Supplementary Information
SupplementaryMethods
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 KH2PO4, 0.5 MgSO4, 0.01 CaCl2, 0.001 FeSO4, 0.1 Na2HPO4, 0.62 CuSO4, 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. Seven-days-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).
Effect of metal ions and various reagents on laccase activity
Different metal ions (1 mM) Al3+, Ca2+, Cd2+, Co2+, Cr2+, Cu2+, Fe2+, Mg2+, Mn2+, Na+, Ni2+, Hg2+, and Zn2+, 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%.
Supplementary Tables
Supplementary Table 1 Summary of purification steps of extracellular laccase from Pleurotus sp. MAK-II
Purification step / Total protein (mg) / Total activity (Units) / Specific activity (Units/mg protein) / Purification (fold) / YieldCulture supernatant / 368.4 / 82,000 / 222.5 / 1 / 100
(NH4)2SO4 Fraction 70% (w/v) / 104 / 44,800 / 430.7 / 1.93 / 54.6
Sephadex G-100 / 36 / 38,000 / 1055.5 / 4.7 / 46.3
DEAE cellulose / 15 / 24,200 / 1613.3 / 7.25 / 29.5
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) / pI / Optimum pH / Optimum temperature (°C) / ReferenceCerrena maxima / 57.0 / 3.5 / 4.0-4.2 / 50 / (Koroleva et al. 2002)
Ganoderma fornicatum / 63.7 / – / 3.0 / 55 / (Huang et al. 2011)
Ganoderma lucidum / 38.3 / 4.56 / 5 / 55 / (Manavalan et al. 2013)
Ganoderma lucidum / 32.18 / 4.79 / – / – / (Manavalan et al. 2012)
Ganoderma lucidum / 42.33 / 4.56 / – / – / (Manavalan et al. 2012)
Ganoderma lucidum / 43.01 / 4.56 / – / – / (Manavalan et al. 2012)
Ganoderma lucidum / 56.04 / 4.71 / – / – / (Manavalan et al. 2012)
Ganoderma lucidum / 57.17 / 4.53 / – / – / (Manavalan et al. 2012)
Lentinula edodes / 58.5 / – / 3.5 / 40 / (Nagai et al. 2009)
Pleurotus eryngii / 60.0 / – / 7 / 50 / (Ueda et al. 2012)
Pleurotus eryngii / 34.0 / – / 3-5 / 70 / (Wang and Ng 2006)
Pleurotus ostreatus / 40.0 / – / 4 / 50 / (Liu et al. 2009)
Pleurotus sajor-caju / 35.0 / – / – / 50 / (Bettin et al. 2011)
Pleurotus sajor-caju / 45.0 / – / – / 50 / (Bettin et al. 2011)
Pleurotus sajor-caju / 70.0 / – / – / 50 / (Bettin et al. 2011)
Pleurotus sajor-caju / 61.0 / – / 5.0 / 40 / (Murugesan et al. 2006)
Pleurotus sajor-caju / 61.0 / 3.2 / 6.0 / 25 / (Zucca et al. 2011)
Pleurotus sp. MAK-II / 43.0 / – / 4.5 / 60 / Present study
Pycnoporus sp. SYBC-L1 / 55.89 / – / 3 / 65 / (Wang et al. 2010)
Pycnoporus sp. SYBC-L1 / 63.07 / – / 2.5 / 70 / (Wang et al. 2010)
Trametes pubescens / 60.0 / – / 6.0 / 60 / (Gaitan et al. 2011)
Trametes pubescens / 120.0 / – / 6.0 / 60 / (Gaitan et al. 2011)
Trametes sp. Ha1 / 62.0 / 3.0 / 5.0 / 70 / (Nakatani et al. 2010)
Trametes sp. Ha1 / 62.0 / 5.9 / 5.0 / 70 / (Nakatani et al. 2010)
SupplementaryTable 3Substrate specificity for purified laccase activity
Substrate / Absorbance at nm / Relative activity (%)ABTS / 436 / 100
Guaiacol / 436 / 98
Pyrogallol / 450 / 82
p-Phenylenediamine / 487 / 55
Catechol / 450 / 38
Ferulic acid / 287 / 18
Tyrosine / 280 / 0
Veratryl alcohol / 310 / 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.
Supplementary Table 4Effect of different metal ions, inhibitors, surfactants, and solvents on purified laccase activity
Chemicals (concentration) / Relative activity (%)Control (None) / 100
Metal ions (1 mM)
Al3+ / 94
Ca2+ / 106
Cd2+ / 75
Co2+ / 83
Cr2+ / 35
Cu2+ / 125
Fe2+ / 96
Mg2+ / 110
Mn2+ / 90
Na+ / 79
Ni2+ / 39
Hg2+ / 6
Zn2+ / 47
Inhibitors (1 mM)
Sodium azide / 0
Cyanide / 0
EDTA / 100
DTT / 27
β-Mercaptoethanol / 35
Surfactants (1%, w/v and v/v)
SDS / 82
Sarkosyl / 89
CTAB / 96
Tween 20 / 98
Tween 80 / 95
Triton-X 100 / 90
Solvents (5%, v/v)
Acetone / 32
Acetonitrile / 11
Butanol / 54
DMSO / 92
DMF / 51
DCM / 67
Ethanol / 98
Methanol / 95
Isopropanol / 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.
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) syringaldehydeused in this study.
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