 and 80oC, respectively (Fig. 4B). Similar stability of laccase from O. vulgaris (OV90) has been reported when incubated for 30 min at 70oC [4] while laccase from Pleurotus ostreatus was almost completely inactivated after 15 min at 70 oC [38]. However, Gonzalez et al. [39] reported 3.7% residual activity of Pycnoporous sanguineus laccase after incubation at 70oC for 1h. Similar to
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our result, Trametes versicolor laccase have also been reported to showed complete inactivation of laccase after 1h of incubation at 80oC [15]. In contrast, laccase from a tree legume, L. leucocephala have been found to show activation upon pre-incubation at 80oC for upto 50 min
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[6]. A bacterial laccase from Thermobifida fusca retained ~95% of its original activity upon incubation at 50oC for 3h [35]. The differences in the temperature optima and thermostability of
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laccases from different sources might be due to the differences in the number of disulphide
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bonds in the protein [40]. 3.4 Effect of pH on papaya laccase activity
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The effect of pH on papaya laccase with the phenolic substrate, catechol was investigated and pH optimum was found to be 8.0 (Fig. 5A). There was a steep increase in activity from pH 6.5 to 8.0
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and at pH values above 8, the
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Fig. 5
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enzyme activity decreased gradually, still retaining 72% of its activity at pH 9.0. However, optimum pH of 6.0 was found with the non-phenolic substrate, ABTS, above which there was a
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decline in activity (data not shown). Thus, the phenolic substrate exhibited alkaline pH optimum while non-phenolic one exhibited acidic pH optimum. The main reason behind the increase and decrease in activity with pH in case of phenolic substrates has been given by Xu [41]. According to him, the pH-dependent decrease in reduction potential of the phenols (due to the phenolphenolate interconversion) is the cause of the increase in activity with pH whereas the hydroxide inhibition of the trinuclear cluster is the cause of decrease in activity with pH. The investigation of pH stability of the purified laccase revealed that it retained more than 75% of initial activity after 24 h incubation at 37oC at pH values from 6.5 to 9.0 (Fig. 5B). This showed its stability under alkaline conditions indicating that it is useful for processes
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involving alkaline conditions such as hair coloring as the swelling of hair swells and penetration of the dyes is enhanced at alkaline pH [22]. Besides, alkaline laccases also find application in textile industry for denim bleaching and in waste water treatment. There have been reports on
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Some alkaline laccases with high activity and stability under alkaline conditions have also been reported [12, 35]. Thus, the ability of papaya laccase to withstand under alkaline conditions
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made this enzyme suitable for various applications.
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3.5 Kinetic constants of papaya laccase
The effect of different phenolic (catechol and hydroquinone) and non-phenolic (ABTS)
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substrates on the activity of purified papaya laccase was determined and various kinetic parameters like Km (substrate affinity), Kcat (turnover number i.e. rate of catalytic process), and
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Kcat/Km (catalytic efficiency i.e. the efficiency of enzyme towards different substrates) were
Table 2
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calculated (Table 2). The Km and Vmax values were found to be 0.04, 1.48 and 11.33 mM, and
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0.04, 0.15 and 0.46 µM min-1ml-1 for ABTS, catechol and hydroquinone, respectively. Thus, the
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affinity of papaya laccase towards various substrates was found to be in the order: ABTS > catechol > hydroquinone. However, Kcat of the enzyme for these substrates were found to be in just opposite order: hydroquinone (3.07 min-1) > catechol (0.99 min-1) > ABTS (0.29 min-1). The catalytic efficiencies of the purified papaya laccase for various substrates (as presented by Kcat/Km) were found to be in the order: ABTS (7.25 mM-1min-1) > catechol (0.67 mM-1 min-1) > hydroquinone (0.27 mM-1 min-1). Thus, the non-phenolic substrate, ABTS was found to be most efficiently oxidized by papaya laccase which is evident from the Km as well as the Kcat/Km values. Catechol was found to be the second efficient substrate; however, the papaya laccase was less reactive towards hydroquinone. Almost similar Km values with ABTS have been reported for laccases from fungi, Ceriolopsis subvermispora (0.042 mM [42]), Daedela quercina (0.038 mM, 12 Page 12 of 32
[43]) and Phlebia radiata (0.049 mM [44]). Lower Km values for ABTS have also been reported with laccases from plant, Populus euramericana (0.026 mM [3]), fungal, Pleurotus pulmunarius (0.21mM [33]), Trichoderma harzianum (0.018 mM [9]), Trametes hirsuta (0.07 mM [36]) and
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bacterial sources such as γ-Proteobacterium JB (0.073 mM [45]). Laccase from plant sources such as Rhus vernicifera, R. succedanea, Amorphophallus campanulatus and L. leucocephala,
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showed Km values of 45, 15, 3.13 and 1.24 mM with catechol, respectively [6, 46, 47]. The Kcat
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value of papaya laccase for ABTS (0.29 min-1) was lower than those found for several other laccases, such as those of L. leucocephala (3.53 min-1 [6], T. hirsuta (197 s-1 [36], P. ostreatus
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(244.32 s-1 [38], Coriolus hirsutus (260 s-1 [48], Trametes pubescens (876 s-1 [49], and Pleurotus pulmonarius (1520 s-1 [33] but higher than those reported from Picnoporus cinnabarinus (920
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min-1 [50] and Phellinus ribis (8.0 x 104 min-1 [32].
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3.6 Effect of various effectors on laccase activity
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The effect of various effectors namely, metal ions, reducing agent (DTT), chelating agent
Table 3
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(EDTA), and detergent (SDS) on papaya laccase activity were studied and are shown in Table 3.
Mn2+, Cd2+, Ca2+and Na+ activated laccase in a concentration-dependent manner (0.1-10 mM) while Na+ exhibited activation of laccase upto 1 mM beyond which it exhibited inhibition of laccase activity. Fe2+, Co2+ and Cu2+ activated laccase in a concentration-dependent manner upto 1 mM. The effect of these metal ions at concentrations beyond 1 mM could not be investigated because of their interference in color development during enzyme assay. A concentrationdependent inhibition of laccase activity was observed with Hg2+ upto 1 mM. Thus, the papaya laccase was found to be fairly tolerant to all the metals tested exhibiting activation with the exception of Hg2+. Some metal tolerant laccases have also been reported from plants like L.
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leucocephala [6], C. pterogonus and Opuntia vulgaris [5]; fungi, Pleurotus ostreatus [38], Trametes versicolor [15] and T. pubescens [49] and bacteria, Streptomyces psammoticus [51]. However, the metal tolerance of papaya laccase was much better than the earlier reports. Thus,
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the highly tolerant activity of papaya laccase towards various metal ions is of great value in terms of its potential industrial application. It is well-known that the catalytic site of laccase
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contains four copper atoms clustered at three types of copper sites (type I, II, and III) which
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perform monoelectronic oxidation of suitable substrates [52]. As reported by Duggleby et al. [53], the activation by the metal ions may be due to the favored conformational modifications
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thus stimulating laccase activity. However, the blockage of the access of the substrate or the transfer of electron at the T1 site results in inhibition in laccase activity [54].
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DTT activated laccase at lower concentration (0.1 mM) while inhibited the enzyme at
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concentrations beyond 0.1 mM in a concentration-dependent manner with complete inhibition in
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activity at 10 mM. Similar activation of laccase at lower and concentration of DTT with L. leucocephala laccase has been reported [6]. The gradual inhibition in laccase activity with
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increasing concentration of DTT indicated the role of thiol groups in laccase activity [55, 56]. SDS and EDTA also inhibited laccase activity similar to those reported from peach, O. vulgaris, C. pterogonus, Trametes versicolor, Streptomyces cyaneus and S. psammoticus [5, 6, 15, 51, 56, 57]. The inhibition of laccase activity by EDTA might be due to the metal chelation of Cu in the catalytic site of the enzyme [14]. However, the change in the conformation of the protein structure might be the cause of the inhibition of activity by SDS. 4. Conclusion A laccase from papaya was purified to homogeneity in a two step process, namely heat treatment and Con-A affinity chromatography with good enzyme yield. It was found to be a hexamer with pH and temperature optima of 8.0 and 70oC, respectively. The enzyme was found to be fairly 14 Page 14 of 32
tolerant towards heat, pH and metal ions. In addition, the papaya laccase showed enhancement in activity in the presence of most of the metal ions. Such characteristics indicate the potential suitability of papaya laccase for use in bioremediation, textile, pulp and paper industries.
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Acknowledgements
Financial support from UGC, New Delhi, India, in the form of Dr. D.S. Kothari Postdoctoral
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Fellowship to NJ is gratefully acknowledged. Financial supports from Department of Higher
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Education, Govt. of Uttar Pradesh, India under Centre of Excellence in Bioinformatics, Department of Biotechnology, Govt. of India under BIF Scheme, New Delhi and Department of
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Science and Technology, New Delhi under Promotion of University Research and Scientific Excellence (DST-PURSE) programme for providing infrastructure facilities are also gratefully
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acknowledged.
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[13] K.K. Sharma, B. Shrivastava, V.R.B. Sastry, N. Sehgal, R.C. Kuhad, Middle-redox potential laccase from Ganoderma sp.: its application in improvement of feed for monogastric animals, Sci Reports (2013) DOI: 10.1038/srep01299.
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ip t
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cr
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te
783.
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Ac ce p
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Decolourization of
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ip t
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cr
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Chaetomium thermophilium and its role in humification, Appl. Environ. Microbiol. 64 (1998) 3175-3179.
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[30] S.B. Younes, S. Sayadi, Purification and characterization of a novel trimeric and thermotolerant laccase produced from the ascomycete Scytalidium thermophilum strain, J. Mol. Catal. B: Enzym. 73 (2011) 35-42.
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[31] A.V. Lisov, A.G. Zavarzina, A.A. Zavarzin, A.A. Leontievsky, Laccases produced by lichens of the order peltigerales, FEMS Microbiol. Lett. 275 (2007) 46-52. [32] K.L. Min, Y.H. Kim, Y.W. Kim, H.S. Jung, Y.C. Hah, Characterization of a novel laccase
ip t
produced by the wood-rotting fungus Phellinus ribis. Arch. Biochem. Biophys. 392 92001) 279286.
cr
[33] C.G.M. DeSouza, R.M. Peralta, Purification and characterization of the main laccase
us
produced by the white rot fungus Pleurotus pulmonarius on wheat bran solid state medium. J. Basic Microbiol. 43 (2003) 278-286.
an
[34] T.B. Ng, H.X. Wang, A homodimeric laccase with unique characteristics from the yellow mushroom Cantharellus cibarius, Biochem. Biophys. Res. Commun. 313 (2004) 37-41.
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[35] C.-Y. Chen, Y.-C. Huang, C.-M. Wei, M. Meng, W.-H. Liu, C.-H. Yan,g Properties of the
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newly isolated extracellular thermo-alkali-stable laccase from thermophilic actinomycetes,
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Thermobifida fusca and its application in dye intermediates oxidation, AMB Express, 3 (2013) 49.
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[36] Z. Haibo, Z. Yinglong, H. Feng, G. Peiji, C. Jiachuan, Purification and characterization of a thermostable laccase with unique oxidative characteristics from Trametes hirsuta, Biotechnol. Lett. 31 (2009) 837-843.
[37] D. D’Souza-Ticlo, D. Sharma, C. Raghukumar, A thermostable metal-tolerant laccase with bioremediation potential from a marine-derived fungus. Mar. Biotechnol. 11 (2009) 725-737 [38] H. Patel, S. Gupte, M. Gahlout, A. Gupte, Purification and characterization of an extracellular laccase from solid-state culture of Pleurotus ostreatus HP-1. 3 Biotech (2013) doi: 10.1007/s13205-013-0129-1.
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[39] E.D. Gonzalez, O.V. Vallejo, C.M. Anaya, M.M. Sanchez, M.C. Gonzalez, L.A. Palomares, J.F. Mallol, Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporous sanguineus isolated from an oil-polluted tropical habitat, Int. Microbiol. 11 (2008) 163-169.
ip t
[40] F. Xu, W. Shin, S.H. Brown, J.A. Wahleithner, U.M. Sundaram, E.I. Solomon, A study of a series of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in
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redox potential, substrate specificity, and stability, Biochem. Biophys. Acta 1292 (1996) 303-
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[41] F. Xu, Effects of redox potential and hydroxide inhibition on the pH activity profile of
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fungal laccases. J. Biol. Chem. 272 (1997) 924-928.
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chrysosporium with
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oxidase
activity, Appl.
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Microbiol. 69 (2003) 6257-6263.
ferroxidase
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[43] P. Baldrian, Increase of laccase activity during interspecific interactions of white-rot fungi, FEMS Microbiol. Ecol. 50 (2004) 245-253.
Ac ce p
[44] S. Kaneko, M. Cheng, S. Murai, S. Takenaka, S. Murakami, K. Aoki, Purification and characterization of an extracellular laccase from Phlebia radiata strain BP-11-2 that decolorizes fungal melanin, Biosci. Biotechnol. Biochem. 73 (2009) 939-942. [45] M. Asgher, S. Kamal, H.M.N. Iqbal, Improvement of catalytic efficiency, thermo-stability and dye decolorization capability of Pleurotus ostreatus IBL-02 laccase by Hydrophobic Sol Gel Entrapment, Chem. Cent. J. 6 (2012)110. [46] T. Omura, Studies on laccases of lacquer trees. I. Comparison of laccases from Rhus vernicifera and Rhus succedanea, J. Biochem. 50 (1961) 264-272.
20 Page 20 of 32
[47] P.S. Paranjpe, M.S. Karve, S.B. Padhye, Characterization of tyrosinase and accompanying laccase from Amorphophallus campanulatus, Ind. J. Biochem. Biophys. 40 (2003) 40-45. [48] K.S. Shin, Y.J. Lee, Purification and characterization of a new member of the laccase family
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from the white-rot basidiomycete Coriolus hirsutus. Arch. Biochem. Biophys. 384 (2000) 109115.
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[49] J. Si, F. Peng, B. Cui, Purification, biochemical characterization and dye decolorization
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capacity of an alkali-resistant and metal-tolerant laccase from Trametes pubescens, Biores.Technol. 128 (2013) 49-57.
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[50] C. Eggert, U. Temp, K.E. Eriksson, The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase, Appl. Environ.
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Microbiol. 62 (1996) 1151-1158.
d
[51] K.N. Niladevi, N. Jacob, P. Prema, Evidence for a halotolerant alkaline laccase in
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Streptomyces psammoticus: purification and characterization, Proc. Biochem. 43 (2008) 654-60. [52] M. Frasconi, G. Favero, H. Boer, A. Koivula, F. Mazzei, Kinetic and biochemical properties
Ac ce p
of high and low redox potential laccases from fungal and plant origin, Biochim. Biophys. Acta 1804 (2010) 899-908.
[53] R.G. Duggleby, Experimental designs for estimating the kinetic parameters for enzymecatalysed reactions, J. Theor. Biol. 81 (1979) 671-684. [54] Z.M. Fang, T.L. Li, F. Chang, P. Zhou, W. Fang, Y.Z. Hong, X.C. Zhang, H. Peng, Y.Z. Xiao, A new marine bacterial laccase with chloride-enhancing, alkaline dependent activity and dye decolorization ability, Biores. Technol. 111 (2012) 36-41. [55] C. Johannes, A. Majcherczyk, Laccase activity tests and laccase inhibitors, J. Biotechnol. 78 (2000) 193-199.
21 Page 21 of 32
[56] M. Lorenzo, D. Moldes, S. Rodríguez Couto, M.A. Sanromán, Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds, Chemosphere 60 (2005) 1124-1128.
ip t
[57] M.E. Arias, M. Arenas, J. Rodriguez, J. Soliveri, A.S. Ball, M. Hernandez, Kraft pulp biobleaching and mediated oxidation of a nonphenol substrate by laccase from Streptomyces
Ac ce p
te
d
M
an
us
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cyaneus CECT3335, Appl. Environ. Microbiol. 69 (2003) 1953-1958.
22 Page 22 of 32
Figure Captions Fig. 1. A: Native-PAGE analysis and silver staining of purified papaya laccase during
ip t
purification. B: In-gel activity staining of purified papaya laccase. The gel after Native-PAGE was immersed in 50 mM catechol solution until the brown-colored band appeared.
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Fig. 2. Calibration plot for the determination of the native molecular weight of purified papaya
us
laccase using gel filtration chromatography. The standard protein markers (1.0 mg/ml) used
albumin (67 kDa) and lysozyme (14.3 kDa).
an
were: catalase (240 kDa), β-amylase (200 kDa), phosphorylase B (97.4 kDa), bovine serum
Fig. 3. A: SDS-PAGE and silver staining of purified papaya laccase. Lane 1: purified laccase,
M
Lane 2: molecular weight markers. Standard protein markers containing myosin (205 kDa),
te
anhydrase (29 kDa) were used.
d
phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), and carbonic
B: Calibration plot for the determination of the subunit molecular weight of purified papaya
Ac ce p
laccase.
Fig. 4. A: Effect of temperature on the activity of papaya laccase. The enzyme was incubated at different temperature (10-90 oC) and activity was measured under standard assay conditions. B: Thermal stability of papaya laccase at different temperatures. Percent relative activity represents the enzyme activity relative to the control (0 min), which was set at 100%. Fig. 5. A: Effect of pH on the activity of papaya laccase. The activity was assayed at different pH (6.5-9.0) under standard assay conditions. B: pH stability of papaya laccase. The enzyme was incubated for 24 h at different pH (6.5-9.0) and activity assayed using a suitable pre-incubated enzyme aliquot under standard assay
23 Page 23 of 32
conditions. Percent relative activity represents enzyme activity calculated by setting the activity,
Ac ce p
te
d
M
an
us
cr
ip t
at optimum pH, as 100%.
24 Page 24 of 32
Total Activity
Total Protein
Specific Activity
(U)
(mg)
(U/mg)
Crude extract
17
571.50
0.03
100
Heat treatment
13.44
20.80
0.65
79
21.70
10.45
0.25
41.30
cr
Table 1. Summary of laccase purification from papaya leaves (20 g)
1376.70
o
(70 C, 30 min) Con-A affinity
Fold Purification 1
61.5
Ac ce p
te
d
M
an
us
chromatography
Yield (%)
ip t
Steps
25 Page 25 of 32
Table 2. Kinetic properties of papaya laccase with various phenolic and non-phenolic substrates Km (mM)
Vmax (µM min-1 ml-1)
Kcat (min-1)
Kcat/Km (mM-1 min-1)
ABTS
0.04
0.04
0.29
7.25
Catechol
1.48
0.15
0.99
Hydroquinone
11.33
0.46
3.07
ip t
Substrates
0.67
Ac ce p
te
d
M
an
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cr
0.27
26 Page 26 of 32
Table 3. 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
0.1
0.5
1
3
Mn2+
100
115±1.7
185±1.5
231±2.0
346±2.2
Cd2+
100
144±1.5
173±1.5
188±1.8
221±2.4
243±2.2
265±2.1
Ca2+
100
109±1.6
119±1.4
130±1.7
175±1.8
192±2.1
222±2.0
Mg2+
100
110±1.8
114±1.3
118±1.6
133±1.8
138±2.0
151±1.8
Na+
100
167±1.3
174±1.7
171±1.7
161±2.0
150±1.5
130±2.0
Fe2+
100
116
148
176
*
*
*
Co2+
100
117±1.3
136±1.2
153±1.4
*
*
*
Cu2+
100
118±1.7
130±1.6
147±1.8
*
*
*
Hg2+
100
95±1.4
d
80±1.3
*
*
*
DTT
100
138±1.8
88±1.4
31±2.1
21±1.5
9±1.5
0
SDS
100
94±1.5
84±1.8
73±1.7
61±1.6
51±2.3
37±2.1
100
97±1.8
89±1.5
84±1.8
73±1.5
66±1.6
51±1.8
EDTA
us
an
M
te
89±1.4
5
10
385±2.0
412±2.5
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0
Ac ce p
Effectors
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Concentration (mM)
values are presented as the mean ± SD of triplicate tests.
* salts of Fe2+, Co2+, Cu2+ and Hg2+ at concentrations beyond 1 mM interfered with the color development during enzyme activity assay and hence the effect could not be measured.
27 Page 27 of 32
i
Figure 1
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
Ac
ce pt
ed
M an
us
cr
Int. J. Biol. Macromol.
A
B
Fig. 1
Page 28 of 32
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Figure 2
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
us
cr
Int. J. Biol. Macromol.
M an
Lysozyme
BSA
ed
Phosphorylase B
ce pt
Beta amylase Catalase
Ac
Laccase
Fig. 2
Page 29 of 32
cr
i
Figure 3
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
2
205 kDa
Band 2
45 kDa
Band 3
38 kDa
Band 4
25 kDa
Band 2
Band 3
66 kDa
Band 4
ce pt
52 kDa
43 kDa
29 kDa
Ac
Band 1
Band 1
ed
97.4 kDa
Catalase
M an
1
us
Int. J. Biol. Macromol.
A
B Fig. 3
Page 30 of 32
cr
i
Figure4
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
M an
us
Int. J. Biol. Macromol.
120
40
10
0 0
20
ce pt
20
40 60 Temperature (oC)
80
% Residual Activity
ed
30
Ac
Activity (U/ml) x 10-2
100 80
50oC
60
60oC 70oC
40
80oC 20 0
100
0
20
40
60
Time (min)
B
A
Fig. 4
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i
Figure5
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
M an
us
Int. J. Biol. Macromol.
ed
20
10 5 6 A
7
ce pt
15
8
100 80 60 40 20
6
9
7
8
9
pH
pH
Ac
Activity (U/ml) x 10-2
25
% Relative Activity
120
30
Fig. 5
Page 32 of 32
S0141-8130(14)00576-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.08.032 BIOMAC 4556
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
5-5-2014 11-8-2014 12-8-2014
Please cite this article as: N. Jaiswal, V.P. Pandey, U.N. Dwivedi, Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.08.032 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Purification of a thermostable alkaline laccase from papaya (Carica papaya) using affinity chromatography
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Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi*
an
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Department of Biochemistry, University of Lucknow, Lucknow-226007, U.P., India
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*Corresponding author. Tel.: +91 522 2740132; Fax: +91 522 2740132.
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E-mail address: [email protected] (U.N. Dwivedi).
1 Page 1 of 32
Abstract A laccase from papaya leaves was purified to homogeneity by a two step procedure namely, heat treatment (at 70oC) and Con-A affinity chromatography. The procedure resulted in 1386.7-fold
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purification of laccase with a specific activity of 41.3 units mg-1 and an overall yield of 61.5%. The native purified laccase was found to be a hexameric protein of ~260 kDa. The purified
cr
enzyme exhibited acidic and alkaline pH optima of 6.0 and 8.0 with the non-phenolic substrate
us
(ABTS) and phenolic substrate (catechol), respectively. The purified laccase was found to be thermostable upto 70oC such that it retained ~80% activity upon 30 min incubation at 70oC. The
an
Arrhenius energy of activation for purified laccase was found to be 7.7 kJ mol-1. The enzyme oxidized various phenolic and non-phenolic substrates having catalytic efficiency (Kcat/Km) in the
M
order of 7.25 mM-1min-1>0.67 mM-1min-1>0.27 mM-1min-1 for ABTS, catechol and hydroquinone, respectively. The purified laccase was found to be activated by Mn2+, Cd2+, Ca2+,
te
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Na+, Fe2+, Co2+ and Cu2+ while weakly inhibited by Hg2+. The properties such as thermostability, alkaline pH optima and metal tolerance exhibited by the papaya laccase make it a promising
Ac ce p
candidate enzyme for industrial exploitation.
Keywords: Alkaline laccase, Papaya, Thermostability.
2 Page 2 of 32
1. Introduction Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a copper-containing
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enzyme that catalyzes the oxidation of a wide variety of organic and inorganic compounds by coupling it to the reduction of oxygen to water. It is widely distributed in bacteria, fungi, plants,
cr
and insects [1]. Extensive studies have been made on bacterial and fungal laccases as compared
us
to plant laccases. Though laccase was first discovered in exudates of the Japanese lacquer tree, Rhus vernicifera [2], only a few plant laccases have been purified and characterized till date.
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Structurally, laccases can be either monomeric, dimeric or multimeric containing four copper atoms per monomer and varied molecular weight [3-6]. Plant laccases have been reported to
M
show higher molecular mass than fungal laccases which may be due to a higher extent of glycosylation than fungal ones [7]. The glycosylation has been reported play a role in copper
te
d
retention, thermal stability and activity of the enzyme [8]. The temperature, pH optima and substrate specificity of laccases have been found to vary depending on the source [9-13]. Laccase
Ac ce p
is a metalloenzyme requiring four copper atoms for its catalytic activity [14]. Other metal ions such as Na+, Ca2+, Mg2+, Mn2+, Fe2+, Co2+, Hg2+, etc. have been reported to either activate or inhibit the laccases depending upon the source [1, 15]. Laccase has been a focus of attention as since it can be used for diverse biotechnological applications such as dye decolorization, biopulping, biobleaching, degradation of xenobiotics, food processing, biopolymer modification, ethanol production, development of biosensors, drug synthesis and organic synthesis, etc [1, 6]. Most of the laccases isolated so far either exhibit lower yield of enzyme activity or are found sensitive to extreme conditions of temperature, pH, metal ions, etc. which result in loss of catalytic activity limiting their large-scale commercial and industrial applications. Thus, thermostability is one of the general demands of enzymes which 3 Page 3 of 32
not only offers the enzymes to work at high process temperatures with higher reaction rates but also reduces the risk of microbial contamination [16]. Thermostable laccases is of demand in biobleaching of pulp and treatment of colored industrial effluents [17, 18]. Such thermostable
ip t
laccases have generally been reported from thermophilic bacteria and fungi [16]). However, reports on plant laccases as a source of thermostable enzymes are very scanty.
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Besides, other obstacles which impede the practical application of laccases in
us
biotechnological industries is the lowered laccase activity in neutral to alkaline pH as well as in the presence of various metal ions which is generally found in wastewaters discharged from
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textile industries [19, 20]. Thus, use of laccases active at alkaline pH is another ‘green technology alternative’ over chemical treatment as many industrial applications such as the
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textile industry (for denim bleaching), cosmetic industry (for hair coloring), and waste water
d
treatment, etc. occur at neutral to alkaline pH [21, 22]. Thus, there is a need to explore newer
process applications.
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sources of laccases with improved properties to facilitate more bio-catalytic and industrial
Ac ce p
The present study therefore reports purification of a thermostable, alkaline and metaltolerant laccase from the leaves of an easily available and economically important medicinal plant, papaya (Carica papaya) employing heat treatment followed by Con-A affinity chromatography.
2. Experimental
2.1 Chemicals and Plant material Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich, USA and were of certified reagent grade. Fresh green and young papaya leaves growing in the garden of Department of Biochemistry, University of Lucknow, were used as plant material.
4 Page 4 of 32
2.2 Enzyme assay and protein estimation Laccase activity was determined by incubating the reaction mixture containing a suitable enzyme
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aliquot and 10 mM catechol (substrate) in 100 mM Tris-HCl buffer (pH 7.5) for 30 min at 37 oC [23]. The increase in absorbance due to oxidation of catechol to o-benzoquinone was measured
cr
at 390 nm using UV-vis spectrophotometer (Elico SL-177). A parallel control containing all the
us
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
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enzyme activity was defined as the amount of enzyme required to produce 1 μmol of obenzoquinone (ε =1260 M-1cm-1) in 1 min under the specified conditions. Similarly, enzyme
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activity using substrates hydroquinone and ABTS, were determined by measuring increase in absorbance at 390 nm (ε for p-benzoquinone = 2240 M-1cm-1) and 420 nm (ε for ABTS+• =36000
te
d
M-1cm-1), respectively.
Protein concentration was estimated by the Bradford dye binding method using bovine
Ac ce p
serum albumin as the standard [24].
2.3 Enzyme isolation from papaya leaves A 30% crude extract was prepared by homogenizing 20 g papaya leaves in 60 ml Tris-HCl buffer (100 mM, pH 7.5) using an ice-cold blender. Solid PVP (polyvinylpyrrolidone, insoluble; 0.1% (w/v)) and 7 mM β-mercaptoethanol were added at the time of extraction. The homogenate was centrifuged at 8500 x g for 30 min at 4oC using a Sigma 4K15 centrifuge. The clear supernatant that was obtained (crude extract) was subjected to purification. All the operations were done at 4oC, unless and otherwise specified. 2.4 Purification of laccase The following two quick steps were undertaken to purify the crude enzyme preparation. 5 Page 5 of 32
2.4.1 Heat treatment The crude extract (50 ml) was heated at 70oC for 30 min in a water bath and was immediately chilled in crushed ice followed by centrifugation at 8500 x g for 15 min at 4oC using Sigma
ip t
4K15 centrifuge. The supernatant was collected and subjected to further purification step. 2.4.2 Concanavalin-A affinity chromatography
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The heat treated enzyme (48 ml) was loaded onto a Concanavalin-A CL Agarose column (5 cm x
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2.5 cm) (Genei, Bangalore, India) pre-equilibrated with 100 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl, 0.1 mM CaCl2 and 0.1 mM MnCl2. The bound proteins were eluted from
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the column by a linear (50-300 mM) sucrose gradient in the same buffer. The fractions containing high laccase activity were pooled and stored at 4oC for further use.
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2.5 Native PAGE and in-gel acivity staining
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Native polyacrylamide gel electrophoresis (PAGE; 7.5%) was performed in order to check the
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homogeneity of the preparation using resolving gel (7.5%), stacking gel (3%) and Tris-glycine running buffer (pH 8.8) as described by Davis [25]. The enzyme samples were loaded into the
Ac ce p
wells of gel and electrophoresis was carried out at 4oC at constant power of 100 V for 90 min. Ingel activity staining of the laccase was done by immersing the gel (after 10% native PAGE) in a catechol solution (50 mM) containing Tris-HCl buffer (100 mM, pH 7.5) until brown colored band appeared.
2.6 Native and subunit molecular weight determination Native molecular weight of the purified laccase was determined by gel filtration chromatography [26] using Sephadex G-200 column chromatography. The void/unoccupied volume (Vo) of the sephadex column was determined using blue dextran. Catalase (240 kDa), β-amylase (200 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (67 kDa) and lysozyme (14.3 kDa) were
6 Page 6 of 32
used as standard proteins (1.0 mg/ml) and were applied onto the column and the amount of protein in column eluent was estimated by Bradford’s method [24]. The elution volume (Ve) of each standard protein as well as purified laccase was measured. The molecular weight of the
ip t
purified laccase was calculated from a calibration curve obtained by plotting the log molecular weight of the standard proteins against the ratio of the elution volumes of the standard proteins
cr
and the void/unoccupied volume of the column (Ve/Vo).
us
The subunit molecular mass was determined using SDS PAGE carried using a 7.5% resolving gel, 3% stacking gel and Tris-glycine running buffer (pH 8.8) run at a constant power
an
of 100 V for 90 min according to the method of Laemmli [27]. Proteins were visualized by silver staining. Standard protein markers containing myosin (205 kDa), phosphorylase b (97.4 kDa),
M
bovine serum albumin (66 kDa), ovalbumin (43 kDa), and carbonic anhydrase (29 kDa) were
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2.7 Effect of temperature
d
used.
Laccase activity at various temperatures (10-90oC) was investigated under standard assay
Ac ce p
conditions as described earlier in Experimental section. The thermostability of the papaya laccase was determined by incubating the enzyme at different temperatures (50, 60, 70 and 80oC) and assaying the activity at various time intervals. 2.8 Effect of pH
The effect of pH on the enzyme activity was studied over a pH range of 6.5-9.0 with the phenolic substrate, catechol and 5.0-9.0 with the non-phenolic substrate, ABTS. The different buffers (100 mM) used were sodium acetate, pH 6.5; Tris-HCl, pH 7.0, 7.5, 8.0 and 8.5; and sodium borate, pH 9.0. The pH stability of the papaya laccase was determined by estimating the activity after
7 Page 7 of 32
incubation of the enzyme at different pH values (6.5-9.0) for 24 h under standard assay conditions. 2.9 Kinetic constants of papaya laccase
ip t
Kinetic constants of papaya laccase for the phenolic and non-phenolic substrates, namely catechol, ABTS and hydroquinone (0-50 mM) were investigated under standard assay
cr
conditions, respectively. Km and Vmax values were obtained by non-linear regression of a plot of
Prism software.
an
2.10 Effect of various effectors on laccase activity
us
enzyme activity vs substrate concentration (hyperbolic Michaelis-Menten plot) using GraphPad
The effect of varying concentration (0-10 mM) of several effectors, Na+, Mg2+, Ca2+, Mn2+, Cu2+,
M
Co2+, Fe2+, Cd2+, Hg2+, DTT, SDS and EDTA on laccase activity was determined by performing
d
the activity assay under standard assay conditions in the absence (control) as well as presence of
3. Results and Discussion
Ac ce p
3.1 Purification of laccase
te
the individual effectors in the reaction mixture.
For purification of laccase, crude homogenate of papaya leaves were initially subjected to heat treatment at 70oC for 30 min which led to removal of more than 96% proteins with a recovery of ~80% of the laccase activity corresponding to ~22 fold purification of laccase. This partially purified laccase preparation was further subjected to Con-A affinity chromatography which led to ~1377 fold purification of laccase with overall recovery of ~62% and a specific activity of ~41 units/mg (Table 1). Table 1 The homogeneity of the purified laccase was established by running a native PAGE where a
8 Page 8 of 32
single band was obtained (Fig. 1A). The purified laccase was found to be catalytically active as established through in-gel activity staining (Fig. 1B). Laccase from Trichoderma harzianum was purified to 151.7 fold by Con-A affinity chromatography with only 0.39% yield and 130.5 units/
ip t
Fig. 1
mg specific activity [9]. Similarly, laccase from Chaetomium thermophilum was purified to 10-
cr
fold using the same Con-A affinity chromatography with 40% yield and 37 units/mg specific
us
activity [28]. However, the two-step purification method employed in our study resulted in purified papaya laccase with good yield, thus serving for various industrial applications of
an
laccases.
3.2 Native and subunit molecular weight determination of papaya laccase
M
The native molecular weight of the purified papaya laccase was found to be ~260 kDa by gel
te
Fig. 2
d
filtration chromatography (Fig. 2).
For the determination of subunit structure of purified laccase, SDS-PAGE was run where four
Ac ce p
bands were visualized on silver staining (Fig. 3A). For determination of size of these bands, a calibration graph between relative mobility and log molecular weight was plotted (Fig. 3B). Based on this calibration plot, the size of these bands was found to correspond to 52, 45, 38 and 25 kDa, suggesting a hexameric structure (consisting of two subunits each of 52 and 45 while one each of 38 and 25 kDa) for the native laccase. Fig. 3 Most of the plant laccases reported so far was found to be monomeric having subunit molecular within the range of 60-100 kDa [11, 29]. Recently, a heterodimeric laccase from a tree legume, Leucaena leucocephala consisting of two subunits, one of 100 kDa and other of 120
9 Page 9 of 32
kDa, have been reported [6]. Multimeric laccases having a native molecular weight of 137 kDa each have been reported from two xerophytic plant species, Cereus pterogonus (CP137) and Opuntia vulgaris (OV137) [12]. Reports of fungal and bacterial laccases in homodimeric,
ip t
heterodimeric and homotrimeric forms have also been made in literature [30-34]. 3.3 Effect of temperature on papaya laccase activity
cr
The effect of temperature on purified laccase was investigated and it was found that laccase
us
activity increased upto 70oC and afterwards decreased rapidly (Fig. 4A). The optimum Fig. 4
an
temperature for laccases from plants like Leucaena leucocephala, Cereus pterogonus and Opuntia vulgaris have been reported to be 80, 90, and 70°C, respectively [4-6]. Some
M
thermostable bacterial and fungal laccases have also been reported [9, 35, 36]. The Q10 value of
d
papaya laccase was found to be >1 upto 70oC similar to L. leucocephala and Cerrena unicolor
te
laccase suggesting that the reaction rate is temperature-dependent [6, 37]. The energy of activation (Ea) as determined from the slope of the Arrhenius plot was found to be 7.7 kJmol-1
Ac ce p
which was also almost similar to those found from L. leucocephala (6.9 kJmol-1 [6]) and Cerrena unicolor (6.9 kJmol-1 [37]).
Time-dependent thermostability of papaya laccase was also investigated by incubating the enzyme at temperatures 50, 60, 70 and 80oC for different time intervals and it was found that the enzyme lost 25, 18, 47 and 100% activity within 1h of incubation at 50, 60 and 70 and 80oC, respectively (Fig. 4B). Similar stability of laccase from O. vulgaris (OV90) has been reported when incubated for 30 min at 70oC [4] while laccase from Pleurotus ostreatus was almost completely inactivated after 15 min at 70 oC [38]. However, Gonzalez et al. [39] reported 3.7% residual activity of Pycnoporous sanguineus laccase after incubation at 70oC for 1h. Similar to
10 Page 10 of 32
our result, Trametes versicolor laccase have also been reported to showed complete inactivation of laccase after 1h of incubation at 80oC [15]. In contrast, laccase from a tree legume, L. leucocephala have been found to show activation upon pre-incubation at 80oC for upto 50 min
ip t
[6]. A bacterial laccase from Thermobifida fusca retained ~95% of its original activity upon incubation at 50oC for 3h [35]. The differences in the temperature optima and thermostability of
cr
laccases from different sources might be due to the differences in the number of disulphide
us
bonds in the protein [40]. 3.4 Effect of pH on papaya laccase activity
an
The effect of pH on papaya laccase with the phenolic substrate, catechol was investigated and pH optimum was found to be 8.0 (Fig. 5A). There was a steep increase in activity from pH 6.5 to 8.0
M
and at pH values above 8, the
d
Fig. 5
te
enzyme activity decreased gradually, still retaining 72% of its activity at pH 9.0. However, optimum pH of 6.0 was found with the non-phenolic substrate, ABTS, above which there was a
Ac ce p
decline in activity (data not shown). Thus, the phenolic substrate exhibited alkaline pH optimum while non-phenolic one exhibited acidic pH optimum. The main reason behind the increase and decrease in activity with pH in case of phenolic substrates has been given by Xu [41]. According to him, the pH-dependent decrease in reduction potential of the phenols (due to the phenolphenolate interconversion) is the cause of the increase in activity with pH whereas the hydroxide inhibition of the trinuclear cluster is the cause of decrease in activity with pH. The investigation of pH stability of the purified laccase revealed that it retained more than 75% of initial activity after 24 h incubation at 37oC at pH values from 6.5 to 9.0 (Fig. 5B). This showed its stability under alkaline conditions indicating that it is useful for processes
11 Page 11 of 32
involving alkaline conditions such as hair coloring as the swelling of hair swells and penetration of the dyes is enhanced at alkaline pH [22]. Besides, alkaline laccases also find application in textile industry for denim bleaching and in waste water treatment. There have been reports on
ip t
Some alkaline laccases with high activity and stability under alkaline conditions have also been reported [12, 35]. Thus, the ability of papaya laccase to withstand under alkaline conditions
cr
made this enzyme suitable for various applications.
us
3.5 Kinetic constants of papaya laccase
The effect of different phenolic (catechol and hydroquinone) and non-phenolic (ABTS)
an
substrates on the activity of purified papaya laccase was determined and various kinetic parameters like Km (substrate affinity), Kcat (turnover number i.e. rate of catalytic process), and
M
Kcat/Km (catalytic efficiency i.e. the efficiency of enzyme towards different substrates) were
Table 2
d
calculated (Table 2). The Km and Vmax values were found to be 0.04, 1.48 and 11.33 mM, and
te
0.04, 0.15 and 0.46 µM min-1ml-1 for ABTS, catechol and hydroquinone, respectively. Thus, the
Ac ce p
affinity of papaya laccase towards various substrates was found to be in the order: ABTS > catechol > hydroquinone. However, Kcat of the enzyme for these substrates were found to be in just opposite order: hydroquinone (3.07 min-1) > catechol (0.99 min-1) > ABTS (0.29 min-1). The catalytic efficiencies of the purified papaya laccase for various substrates (as presented by Kcat/Km) were found to be in the order: ABTS (7.25 mM-1min-1) > catechol (0.67 mM-1 min-1) > hydroquinone (0.27 mM-1 min-1). Thus, the non-phenolic substrate, ABTS was found to be most efficiently oxidized by papaya laccase which is evident from the Km as well as the Kcat/Km values. Catechol was found to be the second efficient substrate; however, the papaya laccase was less reactive towards hydroquinone. Almost similar Km values with ABTS have been reported for laccases from fungi, Ceriolopsis subvermispora (0.042 mM [42]), Daedela quercina (0.038 mM, 12 Page 12 of 32
[43]) and Phlebia radiata (0.049 mM [44]). Lower Km values for ABTS have also been reported with laccases from plant, Populus euramericana (0.026 mM [3]), fungal, Pleurotus pulmunarius (0.21mM [33]), Trichoderma harzianum (0.018 mM [9]), Trametes hirsuta (0.07 mM [36]) and
ip t
bacterial sources such as γ-Proteobacterium JB (0.073 mM [45]). Laccase from plant sources such as Rhus vernicifera, R. succedanea, Amorphophallus campanulatus and L. leucocephala,
cr
showed Km values of 45, 15, 3.13 and 1.24 mM with catechol, respectively [6, 46, 47]. The Kcat
us
value of papaya laccase for ABTS (0.29 min-1) was lower than those found for several other laccases, such as those of L. leucocephala (3.53 min-1 [6], T. hirsuta (197 s-1 [36], P. ostreatus
an
(244.32 s-1 [38], Coriolus hirsutus (260 s-1 [48], Trametes pubescens (876 s-1 [49], and Pleurotus pulmonarius (1520 s-1 [33] but higher than those reported from Picnoporus cinnabarinus (920
M
min-1 [50] and Phellinus ribis (8.0 x 104 min-1 [32].
d
3.6 Effect of various effectors on laccase activity
te
The effect of various effectors namely, metal ions, reducing agent (DTT), chelating agent
Table 3
Ac ce p
(EDTA), and detergent (SDS) on papaya laccase activity were studied and are shown in Table 3.
Mn2+, Cd2+, Ca2+and Na+ activated laccase in a concentration-dependent manner (0.1-10 mM) while Na+ exhibited activation of laccase upto 1 mM beyond which it exhibited inhibition of laccase activity. Fe2+, Co2+ and Cu2+ activated laccase in a concentration-dependent manner upto 1 mM. The effect of these metal ions at concentrations beyond 1 mM could not be investigated because of their interference in color development during enzyme assay. A concentrationdependent inhibition of laccase activity was observed with Hg2+ upto 1 mM. Thus, the papaya laccase was found to be fairly tolerant to all the metals tested exhibiting activation with the exception of Hg2+. Some metal tolerant laccases have also been reported from plants like L.
13 Page 13 of 32
leucocephala [6], C. pterogonus and Opuntia vulgaris [5]; fungi, Pleurotus ostreatus [38], Trametes versicolor [15] and T. pubescens [49] and bacteria, Streptomyces psammoticus [51]. However, the metal tolerance of papaya laccase was much better than the earlier reports. Thus,
ip t
the highly tolerant activity of papaya laccase towards various metal ions is of great value in terms of its potential industrial application. It is well-known that the catalytic site of laccase
cr
contains four copper atoms clustered at three types of copper sites (type I, II, and III) which
us
perform monoelectronic oxidation of suitable substrates [52]. As reported by Duggleby et al. [53], the activation by the metal ions may be due to the favored conformational modifications
an
thus stimulating laccase activity. However, the blockage of the access of the substrate or the transfer of electron at the T1 site results in inhibition in laccase activity [54].
M
DTT activated laccase at lower concentration (0.1 mM) while inhibited the enzyme at
d
concentrations beyond 0.1 mM in a concentration-dependent manner with complete inhibition in
te
activity at 10 mM. Similar activation of laccase at lower and concentration of DTT with L. leucocephala laccase has been reported [6]. The gradual inhibition in laccase activity with
Ac ce p
increasing concentration of DTT indicated the role of thiol groups in laccase activity [55, 56]. SDS and EDTA also inhibited laccase activity similar to those reported from peach, O. vulgaris, C. pterogonus, Trametes versicolor, Streptomyces cyaneus and S. psammoticus [5, 6, 15, 51, 56, 57]. The inhibition of laccase activity by EDTA might be due to the metal chelation of Cu in the catalytic site of the enzyme [14]. However, the change in the conformation of the protein structure might be the cause of the inhibition of activity by SDS. 4. Conclusion A laccase from papaya was purified to homogeneity in a two step process, namely heat treatment and Con-A affinity chromatography with good enzyme yield. It was found to be a hexamer with pH and temperature optima of 8.0 and 70oC, respectively. The enzyme was found to be fairly 14 Page 14 of 32
tolerant towards heat, pH and metal ions. In addition, the papaya laccase showed enhancement in activity in the presence of most of the metal ions. Such characteristics indicate the potential suitability of papaya laccase for use in bioremediation, textile, pulp and paper industries.
ip t
Acknowledgements
Financial support from UGC, New Delhi, India, in the form of Dr. D.S. Kothari Postdoctoral
cr
Fellowship to NJ is gratefully acknowledged. Financial supports from Department of Higher
us
Education, Govt. of Uttar Pradesh, India under Centre of Excellence in Bioinformatics, Department of Biotechnology, Govt. of India under BIF Scheme, New Delhi and Department of
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Science and Technology, New Delhi under Promotion of University Research and Scientific Excellence (DST-PURSE) programme for providing infrastructure facilities are also gratefully
M
acknowledged.
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Microbiol. 62 (1996) 1151-1158.
d
[51] K.N. Niladevi, N. Jacob, P. Prema, Evidence for a halotolerant alkaline laccase in
te
Streptomyces psammoticus: purification and characterization, Proc. Biochem. 43 (2008) 654-60. [52] M. Frasconi, G. Favero, H. Boer, A. Koivula, F. Mazzei, Kinetic and biochemical properties
Ac ce p
of high and low redox potential laccases from fungal and plant origin, Biochim. Biophys. Acta 1804 (2010) 899-908.
[53] R.G. Duggleby, Experimental designs for estimating the kinetic parameters for enzymecatalysed reactions, J. Theor. Biol. 81 (1979) 671-684. [54] Z.M. Fang, T.L. Li, F. Chang, P. Zhou, W. Fang, Y.Z. Hong, X.C. Zhang, H. Peng, Y.Z. Xiao, A new marine bacterial laccase with chloride-enhancing, alkaline dependent activity and dye decolorization ability, Biores. Technol. 111 (2012) 36-41. [55] C. Johannes, A. Majcherczyk, Laccase activity tests and laccase inhibitors, J. Biotechnol. 78 (2000) 193-199.
21 Page 21 of 32
[56] M. Lorenzo, D. Moldes, S. Rodríguez Couto, M.A. Sanromán, Inhibition of laccase activity from Trametes versicolor by heavy metals and organic compounds, Chemosphere 60 (2005) 1124-1128.
ip t
[57] M.E. Arias, M. Arenas, J. Rodriguez, J. Soliveri, A.S. Ball, M. Hernandez, Kraft pulp biobleaching and mediated oxidation of a nonphenol substrate by laccase from Streptomyces
Ac ce p
te
d
M
an
us
cr
cyaneus CECT3335, Appl. Environ. Microbiol. 69 (2003) 1953-1958.
22 Page 22 of 32
Figure Captions Fig. 1. A: Native-PAGE analysis and silver staining of purified papaya laccase during
ip t
purification. B: In-gel activity staining of purified papaya laccase. The gel after Native-PAGE was immersed in 50 mM catechol solution until the brown-colored band appeared.
cr
Fig. 2. Calibration plot for the determination of the native molecular weight of purified papaya
us
laccase using gel filtration chromatography. The standard protein markers (1.0 mg/ml) used
albumin (67 kDa) and lysozyme (14.3 kDa).
an
were: catalase (240 kDa), β-amylase (200 kDa), phosphorylase B (97.4 kDa), bovine serum
Fig. 3. A: SDS-PAGE and silver staining of purified papaya laccase. Lane 1: purified laccase,
M
Lane 2: molecular weight markers. Standard protein markers containing myosin (205 kDa),
te
anhydrase (29 kDa) were used.
d
phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), ovalbumin (43 kDa), and carbonic
B: Calibration plot for the determination of the subunit molecular weight of purified papaya
Ac ce p
laccase.
Fig. 4. A: Effect of temperature on the activity of papaya laccase. The enzyme was incubated at different temperature (10-90 oC) and activity was measured under standard assay conditions. B: Thermal stability of papaya laccase at different temperatures. Percent relative activity represents the enzyme activity relative to the control (0 min), which was set at 100%. Fig. 5. A: Effect of pH on the activity of papaya laccase. The activity was assayed at different pH (6.5-9.0) under standard assay conditions. B: pH stability of papaya laccase. The enzyme was incubated for 24 h at different pH (6.5-9.0) and activity assayed using a suitable pre-incubated enzyme aliquot under standard assay
23 Page 23 of 32
conditions. Percent relative activity represents enzyme activity calculated by setting the activity,
Ac ce p
te
d
M
an
us
cr
ip t
at optimum pH, as 100%.
24 Page 24 of 32
Total Activity
Total Protein
Specific Activity
(U)
(mg)
(U/mg)
Crude extract
17
571.50
0.03
100
Heat treatment
13.44
20.80
0.65
79
21.70
10.45
0.25
41.30
cr
Table 1. Summary of laccase purification from papaya leaves (20 g)
1376.70
o
(70 C, 30 min) Con-A affinity
Fold Purification 1
61.5
Ac ce p
te
d
M
an
us
chromatography
Yield (%)
ip t
Steps
25 Page 25 of 32
Table 2. Kinetic properties of papaya laccase with various phenolic and non-phenolic substrates Km (mM)
Vmax (µM min-1 ml-1)
Kcat (min-1)
Kcat/Km (mM-1 min-1)
ABTS
0.04
0.04
0.29
7.25
Catechol
1.48
0.15
0.99
Hydroquinone
11.33
0.46
3.07
ip t
Substrates
0.67
Ac ce p
te
d
M
an
us
cr
0.27
26 Page 26 of 32
Table 3. 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
0.1
0.5
1
3
Mn2+
100
115±1.7
185±1.5
231±2.0
346±2.2
Cd2+
100
144±1.5
173±1.5
188±1.8
221±2.4
243±2.2
265±2.1
Ca2+
100
109±1.6
119±1.4
130±1.7
175±1.8
192±2.1
222±2.0
Mg2+
100
110±1.8
114±1.3
118±1.6
133±1.8
138±2.0
151±1.8
Na+
100
167±1.3
174±1.7
171±1.7
161±2.0
150±1.5
130±2.0
Fe2+
100
116
148
176
*
*
*
Co2+
100
117±1.3
136±1.2
153±1.4
*
*
*
Cu2+
100
118±1.7
130±1.6
147±1.8
*
*
*
Hg2+
100
95±1.4
d
80±1.3
*
*
*
DTT
100
138±1.8
88±1.4
31±2.1
21±1.5
9±1.5
0
SDS
100
94±1.5
84±1.8
73±1.7
61±1.6
51±2.3
37±2.1
100
97±1.8
89±1.5
84±1.8
73±1.5
66±1.6
51±1.8
EDTA
us
an
M
te
89±1.4
5
10
385±2.0
412±2.5
cr
0
Ac ce p
Effectors
ip t
Concentration (mM)
values are presented as the mean ± SD of triplicate tests.
* salts of Fe2+, Co2+, Cu2+ and Hg2+ at concentrations beyond 1 mM interfered with the color development during enzyme activity assay and hence the effect could not be measured.
27 Page 27 of 32
i
Figure 1
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
Ac
ce pt
ed
M an
us
cr
Int. J. Biol. Macromol.
A
B
Fig. 1
Page 28 of 32
i
Figure 2
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
us
cr
Int. J. Biol. Macromol.
M an
Lysozyme
BSA
ed
Phosphorylase B
ce pt
Beta amylase Catalase
Ac
Laccase
Fig. 2
Page 29 of 32
cr
i
Figure 3
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
2
205 kDa
Band 2
45 kDa
Band 3
38 kDa
Band 4
25 kDa
Band 2
Band 3
66 kDa
Band 4
ce pt
52 kDa
43 kDa
29 kDa
Ac
Band 1
Band 1
ed
97.4 kDa
Catalase
M an
1
us
Int. J. Biol. Macromol.
A
B Fig. 3
Page 30 of 32
cr
i
Figure4
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
M an
us
Int. J. Biol. Macromol.
120
40
10
0 0
20
ce pt
20
40 60 Temperature (oC)
80
% Residual Activity
ed
30
Ac
Activity (U/ml) x 10-2
100 80
50oC
60
60oC 70oC
40
80oC 20 0
100
0
20
40
60
Time (min)
B
A
Fig. 4
Page 31 of 32
cr
i
Figure5
Nivedita Jaiswal, Veda P. Pandey and Upendra N. Dwivedi
M an
us
Int. J. Biol. Macromol.
ed
20
10 5 6 A
7
ce pt
15
8
100 80 60 40 20
6
9
7
8
9
pH
pH
Ac
Activity (U/ml) x 10-2
25
% Relative Activity
120
30
Fig. 5
Page 32 of 32