Journal of Molecular Catalysis B: Enzymatic 113 (2015) 6875

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Journal of Molecular Catalysis B: Enzymatic

journal homepage: www.elsevier.com/locate/molcatb

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. SDSPAGE followed
by MALDITOF peptide ngerprinting conrmed laccase isozyme with molecular mass of 2466 kDa. Two
laccase isozymes (Glac H1 and Glac L1) were puried from native-PAGE protein purication 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
diversied 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 delignication, paper bleaching, deinking
of news paper, bioremediation, textile industries, biosensors, and
in medical sectors [610].

Corresponding author. Tel.: +91 9996303126.

E-mail address: kekulsharma@gmail.com (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 [1113].
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 [3,1417]. Till date, only three laccase
isozymes from G. lucidum have been puried 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 [1921]. Polysaccharides and some other bioactive
metabolites with antioxidative properties from G. lucidum, Cerrena
unicolor, G. applanatum, Lentinus edodes and Trametes versicolor
have been reported [2224], 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
puried from G. lucidum MDU-7 and their differential antioxidative
properties were demonstrated. Moreover, this is the rst report of
multiple peptide sequences and its specic catalytic role.

A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 6875

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 SDSPAGE, 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 citratephosphate 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 puried laccase isozymes

The basidiocarps were collected from the delignied 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 3.08.0
using suitable buffers. The substrate was prepared in 50 mM of citrate buffer (pH 3.06.0), phosphate buffer (pH 7.08.0), TrisHCl
(pH 9.0) and carbonatebiscarbonate 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 l1 ): 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 dened as the change in absorbance of 0.01 ml1 min1
at 470 nm. The protein content was estimated by Lowrys 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 ask. Each ask 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
(110 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 3.06.0) and phosphate buffer
(pH 7.08.0) for 30 min at 30 C, whereas, the temperature stability was determined by incubating the enzyme samples at various
temperatures (25 C60 C) for different time intervals (18 h).
2.8.3. The kinetic parameters of puried laccase isozymes
MichaelisMenten 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 citratephosphate buffer (pH 4.0). The values of Km and
Vmax were calculated from EadieHofstee 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
nal 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. Purication of laccase isozymes

The culture broth (950 ml) was ltered through Whatman lter
no. 1 and centrifuged at 13,000 g for15 min at 10 C. The protein
extract was concentrated using an Amicon Ultra-15 membrane lter (Millipore, Germany). Partial purication of laccase from the
culture ltrate was carried out by addition of nely ground ammonium sulfate at three different saturation levels, i.e., 020%, 2040%
and 4080%. After overnight incubation at 4 C, the culture ltrate
was centrifuged at 9000 g for 20 min. Precipitates were dissolved
in 20 mM citratephosphate buffer (pH 4.0) and dialyzed overnight
against same buffer at 4 C. Further, laccase isozymes were puried from native-PAGE (12%) according to the modied method as
described earlier by Retamal et al. [30] (Fig. 3c).
2.7. Molecular mass of laccase isoenzymes
Sodium dodecyl sulfatepolyacrylamide gel electrophoresis
(SDSPAGE) and non-denatured PAGE (native PAGE) were used to
determine the molecular mass of partially puried and puried

2.9. UV absorption spectra of puried 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. Identication of laccase isozymes by MALDITOF analysis
The identication of proteins by peptide mass ngerprinting
was carried out by MALDITOF/TOF analysis of partially puried
copper induced laccase proteins by cutting the band obtained
by SDSPAGE analysis. The trypsin digested gel samples and
the resulting peptides were spotted on MALDI target plate to
obtain peptide spectra using ABI SCIEX MALDITOF/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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A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 6875

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

2.11. Antioxidant property of laccase isozymes

A modied 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 citratephosphate 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% SDSPAGE gel (Bio-rad mini, USA) along with markers
(SigmaAldrich, USA), and were visualized by staining with CBB
R-250.
3. Results and discussion
3.1. Isolation and identication of a laccase producing strain of
basidiomycetous fungus
Basidiomycetous fungus was identied 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 6088% (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. Purication and characterization of isozymes
The two laccase isozymes, i.e., Glac H1 and Glac L1 were puried
from native-PAGE (Fig. 3c). Both puried 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 ultraltration,
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 purication have been
demonstrated via. PAGE, achieving 8095% of protein recovery and
also the amino acid sequences of puried proteins have been determined successfully [30].
The zymography of laccase proteins on native page conrmed
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 purication 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 specic 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, 34 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. (

A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 6875

71

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, 18, sample collected on 4th day, than onward after every 48 h up to 15th day. (c) Procedure of isozymes purication from native-PAGE. (d) Puried 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 L1Glac L5, low mol mass laccase; Glac H1, high mol mass laccase.

Fig. 4. (a) Optimal pH of puried 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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A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 6875

Fig. 5. Effects of metal ions and additives on puried 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 puried laccase
isozymes belong to typical blue copper oxidases family.
3.5. Effect of pH on laccase activity
Puried 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 5.07.0) [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., 3.05.0 (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 3040 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
MichaelisMenten 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

A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 6875

73

Fig. 6. (a) SDSPAGE analysis of partially puried laccase. (A) Coomassie staining. M-Marker Lane (14.2200 kDa). (IIV) laccase isozymes conrmation by MALDITOF.

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
signicant 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 puried laccases

Kinetic parameters of puried 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
EadieHofstee 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 afnity, 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 afnity 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. Conrmation of laccase isozymes by MALDITOF

The partially puried laccase from G. lucidum MDU-7 showed
molecular mass of 24 kDa66 kDa (Fig. 6a). Similarly, laccase
enzyme of 4066 kDa [3], 38.3 kDa [17], 62 kDa [4] from Ganoderma
has been reported.
The copper induced partially puried laccase isozymes were
identied by MALDITOF and the peptide ngerprints were compared in NCBI protein database. The peptide sequences of G.
lucidum MDU-7 were matched (low score) with laccase from G.
lucidum, hence conrmed all the isozymes belongs to laccase family
(Fig. 7IIV).

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A. Kumar et al. / Journal of Molecular Catalysis B: Enzymatic 113 (2015) 6875

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 modication 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 proteinprotein 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 specic 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 nding is a
paradigm shift in the eld of molecular catalysis, where substrate
specic reactions are preferred. Further, differential biochemical
and kinetic properties of laccase isozymes support the non-specic
catalytic property of fungal laccases vis--vis diverse biotechnological applications.
Conict of interests
The authors declare no competing nancial interest.
Acknowledgements
Authors would like to acknowledge DST, New Delhi (no.
SR/FT/LS-100/2010) for their nancial support and AIRF, JNU, New
Delhi for providing MALDITOF facility.
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