Supplementary Information
Supplementary Methods
Fungal strain and culture conditions
Stock cultures of Pleurotus sp. (strain ‘MAK-II’) were maintained on the potato dextrose agar
(PDA) slant at 4 °C in the dark. The mycelium from the slant was transferred to PDA plates and incubated at 30 °C for 7 days. Mycelial discs from the peripheral region of actively growing culture were used as inoculum. The production medium contains (g l
-1
): 10.0 mannitol, 2.0 yeast extract, 2.0 L-proline, 0.3 erythromycin, 1.0 KH
2
PO
4
, 0.5 MgSO
4
, 0.01 CaCl
2
, 0.001 FeSO
4
, 0.1
Na
2
HPO
4
, 0.62 CuSO
4
, 0.0275 adenine, and 2.0 biotin in 1 l distilled water (pH 5.0). Laccase production was carried out under submerged fermentation in a laboratory scale bioreactor (5 l working volume capacity). Three liters of the production medium were added to the reactor and sterilized for 20 min at 121 °C at 15 psi. The pre-cultures were prepared in 250-ml Erlenmeyer flasks containing nutrient medium with glucose (20 g l
−1 ) in static conditions at 30 °C. Sevendays-old pre-cultures were homogenized before transferring into the medium in the bioreactor
(20 g l
−1
wet mycelium). The agitator and flow rate of filter-sterilized air were set at 100 rpm and
3 l h
−1
, respectively. The internal temperature of the bioreactor was maintained at 30 °C. After 3 days incubation, vanillic acid (1 mM) was added to the production medium as inducer to enhance laccase production.
Laccase gene amplification using degenerative primers from Pleurotus sp. MAK-II
Laccase gene amplification was performed with primers Cu1F: 5′-CAT(C) TGG CAT(C) GGN
TTT(C)TTT(C) CA-3′ and Cu2R: 5′-G G(A)CT GTG GTA CCAGAA NGT NCC-3′ according
to the method of D'Souza et al. (1996).
1

Effect of metal ions and various reagents on laccase activity
Different metal ions (1 mM) Al 3+ , Ca 2+ , Cd 2+ , Co 2+ , Cr 2+ , Cu 2+ , Fe 2+ , Mg 2+ , Mn 2+ , Na + , Ni 2+ ,
Hg
2+
, and Zn
2+ , or various inhibitors (1 mM; that included cyanide, β-mercaptoethanol, dithiothreitol, ethylenediaminetetraacetic acid, and sodium azide, or various surfactants (1% w/v and v/v; viz.: cetyltrimethylammonium bromide, Sarkosyl, SDS, Tween 20, Tween 80, and
Triton-X 100), or various solvents (5% v/v; that included acetone, acetonitrile, butanol, dimethyl sulfoxide, dimethylformamide, dichloromethane, ethanol, methanol, and isopropanol) were examined on laccase activity by pre-incubating the enzyme in above chemicals (metal ions, inhibitors, surfactants, and solvents ) for 1 h and the remaining enzyme activity was assayed under standard assay condition using 1 mM ABTS as substrate in sodium acetate buffer (100 mM, pH 4.5) at 30 °C. The activity assayed in the absence of any additives (metal ions or inhibitors or surfactants or solvents) was defined as 100%.
2

Supplementary Tables
Supplementary Table 1 Summary of purification steps of extracellular laccase from Pleurotus sp. MAK-II
Yield Purification step Total protein Total activity
(mg)
Culture supernatant 368.4
(Units)
82,000
104 44,800
Specific activity
(Units/mg protein)
222.5
430.7
Purification
(fold)
1
1.93
100
54.6 (NH
4
)
2
SO
4
Fraction
70% (w/v)
Sephadex G-100
DEAE cellulose
36
15
38,000
24,200
1055.5
1613.3
4.7
7.25
46.3
29.5
3

Supplementary Table 2 Comparison of physicochemical properties of laccase from Pleurotus sp. MAK-II and laccases from other white-rot fungi
Name of fungi Molecular mass (kDa)
Cerrena maxima 57.0 pI
3.5
Optimum pH Optimum
4.0-4.2 temperature
(°C)
50
Reference
–
3.0 55
(Koroleva et al.
2002)
(Huang et al. 2011)
Ganoderma fornicatum
Ganoderma lucidum
Ganoderma lucidum
Ganoderma lucidum
Ganoderma lucidum
Ganoderma lucidum
63.7
38.3
32.18
42.33
43.01
56.04
Ganoderma lucidum
57.17
Lentinula edodes 58.5
Pleurotus eryngii 60.0
Pleurotus eryngii 34.0
4.56
4.79
4.56
4.56
4.71
4.53
–
–
–
5
–
–
–
–
–
3.5
7
3-5
55
–
–
–
–
–
40
50
70
40.0 – 4 50
(Manavalan et al.
2013)
(Manavalan et al.
2012)
(Manavalan et al.
2012)
(Manavalan et al.
2012)
(Manavalan et al.
2012)
(Manavalan et al.
2012)
(Nagai et al. 2009)
(Ueda et al. 2012)
(Wang and Ng
2006)
(Liu et al. 2009)
Pleurotus ostreatus
Pleurotus sajorcaju
Pleurotus sajorcaju
Pleurotus sajorcaju
Pleurotus sajor-
35.0
45.0
70.0
61.0
–
–
–
–
–
–
–
5.0
50
50
50
40
(Bettin et al. 2011)
(Bettin et al. 2011)
(Bettin et al. 2011)
(Murugesan et al.
4

caju
Pleurotus sajorcaju
Pleurotus sp.
MAK-II
Pycnoporus sp.
SYBC-L1
Pycnoporus sp.
SYBC-L1
61.0
43.0
55.89
63.07
Trametes pubescens
Trametes pubescens
60.0
120.0
Trametes sp. Ha1 62.0
Trametes sp. Ha1 62.0
–
–
–
–
3.2
–
3.0
5.9
5
6.0
6.0
5.0
5.0
6.0
4.5
3
2.5
70
70
60
60
65
70
25
60
2006)
(Zucca et al. 2011)
Present study
(Wang et al. 2010)
(Wang et al. 2010)
(Gaitan et al. 2011)
(Gaitan et al. 2011)
(Nakatani et al.
2010)
(Nakatani et al.
2010)

Supplementary Table 3 Substrate specificity for purified laccase activity
Substrate
ABTS
Guaiacol
Pyrogallol p -Phenylenediamine
Catechol
Absorbance at nm Relative activity (%)
436 100
436 98
450
487
450
82
55
38
Ferulic acid
Tyrosine
Veratryl alcohol
287
280
310
18
0
0
Values represent the mean of three independent experiments, with a maximal mean deviation of ± 5 %. Enzyme activity was measured under standard assay conditions, using ABTS as substrate.
6

Supplementary Table 4 Effect of different metal ions, inhibitors, surfactants, and solvents on purified laccase activity
Chemicals (concentration)
Control (None)
Mg 2+
Mn 2+
Na +
Ni 2+
Hg 2+
Zn 2+
Metal ions (1 mM)
Al 3+
Ca 2+
Cd 2+
Co 2+
Cr 2+
Cu 2+
Fe 2+
Inhibitors (1 mM)
Sodium azide
Cyanide
EDTA
DTT
β-Mercaptoethanol
Surfactants (1%, w/v and v/v)
SDS
Sarkosyl
CTAB
Tween 20
Tween 80
Triton-X 100
Solvents (5%, v/v)
Acetone
Acetonitrile
Butanol
Relative activity (%)
100
96
110
90
79
39
6
47
94
106
75
83
35
125
82
89
96
98
95
90
32
11
54
0
0
100
27
35
7

DMSO
DMF
DCM
Ethanol
92
51
67
98
Methanol
Isopropanol
95
97
Values represent the mean of three independent experiments, with a maximal mean deviation of ± 5 %. Enzyme activity was measured under standard assay conditions, using ABTS as substrate.
8

Supplementary Fig. 1 Chemical structures of the different dyes (a) Congo Red (diazo dye), (b)
Remazol Brilliant Blue R (anthraquinone dye) and redox mediators, (c) 1-hydroxybenzotriazole,
(d) p-coumaric acid, (e) violuric acid, and (f) syringaldehyde used in this study.
9

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