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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].
69
2.2. Microorganism
2.1. Chemicals
70
Fig. 1. Phylogenetic tree using internal transcribing spacers (ITS) regions of G. lucidum MDU-7.
Fig. 2. Biomass estimation and laccase of G. lucidum MDU-7 and zymogram of crude
) Biomass; (
) Laccase activity.
protein collected on different hours. (
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.
(
72
Fig. 5. Effects of metal ions and additives on puried laccase isozymes (a,b) Glac H1 and (c,d) Glac L1. (
) 1 mM; (
) 3 mM; (
) 6 mM; (
) 9 mM.
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)
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
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.
74
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).
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].
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