Laccase isozymes from Ganoderma lucidum MDU-7: Isolation, characterization, catalytic properties and differential role during oxidative stress

Journal of Molecular Catalysis B: Enzymatic 113 (2015) 68–75

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Laccase isozymes from Ganoderma lucidum MDU-7: Isolation, characterization, catalytic properties and differential role during oxidative stress Amit Kumar a , Krishna Kant Sharma a,∗ , Pramod Kumar b , Nirala Ramchiary b a Laboratory of Enzymology and Recombinant DNA Technology, Department of Microbiology, Maharshi Dayanand University, Rohtak 124001, Haryana, India b School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

a r t i c l e

i n f o

Article history: Received 8 October 2014 Received in revised form 15 January 2015 Accepted 16 January 2015 Available online 28 January 2015 Keywords: Laccase isozymes Native-PAGE Catalytic properties Antioxidant

a b s t r a c t Strain of Ganoderma lucidum MDU-7 produce multiple extracellular isoforms of laccase in submerged culture condition using malt extract as a carbon source and copper sulfate as an inducer. SDS–PAGE followed by MALDI–TOF peptide fingerprinting confirmed laccase isozyme with molecular mass of 24–66 kDa. Two laccase isozymes (Glac H1 and Glac L1) were purified from native-PAGE protein purification method and a comparative catalytic and antioxidant study has been performed. Both of the laccase isozymes have optimum temperature and pH at 50 ◦ C and 4.0, respectively. Glac L1 has higher stability in comparison to Glac H1, over wide range of temperature, pH, divalent metal ions and surfactants. The Km values of Glac L1 and Glac H1 determined for guaiacol, ABTS and O-tolidine were 98 ␮M, 26 ␮M, 320 ␮M and 281 ␮M, 29 ␮M, 338 ␮M, respectively. Glac H1, irrespective to its laccase activity and stability, acts as a better antioxidant than Glac L1. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Ganoderma lucidum has been used as medicinal mushroom for treatment of various human diseases [1]. Recent, whole genome sequence of G. lucidum has revealed complete set of ligninolytic peroxidases, laccases and cellobiose dehydrogenases, which makes it excellent model for ligninolytic enzymatic studies [2]. Earlier, Ganoderma has been reported with different lignin modifying enzymes, such as, manganese-dependent peroxidase (MnP) and laccase [3,4]. Laccases (p-diphenol: dioxygen oxidoreductase; EC 1.10.3.2) are polyphenol oxidases that catalyze the oxidation of phenolic compounds with a concomitant reduction of oxygen to water [5]. Laccases are present in fungi, plants, insects and bacteria with diversified functions, such as, polymerization in plants, depolymerization and pathogenicity in fungi and bacteria [6,7]. Several studies have been focused on laccase production because of its potential application in delignification, paper bleaching, deinking of news paper, bioremediation, textile industries, biosensors, and in medical sectors [6–10].

∗ Corresponding author. Tel.: +91 9996303126. E-mail address: [email protected] (K.K. Sharma). http://dx.doi.org/10.1016/j.molcatb.2015.01.010 1381-1177/© 2015 Elsevier B.V. All rights reserved.

Multiple isoforms of laccase have been reported depending on the fungal species and different environmental factors [11–13]. Many aromatic compounds and metals ion have been reported which regulate the production of differential laccase isozymes [14,15]. Laccase isozymes from different fungal strains have been characterized previously [3,14–17]. Till date, only three laccase isozymes from G. lucidum have been purified and characterized. Wood decay fungi via. fenton reaction produces reactive oxygen species (ROS) i.e., hydroxyl (• OH) radical and other secondary radicals (ROO• and • OOH), which involved in wood decay [18]. ROS are generated during normal cellular metabolism or in some stress conditions, commonly responsible for the aging, DNA damage, and protein damage [19–21]. Polysaccharides and some other bioactive metabolites with antioxidative properties from G. lucidum, Cerrena unicolor, G. applanatum, Lentinus edodes and Trametes versicolor have been reported [22–24], whereas, laccase has been reported with antiproliferative [25,26], prooxidant [23] and HIV reversetranscriptase inhibitor activity [27]. Earlier, laccase gene from G. lucidum has been cloned and studied for its antioxidant properties [28]. In the present study, Cu2+ induced laccase isozymes were purified from G. lucidum MDU-7 and their differential antioxidative properties were demonstrated. Moreover, this is the first report of multiple peptide sequences and its specific catalytic role.

A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 68–75

2. Materials and methods

69

2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), guaiacol, O-tolidine and coomassie brilliant blue G-250 were purchased from Sigma Co., (St. Louis, MO, USA). All other media components and chemicals were of highest purity grade available commercially.

laccases, respectively. The gel preparation and sample loading of SDS and native-PAGE were performed according to the standard method [31]. In SDS–PAGE, the protein bands were visualized with Coomassie Brilliant Blue G-250 colloidal staining and destained with solution containing 25% methanol as described elsewhere [32]. After nativePAGE the gel was stained with 0.1 M citrate–phosphate buffer (pH 4.0) containing 2 mM O-tolidine and incubated at 30 ◦ C in dark condition [16].

2.2. Microorganism

2.8. Characterization of purified laccase isozymes

The basidiocarps were collected from the delignified biomass and processed by following standard isolation protocol. Primer sequences ITS1 – 5 -TCC GTA GGT GAA CCT GCG G-3 (forward) and ITS4 – 5 -TCC TCC GCT TAT TGA TAT-3 (reverse) were used to identify laccase producing basidomycetous fungi. The sequence, thus obtained was analyzed using NCBI BLAST and sequence was submitted to GenBank. The phylogenetic tree was constructed to locate the taxonomic position of the fungus using the MEGA analysis tool v 6.

2.8.1. Effect of pH and temperature on laccase activity The enzyme activity was assayed in the pH range of 3.0–8.0 using suitable buffers. The substrate was prepared in 50 mM of citrate buffer (pH 3.0–6.0), phosphate buffer (pH 7.0–8.0), Tris–HCl (pH 9.0) and carbonate–biscarbonate buffer (pH 10.0). The enzyme activity was expressed as percent relative activity with respect to maximum activity, which was considered as 100%. Effect of temperature on isozymes activity was determined by incubating the reaction mixture at different temperature varying from 25 ◦ C to 60 ◦ C under standard assay conditions.

2.1. Chemicals

2.3. Culture conditions G. lucidum MDU-7 was maintained on malt extract agar (MEA) containing (g l−1 ): malt extract 20.0, KH2 PO4 0.5, MgSO4 ·7H2 O 0.5, Ca(NO3 )2 ·4H2 O 0.5, agar 20.0 (pH 5.2) at 30 ◦ C [4]. Pure culture was maintained on MEA slants at 30 ◦ C and sub-cultured fortnightly. 2.4. Analytical procedure Guaiacol was used as a substrate for assaying laccase activity following the method as described earlier [4]. One unit (U) of laccase was defined as the change in absorbance of 0.01 ml−1 min−1 at 470 nm. The protein content was estimated by Lowry’s method using bovine serum albumin (BSA) as a standard [29]. 2.5. Production of laccase isozyme Laccase production was carried out in 25 ml malt extract broth (MEB) in 250 ml Erlenmeyer flask. Each flask was inoculated with four fungal discs (8 mm dia each) from the periphery of 5 days old culture of G. lucidum MDU-7. Thereafter, the culture medium was induced on 3rd day with different concentration of CuSO4 (1–10 mM).

2.8.2. Thermostability and pH stability of laccase isozymes The pH stability was determined by incubating the enzyme in buffers of 50 mM of citrate buffer (pH 3.0–6.0) and phosphate buffer (pH 7.0–8.0) for 30 min at 30 ◦ C, whereas, the temperature stability was determined by incubating the enzyme samples at various temperatures (25 ◦ C–60 ◦ C) for different time intervals (1–8 h). 2.8.3. The kinetic parameters of purified laccase isozymes Michaelis–Menten constant (Km ) and the maximum rate of reaction (Vmax ) were determined by using guaiacol (470 nm), ABTS (420 nm) and O-tolidine (627 nm) as substrate at concentration ranging from 0.5 to 3.5 mM, 0.1 to 0.9 mM, and 0.2 to 1 mM, respectively, in citrate–phosphate buffer (pH 4.0). The values of Km and Vmax were calculated from Eadie–Hofstee plot. 2.8.4. Effect of metal ions and additives on enzyme activity The effect of various bivalent metal ions and additives, viz. sodium azide, SDS, Triton X-100, EDTA and CTAB on activity of laccase isozymes were determined by incubating the isozymes at a final concentration ranging from 1 to 9 mM for 30 min at 30 ◦ C and measuring the residual activity under the standard assay conditions.

2.6. Purification of laccase isozymes The culture broth (950 ml) was filtered through Whatman filter no. 1 and centrifuged at 13,000 × g for15 min at 10 ◦ C. The protein extract was concentrated using an Amicon Ultra-15 membrane filter (Millipore, Germany). Partial purification of laccase from the culture filtrate was carried out by addition of finely ground ammonium sulfate at three different saturation levels, i.e., 0–20%, 20–40% and 40–80%. After overnight incubation at 4 ◦ C, the culture filtrate was centrifuged at 9000 × g for 20 min. Precipitates were dissolved in 20 mM citrate–phosphate buffer (pH 4.0) and dialyzed overnight against same buffer at 4 ◦ C. Further, laccase isozymes were purified from native-PAGE (12%) according to the modified method as described earlier by Retamal et al. [30] (Fig. 3c). 2.7. Molecular mass of laccase isoenzymes Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and non-denatured PAGE (native PAGE) were used to determine the molecular mass of partially purified and purified

2.9. UV absorption spectra of purified laccase isozymes The laccase UV-absorbance spectrum was scanned from 200 to 800 nm at room temperature on a Shimadzu UV-1800 spectrophotometer. 2.10. Identification of laccase isozymes by MALDI–TOF analysis The identification of proteins by peptide mass fingerprinting was carried out by MALDI–TOF/TOF analysis of partially purified copper induced laccase proteins by cutting the band obtained by SDS–PAGE analysis. The trypsin digested gel samples and the resulting peptides were spotted on MALDI target plate to obtain peptide spectra using ABI SCIEX MALDI–TOF/TOF 5800. The peptides were then analyzed by comparing mass spectrometry data in the National Center for Biotechnology Information (NCBInr) protein database using the Mascot search algorithm [33].

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Fig. 1. Phylogenetic tree using internal transcribing spacers (ITS) regions of G. lucidum MDU-7.

2.11. Antioxidant property of laccase isozymes A modified method of BSA protection assay was done as described earlier by Joo et al. [28]. BSA (50 ␮g) was added in 0.1 ml of 0.1 M citrate–phosphate buffer, pH 5.20 containing 100 ␮M copper and 2.5 mM hydrogen peroxide. Laccase isozymes (1 and 5 unit) were added and incubated for 2 h at 37 ◦ C. Ascorbate and lysozyme (50 ␮g/0.1 ml) were used as a control in the absence and presence of laccase isozymes. BSA (2 ␮g) from each reaction mixture was run on 12% SDS–PAGE gel (Bio-rad mini, USA) along with markers (Sigma–Aldrich, USA), and were visualized by staining with CBB R-250. 3. Results and discussion 3.1. Isolation and identification of a laccase producing strain of basidiomycetous fungus Basidiomycetous fungus was identified as G. lucidum MDU-7 (GenBank accession no. KF549493.1). The neighbor joining phylogenetic tree of ITS conserved sequence of G. lucidum MDU-7 showed moderate similarity with G. lucidum MDU-3, but shared clade with other G. lucidum strains, and the boot strap analysis support phylogenetic relationship with score value of 60–88% (Fig. 1).

[3,16]. Copper metal ions have a substantial effect on induction of laccase isozymes, their stabilization and on transcript regulation [14,17,36,37]. 3.4. Purification and characterization of isozymes The two laccase isozymes, i.e., Glac H1 and Glac L1 were purified from native-PAGE (Fig. 3c). Both purified laccase isozymes showed a single band in native gel electrophoresis followed by activity staining (Fig. 3d). Earlier, two thermostable laccase isozymes have been reported from Pycnoporus sanguineus by using ultrafiltration, ion exchange and hydrophobic interaction chromatography [38]. High throughput techniques currently used to purify proteins are sometimes cost intensive, time consuming and practically impossible. So, the numbers of different protein purification have been demonstrated via. PAGE, achieving 80–95% of protein recovery and also the amino acid sequences of purified proteins have been determined successfully [30]. The zymography of laccase proteins on native page confirmed molecular mass in range between 30 and 70 kDa (Fig. 3d). Similarly, Sharma et al. [4] reported laccase enzyme on native PAGE with a molecular mass of 68 kDa from Ganoderma sp. Thus, the above procedure for protein purification used in our study was unique and cost competitive, which can be further used

3.2. Production of laccase isozymes Laccase production from G. lucidum MDU-7 under liquid fermentation condition started after 48 h of incubation (3.9 U/ml), showed highest laccase activity on 96 h (8.3 U/ml) and declined thereafter (Fig. 2). The laccase activity correlations with biomass are species specific and behave independently [13]. Zymogram study of culture supernatant at different time intervals showed differential patterns of constitutive laccase isozymes (Fig. 2). Earlier, nine active enzymes have been reported by overexpression of the non-allelic 17 laccase genes from Coprinopsis cinerea under the control of a constitutive promoter [34]. Differential production of laccase isozymes in time dependent manner may be due to different physiological role. Thus, further understanding of physiological role of laccase isozymes in different fungi is important for basic and applied purposes [35]. 3.3. Effect of copper on laccase isozyme production Laccase activity in copper induced G. lucidum MDU-7 was found to be highest on 336 h (771 U/ml). Activity staining of nativePAGE showed six laccase isozymes from G. lucidum MDU-7, when induced with copper sulfate (Fig. 3b). Earlier, 3–4 isozymes have been reported from G. lucidum using different induction medium

Fig. 2. Biomass estimation and laccase of G. lucidum MDU-7 and zymogram of crude ) Biomass; ( ) Laccase activity. protein collected on different hours. (

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Fig. 3. Laccase production and isozyme study from G. lucidum MDU-7. (a) Laccase activity after inducing culture medium with CuSO4. (b) Laccase isozymes activity staining with O-tolidine, 1–8, sample collected on 4th day, than onward after every 48 h up to 15th day. (c) Procedure of isozymes purification from native-PAGE. (d) Purified isozymes; activity staining with I, II, ABTS; III, IV, guaiacol; V, VI, O-tolidine; I, III, V, Glac L1; II, IV, VI, Glac H1. Glac L1–Glac L5, low mol mass laccase; Glac H1, high mol mass laccase.

Fig. 4. (a) Optimal pH of purified laccase isozymes (Glac H1 and Glac L1) from G. lucidum MDU-7. (b) The pH stability of laccase isozymes (Glac H1 and Glac L1). (c) Optimal ) Glac H1; ( ) Glac L1; temperature for laccase isozymes (Glac H1 and Glac L1) was determined. Thermostability of laccase isozymes (d) Glac H1 and (e) Glac L1. ( ) 30 ◦ C; ( ) 40 ◦ C; ( ) 50 ◦ C. (

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Fig. 5. Effects of metal ions and additives on purified laccase isozymes (a,b) Glac H1 and (c,d) Glac L1. (

to understand the biochemical properties of proteins and their applicability. The UV spectrum studies of both laccase isozymes (Glac H1 and Glac L1) showed a broad peak around 600 nm region due to the presence of type I copper atom [5]. Thus, the purified laccase isozymes belong to typical blue copper oxidases family. 3.5. Effect of pH on laccase activity Purified laccase isozymes (Glac H1 and Glac L1) were active over acidic pH, with optimum activity at pH 4.0 (Fig. 4a), which was similar to the earlier reports from G. lucidum [16], Ganoderma sp. [4], Albatrellus dispansus [27] and Lentinula edodes [39]. The pH optima of laccase varied with substrates, such as, acidic for ABTS and guaiacol (lower than pH 5.0) and slightly higher for DMP and syringaldazine (pH 5.0–7.0) [4]. In the present study, both of the laccase isozymes (Glac H1 and Glac L1) were found to be stable over a wide acidic pH range, i.e., 3.0–5.0 (Fig. 4b). Similarly, pH stability of laccase enzyme from Ganoderma sp. was reported in acidic conditions [4]. Increase in pH cause conformational change

) 1 mM; (

) 3 mM; (

) 6 mM; (

) 9 mM.

in laccase structure and specially on catalytic site, thus internal electron transfer of laccase get inhibited and reaction product may be differed [4]. 3.6. Effect of temperature on laccase activity Both the laccase isozymes (Glac H1 and Glac L1) have temperature optima at 50 ◦ C (Fig. 4c), and stable in 30–40 ◦ C range. Glac H1 and Glac L1 lost 50% of residual activity at 50 ◦ C after 1 h and 4 h of incubation, respectively (Fig. 4d and e). The temperature optima of laccase isozymes were similar to the previous reports [4]. Higher optimum temperature of laccase isozymes makes them widely useful for many biotechnological applications. 3.7. Effect of metal ions and additives The effects of several metal ions and surfactants on the activity of Glac H1 and Glac L1 were examined using guaiacol as a substrate at pH 4.0. The isozymes Glac H1 and Glac L1 were inhibited by Cr2+ , Hg2+ , Fe2+ and NaN3 in a concentration dependent manner. Both

Table 1 Michaelis–Menten kinetic constants of studied laccase isozymes from G. lucidum MDU-7 at their optimal pH with different substrates. Laccase isozymes

ABTS Km (␮M)

Glac H1 R2 Glac L1 R2

29 26

O-tolidine Vmax (␮mol/ml/min) 0.152 0.974 0.780 0.993

Guaiacol

Km (␮M)

Vmax (OD/min/unit enzyme)

Km (␮M)

Vmax (␮M/ml/min)

338

0.44 0.946 1.7 0.985

281

180,000 0.970 320,000 0.985

320

98

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Fig. 6. (a) SDS–PAGE analysis of partially purified laccase. (A) Coomassie staining. M-Marker Lane (14.2–200 kDa). (I–IV) laccase isozymes confirmation by MALDI–TOF.

isozymes were relatively stable in the presence of several metal ions, such as, Ca2+ , Ba2+ , Cd2+ , Al2+ , and Mo2+ . Interestingly, more than 100% residual activity was observed in the presence of Cu2+ and Zn2+ (Fig. 5a and c). Both of the isozymes were highly stable in the presence of metal ions and similar in characteristics to earlier reported laccase enzymes from Ganoderma sp. [4,40]. Isozymes, Glac H1 and Glac L1 also showed stability with anionic surfactants (SDS) and metal ion chelators (EDTA). At higher concentration, non-ionic (Triton X-100) and cationic (CTAB) surfactant showed significant loss in laccase activity. Further, Glac L1 was found to be more stable than Glac H1, in the presence of Triton X-100 and CTAB (Fig. 5b and d). Interestingly, Glac L1 was found to be more stable during zymogram study (Fig. 3b).

3.8. The kinetic parameters of purified laccases Kinetic parameters of purified laccase isozymes, Glac H1 and Glac L1 were studied with different substrates. The apparent Km value of the Glac L1 and Glac H1 for guaiacol determined from the Eadie–Hofstee plot was estimated to be 98 ␮M and 281 ␮M, respectively. The Km values of Glac L1 and Glac H1 determined for ABTS and O-tolidine were 26 ␮M, 29 ␮M and 320 ␮M, 338 ␮M, respectively (Table 1). Our results suggest that isozymes have different

substrate binding affinity, i.e., ABTS should be an effective substrate for Glac H1 and Glac L1, whereas guaiacol for Glac L1. Both isozymes showed the lower Km value for O-tolidine than the earlier reports [16]. The substrate affinity of other fungal laccase was much higher, i.e., 1300 ␮M [26] and 966.5 ␮M [41] for ABTS and 2270 ␮M [26] and 1112.2 ␮M [41] for guaiacol respectively. Earlier, Manavalan et al. [17] reported laccase from G. lucidum with Km of 47 ␮M for ABTS, which was higher than Glac H1 and Glac L1. The differences in kinetic properties suggest broad biochemical role and diverse biotechnological applications.

3.9. Confirmation of laccase isozymes by MALDI–TOF The partially purified laccase from G. lucidum MDU-7 showed molecular mass of 24 kDa–66 kDa (Fig. 6a). Similarly, laccase enzyme of 40–66 kDa [3], 38.3 kDa [17], 62 kDa [4] from Ganoderma has been reported. The copper induced partially purified laccase isozymes were identified by MALDI–TOF and the peptide fingerprints were compared in NCBI protein database. The peptide sequences of G. lucidum MDU-7 were matched (low score) with laccase from G. lucidum, hence confirmed all the isozymes belongs to laccase family (Fig. 7I–IV).

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Fig. 7. Antioxidant properties of Glac H1 and Glac L1 against BSA and Cu2+ /H2 O2 model system. (A) 50 ␮g of BSA was added to 0.1 ml of dH2 O; (B) 50 ␮g of BSA was added 0.1 ml of 100 ␮M copper and 2.5 mM H2 O2 ; (C) 50 ␮g of ascorbate was added to 0.1 ml of 50 ␮g of BSA, 100 ␮M copper and 2.5 mM H2 O2 ; (D and E) 1 U and 5 U of Glac H1 were added to (C), respectively; (F and G) 1 U and 5 U of Glac L1 were added to (C), respectively; (H) 50 ␮g of lysozyme was added to (B); (I) 50 ␮g of ascorbate was added to (H); (J) 5 U of Glac L1 was added to (I); and (K) 5 U of Glac H1 was added to (I).

3.10. Antioxidative properties of laccase isozymes

4. Conclusion

Both the laccase isozymes (Glac H1 and Glac L1) revealed antioxidant properties with BSA as a reference protein. These isozymes showed an antioxidant property, however, Glac H1 was found to be a stronger antioxidant than Glac L1. Further, Glac H1 was produced in the stationary growth phase by G. lucidum MDU-7, when grown on metal (Cu2+ ) induced stress conditions (Fig. 3b). So, the possible role of Glac H1 isozyme was predicted to have an antioxidant. Our studies showed that laccase isozyme Glac H1 act as scavenger for free radical and protect BSA from oxidative degradation (Fig. 7D, E, and K). The scavenging property of laccase isozyme competes with ascorbate for Cu2+ metal ion, thus protect BSA from free radical degradation/toxicity (Fig. 7F, G, and J). Alternately, the hydroxyl radical scavenging properties of isozymes, which react with free radical and stabilize the molecules by the generation of intermediate biomolecules, which act as an antioxidant for • OH radicals [42]. Alteration in reaction conditions also effect the fate of enzyme i.e., laccase in monophasic system with dioxane as a solvent leads to the formation of ␤-5 dimer of ferulic acid, which act as a better antioxidant than the substrate itself. While, ethanol as a co-solvent leads to the formation of ␤–␤ dimer, having lower antioxidant properties even than from the substrate i.e., ferulic acid [43]. Earlier reports suggest that the free radicals did not react with glycosylated proteins and lead to the formation of reaction product i.e., corresponding dimer of the compounds generating free radicals. Also, the carbohydrate content of the proteins helps in the protection of the polypeptides from modification by free radicals [44]. BSA at higher concentration gets degraded in the presence of copper and hydrogen peroxide in a time dependent manner [45]. Ascorbate at higher concentration in Cu2+ /H2 O2 model system produces • OH radicals which ultimately degrade the BSA protein [45] (Fig. 7C). Moreover, lysozyme shows protein–protein interaction with BSA protein [46]. Therefore, a faint band of BSA was observed in the presence of lysozyme, while BSA was completely degraded in the absence of lysozyme (Fig. 7). BSA has maximum stability at pH 7.0, but it is also quite stable at pH 5.0. [47]. In the present study, the antioxidant property of laccase isozymes was studied at pH 5.2 because both the isozymes were found to be highly active and more stable at acidic pH conditions. Earlier, antioxidant property of recombinant laccase (GLlac1) at pH 7.3, from G. lucidum was reported by Joo et al. [28].

Present study highlights the specific role of laccase isozymes produced under different environmental conditions. For certain biochemical reactions and synthetic chemistry, laccase has been reported to perform unique reaction. Our current finding is a paradigm shift in the field of molecular catalysis, where substrate specific reactions are preferred. Further, differential biochemical and kinetic properties of laccase isozymes support the non-specific catalytic property of fungal laccases vis-à-vis diverse biotechnological applications. Conflict of interests The authors declare no competing financial interest. Acknowledgements Authors would like to acknowledge DST, New Delhi (no. SR/FT/LS-100/2010) for their financial support and AIRF, JNU, New Delhi for providing MALDI–TOF facility. References [1] P. Batra, A.K. Sharma, R. Khajuria, Int. J. Med. Mushrooms 15 (2013) 127–143. [2] S. Chen, J. Xu, C. Liu, Nat. Commun. 3 (2012) 913. [3] T.M. D’Souza, C.S. Merritt, C.A. Reddy, Appl. Environ. Microbiol. 65 (1999) 5307–5313. [4] K.K. Sharma, B. Shrivastava, V.R. Sastry, N. Sehgal, R.C. Kuhad, Sci. Rep. 3 (2013) 1299. [5] C.F. Thurston, Microbiology 140 (1994) 19–26. [6] K.K. Sharma, R.C. Kuhad, Indian J. Microbiol. 48 (2008) 309–316. [7] D. Singh, K.K. Sharma, M.S. Dhar, J.S. Virdi, Biochem. Biophys. Res. Commun. 449 (2014) 157–162. [8] C.S. Rodríguez, J.L.T Herrera, Biotechnol. Adv. 24 (2006) 500–513. [9] R.C. Kuhad, G. Mehta, R. Gupta, K.K. Sharma, Biomass Bioenerg. 34 (2010) 1189–1194. [10] D. Singh, K.K. Sharma, S. Jacob, S.K. Gakhar, Water Air Soil Pollut. (2014) 225–2175. [11] P. Giardina, G. Palmieri, A. Scaloni, B. Fontanella, V. Farazo, G. Cennamo, G. Sannia, Biochem. J. 341 (1999) 655–663. [12] J.L. Dong, Y.W. Zhang, R.H. Zhang, W.Z. Huang, Y.Z. Zhang, J. Basic Microbiol. 45 (2005) 190–198. ˜ N.I. Sanabria, L.L. Villalba, P.D. Zapata, BioResources 8 [13] M.I. Fonseca, J.I. Farina, (2013) 2855–2866. [14] C. Galhaup, S. Goller, C.K. Peterbauer, J. Strauss, D. Haltrich, Microbiology 148 (2002) 2159–2169.

A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 68–75 [15] Y.Z. Xiao, Q. Chen, J. Hang, Y.Y. Shi, Y.Z. Xiao, J. Wu, Y.Z. Hong, Y.P. Wang, Mycologia 96 (2004) 26–35. [16] E.M. Ko, Y.E. Leem, H.T. Choi, Appl. Microbiol. Biotechnol. 57 (2001) 98–102. [17] T. Manavalan, A. Manavalan, K.P. Thangavelu, K. Heese, Biochem. Eng. J. 70 (2013) 106–114. [18] K.E. Hammel, A.N. Kapich, K.A. Jensen Jr., Z.C. Ryan, Enzyme Microb. Technol. 30 (2002) 445–453. [19] B.A. Ames, M.K. Shingenaga, E.M. Park, Oxidation Damage and Repair: Chemical. Biological and Medical Aspects, Pergamon, Oxford, 1991. [20] N.A. Simonian, J.T. Coyle, Annu. Rev. Pharmacol. Toxicol. 36 (1996) 83–106. [21] E.R. Stadtman, Ann. N. Y. Acad. Sci. 928 (2001) 22–38. ´ M.M. Vrvic, ´ N. Todorovic, ´ D. Jakovljevic, ´ L.J.L.D. [22] M. Kozarski, A. Klaus, M. Nikˇsic, Van Griensvend, J. Food Compos. Anal. 26 (2012) 144–153. ´ [23] M. Jaszek, M. Osinska-Jaroszuk, G. Janusz, A. Matuszewska, D. Stefaniuk, J. Sulej, J. Polak, M. Ruminowicz, K. Grzywnowicz, A. Jarosz-Wilkołazka, BioMed Res. Int. 2013 (2013). ´ [24] M. Osinska-Jaroszuk, M. Jaszek, M. Mizerska-Dudka, A. Błachowicz, T. Piotr Rejczak, G. Janusz, J. Wydrych, J. Polak, A. Jarosz-Wilkołazka, M. Kandefer´ BioMed Res. Int. 2014 (2014). Szerszen, [25] M. Li, G. Zhang, H. Wang, T. Ng, J. Microbiol. Biotechnol. 20 (2010) 1069–1076. [26] J. Sun, C. Qing-Jun, Z. Meng-Juan, W. He-Xiang, Z. Guo-Qing, J. Mol. Catal. B: Enzym. 99 (2014) 20–25. [27] H.X. Wang, T.B. Ng, Biochem. Biophys. Res. Commun. 315 (2004) 450–454. [28] S.S. Joo, I.W. Ryu, J.K. Park, Y.M. Yoo, D.H. Lee, K.W. Hwang, H.T. Choi, C.J. Lim, D. Lee, K. Kim, Mol. Cells 25 (2008) 112–118. [29] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265–275. [30] C.A. Retamal, P. Thiebaut, E.W. Alves, Anal. Biochem. 268 (1999) 15–20.

75

[31] J. Sambrook, D.W. Russel, Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, New York, 2001. [32] V. Neuhoff, N. Arold, D. Taube, W. Ehrhardt, Electrophoresis 9 (1988) 255–262. [33] D.N. Perkins, D.J. Pappin, D.M. Creasy, J.S. Cottrell, Electrophoresis 20 (1999) 3551–3567. [34] S. Kilaru, P.J. Hoegger, U. Kues, Curr. Genet. 50 (2006) 45–60. [35] C. Gu, F. Zheng, L. Long, J. Wang, S. Ding, PLOS ONE 9 (2014) e93912. [36] G. Palmieri, P. Giardina, C. Bianco, B. Fontanella, G. Sannia, Appl. Environ. Microbiol. 66 (2000) 920–924. [37] V. Faraco, P. Giardina, G. Sannia, Microbiology 149 (2003) 2155–2162. [38] L.I. Ramírez-Cavazos, C. Junghanns, N. Ornelas-Soto, D.L. Cárdenas-Chávez, C. Hernández-Luna, P. Demarche, E. Enaud, R. García-Morales, S.N. Agathos, R. Parra, J. Mol. Catal. B: Enzym. 108 (2014) 32–42. [39] H.X. Wang, T.B. Ng, Biochem. Biophys. Res. Commun. 322 (2004) 17–21. [40] H.L. Kumari, M. Sirsi, Arch. Mikrobiol. 84 (1972) 350–357. [41] J. Sun, R.H. Peng, A.S. Xiong, Y. Tian, W. Zhao, H. Xu, D.T. Liu, J.M. Chen, Q.H. Yao, Mol. Biol. Rep. 39 (2012) 3807–3814. [42] K. Suyeon, A. Cavaco-Paulo, Appl. Microbiol. Biotechnol. 93 (2012) 585–600. [43] O.E. Adelakun, T. Kudangaa, A. Parker, I.R. M. Green, l. Roes-Hill, S.G. Burton, J. Mol. Catal. B: Enzym. 74 (2012) 29–35. ˇ ´ M. Sulc, [44] V. Martínek, J. Sklenáˇr, M. Draˇcínsky, K. Hofbauerová, K. Bezouˇska, E. Frei, M. Stiborová, Toxicol. Sci. 117 (2010) 359–374. [45] J.C. Mayo, D.X. Tan, R.M. Sainz, M. Natarajan, S. Lopez-Burillo, R.J. Reiter, Biochim. Biophys. Acta 1620 (2003) 139–150. [46] S. Poole, S.I. West, C.L. Walters, J. Sci. Food Agric. 35 (1984) 701–711. [47] T. Estey, J. Kang, S.P. Schwendeman, J.F. Carpenter, J. Pharm. Sci. 95 (2006) 1626–1639.