Purification, Peptide Sequencing and Study of Antiproliferative Activity of

Laccase against Liver cancer cell line Hep G2 and human breast cancer cell line
MCF 7
Author
Antik Kiron Bose

See Affilations

Senior Scientist and Project Advisor, Fred Hutchinson Cancer Research Center,
1100 Fairview Avenue N, Seattle, WA 98109

Summary
Three isozymes of laccase LCC 1, LCC 2 and LCC 3 have been purified from Pleurotus ostreatus strain V-184
using Toyopearl DEAE-650 Isoelectric focusing, ConA-sepharose 4B chromatography, Superdex-75 gel filtration, PD10
desalting column and Vydac C-18 Reverse phase HPLC chromatography with 950, 1220 and 935 folds of purification for
LCC 1, 2 and 3 respectively. The native molar mass of LCC 1 and 2 heterodimer was calculated to be 130Kda by gel
filtration and by 6% SDS-PAGE bands of 65, 60 and 80Kda were obtained for LCC 1, 2 and 3 respectively. Number of SH
groups in LCC 1 was calculated to be 4.68 and its full peptide sequencing gave a 533aa protein which was deposited in
Protein public database Uniprot with Acc. no. Q12739. IC50 of LCC 1 on Hepatocellular Carcinoma human (Hep G2) and
human brest cancer cell lines (MCF 7) were found to be 2.8 and 3.3 M respectively. 1 to 5M Menadione with 4.6M
LCC 1 was found to kill Hep G2 cells by production of hydroxyl radical from semiquinone produced by laccase catalysed
Quinone-redox cycle. For elucidation of mode of action of laccase on killing Hep G2 cells; a patent has been registered
with application no. 3299/DELP/2011 A at CGPDTM, West Bengal. The temperature and pH optima for all isozymes were
found to be 30C and 5.0 respectively. Spectral analysis showed presence of Type 3 and Type 1 Cu (II) in LCC 1 and
reduction of Type 1 during catalysis of Guaiacol. K
M
and V
max
for 3 isozymes with Guaiacol was found to be 84.034M,
714.25 U/min; 74.074M, 204.0816 U/min; 158.73M, 714.3U/min respectively for LCC 1, 2 and 3.
Introduction
Laccases, EC 1. 10. 3. 2, p-diphenol: dioxygen oxidoreductase, are part of a larger group of enzymes termed the multi-
copper enzymes, which includes among others ascorbic acid oxidase and ceruloplasmin. Laccase was first described by
Yoshida (1883) and was characterized as a metal containing oxidase by Bertrand(1985). This makes it one of the oldest
enzymes ever described.

Laccases can be roughly divided into two major groups which show clear differences, i.e. those from higher plants and
those from fungi (Harvey and Walker, 1999; Mayer and Harel, 1979; Solomon et. al.; 1996). The presence of laccase-like
enzymes has been reported in bacteria (Claus and Filip, 1997; Givaudan et. al.; 1993) as well as in insects (Diamantidis
et.al.; 2001; Hopkins and Kramer, 1992; Kramer et.al., 2001); however, some of the reports on the presence of laccases
in insects must be treated with caution; e.g. the enzyme present in the hemolymph of Anopheles is probably not a
laccase (Sidjanski et. al., 1997). The reviews on laccase by Messerschmidt (1993, 1997) and by Solomon et. al.(1996)
provide excellent summaries of the enzymology and electron transfer mechanism of the laccases, and a book edited by
Messerschmidt (1997) contains a series of articles dealing with different aspects of laccase lunctics and mechanism of
action and possible roles of the enzymes. The relationship between laccases and other multi-copper enzymes is stressed.
Spectroscopic techniques have provided further insights into the molecular mechanisms of copper proteins (Randall et.
al., 2001; Solomon et. al., 1998). Laccase from Coprinus cinereus, expressed in Aspergillus oryzae has been crystallized
and its three-dimensional structure was determined (Ducros et. al., 2001). Fungal laccases have been reviewed by
Thurston (1994) and their role in delignification by the white rot fungi has also been discussed (Eggert et. al., 1996; Youn
et. al., 1995). Laccases can degrade lignin in the absence of lignin peroxidase and Manganese peroxidase. It is fairly
obvious that the laccases are very ancient enzymes from an evolutionary point of view and that the enzyme activity
linked to 3 different copper sites must have been a very early evolutionary process. At the same time it should be
remembered that the properties of different laccases show a great deal of divergence, hence it can be debated whether
they should all be lumped together on the basis of their catalytic sites or whether some separation into different groups
is indicated.
Laccases occur widely in fungi and reports of their presence in more and more fungal species have been
published (Thurston, 1994). Their presence has been reported less frequently in higher plants. All laccases described to
date are glycoproteins. The classical demonstration of laccase in Rhus vernicifera is well documented and the enzyme
has been characterized in great details (Huttermann et. al., 2001). In addition, entire family of the Anacardiaceae, of
which the lacquer tree is a member appear to contain laccase in resin ducts and in the secreted resin (Huttermann et.
al., 2001). Reports on the presence of laccase in other plant species are more limited. Cell cultures of Acer
pseudoplatanus have been shown to produce and secrete laccase (Bligny and Douce, 1983; Tezuka et. al., 1993) and
Pinus taeda tissue has been shown to contain eight laccases, all expressed predominantly in xylem tissue (Sato et. al.,
2001). Other reports are those of Wosilait et. al. (1954) on presence of laccase in leaves of Aesculus parviflora and in
green shoots of tea (Gregory and Bendall, 1966). Five distinct laccases have been shown to be present in the xylem
tissue of Populus euramericana (Ranocha et. al., 1999). In contrast to the very many reports on the fungal laccases,
surveys of the occurance of laccase in green plants seem to have been badly neglected. They may be bound to cell walls
in some higher plants. It is difficult to determine their activity in crude extracts against a background of polyphenol
oxidase and peroxidase activity.
The presence of laccase has been documented in virtually every fungus examined for it. The genes for
numerous laccases have been cloned and the sequences deposited in the appropriate gene register. In fungal species
presence of both constitutive and inducible laccases has been reported. Usually the enzymes originate in the cytoplasm,
but many instances of secretion of laccases have been reported. Fungal laccases can oxidise quinol with uptake of
oxygen, develop pigmentation of fungal spores, regeneration of tobacco protoplasts, act as fungal virulence factor, help
in lignifications of cell wall and delignify cell wall during white rot of wood. They are commonly used to delignify woody
tissues, produce ethanol and to distinguish between morphine and codeine (Alfred M. Mayer, Richard C. Staples, 2002).
Laccases help to polymerize natural phenols to develop new cosmetic pigments, hair dyeing materials, deodorants,
toothpastes, mouth washes (Upendra N. Dwivedi, Priyanka Singh, Veda P. Pandey, Anoop Kumar, 2011). Laccases can
degrade polyaromatic hydrocarbonyl (PAH) like benzo () pyrene. They catalyse 1-electron oxidation of phenolic
substrates or aromatic amines to form quinines or polymerization products. In presence of low molecular mass
substrates called mediators, laccases can oxidise much wider range of compounds including PAHs (Laccase Mediator
System LMS). Mediators act as electron carriers. They can reduce products of anthracene biodegradation. They oxidize
anthracene to 9, 10 anthraquinone (Miguel Alcalde, Thomas Bulter, Francis H. Arnold, 2002).
The range of substrates which various laccases can attack is very wide. Basically any substrate with
characteristics similar to p-diphenol will be oxidized by laccases like Dihydroxybenzaldehyde, Guaiacol, Pyrogallol, Gallic
acid, o-tolidine, Dichlorophenol indophenols (DCPIP), Azino-bis(3-ethyl benzthia zoline-6-sulfonic acid) (ABTS), Violuric
acid (VIA), Poly R- 478, Coomassie Brilliant Blue (CBB) and Methyl green. In addition at least some of the fungal laccases
can oxidise monophenols such as cresols and some are able to oxidise ascorbic acid. But inadequate experimentation
has been carried out to differentiate between laccases and peroxidases and laccases and other polyphenoloxidases. An
indication of the complexity of thus problem is provided by the work of Schever and Fischer (1998). They purified a
laccase II from Aspergillus nidulans. Sequencing of the enzyme, despite its substrate specificity as a laccase and the high
degree of purification indicated more similarity to peroxidase than to laccase.

Materials Required
Fungal material- Fruit body of Pleurotus ostreatus strain V-184.
Instruments:- Morter and Pestle, Systronics- Photoelectric colorimeter- 113 (8 filters), wavelength range (430-700
nm),Systronics- Spectrophotometer LB5SA, wavelength range (240-690 nm),C24 REMI cooling centrifuge (rotor diameter
9cm),Chromatographic Glass Column, loading needle, stand.,Digital balance,Glass oven,Dialysis bag [Dialysis membrane
50, batch no. 0809353, AV diameter 14.3mm, capacity 0.61ml/m, Himedia 23, Vadani Insdustrial Estate LBS Marg,
Mumbai- 86],Gel apparatus and power pack,Bio-Rad Mini-Trans blot cell electrophoretic blotting apparatus, Perkin-
Elmar Applied Biosystems 477A pulsed-liquid protein sequencer equipped with model 120A phenylthiohydantoin
analyser, Zitex
R
G-filter membrane (pore size 5-6m; Saint Gobain performance plastic),Incubator and Electronic wath
bath, Phase Contrast Microscope (National Model 162-PH, Binocular version), Polyvinylidine fluoride (PVDF) membrane,
Fraction collector Gilsons Prep FC collector
Chemicals:-Tris-HCl, Na
2
EDTA, -marcaptoethanol, Benzamidine hydrochloride hydrate, 98% (catalogue no.- 206752-36-
5 B6506, Sigma-Aldrich), Leupeptin (Trade name Chemicon, Millipore, catalogue no.18, Ac-Leu_Leu Arginal
hemisulfated form, unot 843g/mg), Guaiacol, NaOH, (NH
4
)
2
SO
4
, Na
2
CO
3
, Folin-Ciocalteau reagent, CuSO
4
, Toyopearl
DEAE-650 (M-grade,weak anion exchanger, meanpore size 1000, particle size 65m, pressure rating 3bar, shipping
buffer- 20%, ethanol, pH stability 2-13, shelf life-10 years, Tosoh Biosciences Gm bH Zettachring 6, 70567, Stuttgart,
Germany,(Email info-tbg@tosoh.com), Sodium-2,2-dimethyl succinate, MES buffer, HCl, KCl, 20% ampholytes (pH range
2.5-9.0, Bio-Rad, Milan, Italy), glycerol, bromophenol blue, glycine, Tris, SDS(Sodium dodecyl sulfate), ConA coupled to
Sepharose 4B via CNBr (GE health care Biosciences, 800centennial Avenue, P.Box 1327, Piscataway, NJ, (Binding capacity
30mg/ml, coupling method CNBr activation, Matrix- 4% Agarose, Paticle size- 45-165m, pH stability 4-9, catalog no.17-
0440-03), CaCl
2,
MnCl
2
, NaCl, MgCl
2
, Glucose, Na
2
HPO
4
, KH
2
PO
4
, Tween 20, anti IgG Ab conjugated to Horse radish
peroxidase (Abazyme), monoclonal Ab against copper binding region II of 60KD laccase from Agaricus bisporus
(Abbiotech LLC), H
2
O
2
, 3, 3, 5, 5- tetramethyl benzidine ( Litton Bionetics, Kensington), Superdex- 75 (separation
range- 3,000 7,00,000; Matrix spherical composite of cross linked agarose and dextran, average particle size- 13m,
pressure 18bar, GF Health Care Lifesciences USA), Epichlorohydrin, dimethyldichlorosilane, Blue dextran- 2000, MW
markers [Bovine Pancreatic Chymotrypsin(20.6KD), chicken lactate dehydrogenase (H) (150 KD), Horse liver catalase
(222KD), Pleurotus sajor-caju urease(450KD), Squid heamocyanin (612 KD), Mitochondrial heat shock chaperone(60KD),
Regulatory subunit of ser/Thr PP2A(65KD), Human tyrosine kinase(80 KD), Phosphorylase b(97.4 KD), ovalbumin(45 KD),
Cyt oxidase of Pseudomonas aeruginosa (89.8 KD), Bovine serum albumin (66.2 KD), carbonic anhydrase (31KD) from
Sigma-Aldrich, USA], Ellmans Reagent [5,5 dithiobis (2-nitrobenzoic acid)], Urea, Dithiothreitol, iodoacetamide,
Ammonium bicarbonate, PD 10 desalting column ( G.E Healthcare lifesciences, inner diameter 1.5cm, length 7.4cm,
column material polypropylene, Frit material polyethylene, pore size- 20-80m), Trypsin (100U/ml, Sigma-Aldrich), N-
Glycosidase F (PNGaseF, 0.15U/ml) and 10X Glycoprotein denaturing buffer (New England Biolabs), Tergitol-type NP-40,
Endoproteinase Lys-C (Sigma-Aldrich), Endoproteinase Glu-C(P8100; New England Biolabs), acetonitrile, Trifluoroacetic
acid (TFA), Phenylisothiocyanate, Hep G2 (human liver carcinoma) cell line (ATCC NO. HB-8065, Abcam, USA), Dulbeecos
Modified Eagles Medium (DMEM) (100mg/dl glucose + 350mg/dl Glucose, GIBCO BRL, catalogue no.31600, Grand
Island, NY), Fetal bovine serum (FBS), insulin like growth factor (IGF-) (Sigma-Aldrich), MCF-7 (Michigan Cancer
Foundation-7, human breast cancer cell line Lonza AG, USA), 2-methyl-1,4-napthoquinone (Menadione), dimethyl
sulfoxide (DMSO), Urease Assay Kit (purchased from Worthington, Biochemical Pvt. Ltd., 730 Vassar Avenue, Lakewood,
New Jersey- 08701), chicken lactate dehydrogenase (H) assay kit (Bio Scientific, 3913 Todd Lane, Suite 312, Austin,
TX78744, USA, Fax no. (512)707-8122, Horse liver catalase assay kit (Calbiochem Biosciences Inc; Catalogue no. 219265,
10394, Pacific Center Court, San Diego, CA92121, USA), Assay kit for Bovine Pancreatic Chymotrypsin (catalog no.
FGAP034, Warthington Biochemical Pvt. Ltd., 730 Vassar Avenue, Lakewood, NJ-08701).

Procedure & Results
Purification of laccase
Pleurotus ostreatus strain V-184 was propagated on 2% Malt-extract agar using liquid culture media, described
by Mansur et. al. (1997) at pH 4.5 with 20mM sodium-2,2-dimethyl succinate and 50mM MES buffer foe 16 days
at 28C. The fruit body was used as the source of laccase. Strain identification was done by Dr. Andrii Grygansky,
Department of Biology, Duke University, Durham, NC27708, USA. Strain identification was based on
precomputed BLAST of flanking sequences of Tandem Repeats (TR) from strain against TR database. The enzyme
was obtained with 80% ammonium sulfate cut showing a recovery of 100% and 2folds of purification. The cut
solution was dialysed against dialysis buffer with 2.82 folds of purification and recovery of 99%. The enzyme was
eluted on Toyopearl DEAE-650 (M-grade) anion exchanger with 300mM KCl in assay buffer (pH 5.0) as a single
peak (tube 3) with 750713 folds of purification and 20.604% recovery. Fraction with activity was pooled and
dialysed for 24hrs with 96.159 folds of purification and 16.223% of recovery. The dialysed fraction was applied
to Iso-electric focusing on Toyopearl DEAE-650 anion exchanger with 20% ampholytes (range pH- 2.5-9.0). A
fraction with laccase activity with pI 3.0 was eluted with 300mM KCl prepared in 50mM Tris-HCl buffer (pH 2.5).
A separate fraction with laccase activity with pI 4.7 was eluted with laccase 300mM KCl elute prepared in 50mM
Tris-HCl buffer (pH 4.5) with 121.334 and 349.515 folds of purification and 8.242% and 4.12% of recoveries for
the fractions were separately dialysed for 24hrs with 145.60 and 388.35 folds of purification. Dialysed fraction
with pI 3.0 was applied to Concanavalin A-sepharose 4B affinity chromatography. Two fractions with laccase
activities were eluted with 200 and 300mM Glucose in assay buffer (pH 5.0) with 912.62 and 1096.1 folds of
purification and recoveries of 4.12% and 3.3% respectively. Similarly dialysed fraction with pI 4.7 was applied to
ConA-sepharose 4B column and eluted as a single peak with 300mM Glucose in assay buffer (pH 5.0) with
893.203 folds of purification and recovery of 4.12%. 200mM elute with pI 3.0 was named as LCC 1, 300mM elute
with pI 3.0 as LCC 2 and 300mM elute with pI 4.7 as LCC 3. The 3 isozymes were separately dialysed for 24hrs
with 950, 1220 and 935 folds of purification for isozyme 1 (LCC 1), isozyme 2 (LCC 2) and isozyme 3 (LCC 3)
respectively. 300mM KCl elute (pI 3.0) from Toyopearl DEAE-650 (M-grade) Isoelectric-focussing containing LCC
1 and LCC 2 was applied to Superdex-75 Gel filtration column and eluted as a single peak with 50mM Tris-HCl
buffer with 5713 fold of purification and 1.64% recovery and the final enzyme activity was measured to be
285.6707 U/g protein/min. Laccase was purified until apparent electrophoretic homogenecity was reached
showing monomers of 65, 60 and 80kDa for LCC 1, LCC 2 and LCC 3 respectively.

Steps of purification Activity
(U/min)
Protein
content
(g/ml)
Specific
Activity
(U/g
protein/min)
Fold of
purification
Yield
(%)
A. crude 5
100
0.05 Nil
100
B. 80%
(NH
4
)
2
SO
4
cut
10
100
0.10 2
100
C. Dialysis (24
hrs)
14
99
0.1414 2.82
99
D. Toyopearl
DEAE-650(M
grade)-
chromatograp
hy 300mM
KCl elute tube
3


78



20.604


3.786



75.713



20.604
E. Dialysis
(24hrs)

78

16.223
4.808
96.159

16.223
F. Isoelectric
focusing
i.Isozyme (pI
3.0) in 300ml
KCl (pH 2.5)
tube 4
ii. Isozyme
(pI- 4.7) in
300mM KCl
elute (pH 4.5)
tube 3




50




72




8.24175




4.12



6.067



17.476




121.334




349.525




8.242




4.12
G. Dialysis
isozyme

(pI 3.0)


(pI 4.7)




60



80




8.24175



4.12



7.28


19.417




145.60



388.35




8.241



4.12
H. Con A-
sepharose 4B
affinity
chromatograp
hy
200mM
Glucose elute
of isozyme
LCC 1 (pI-3.0)
( tube 2)

300mM
Glucose elute
of isozyme
LCC 2 (pI-3.0)
(tube 2)

300mM
Glucose elute
of isozyme
LCC 3 (pI 4.7)
( tube 2)








188







181






184







4.12







3.30






4.12






45.63






54.84





44.66







912.62







1096.1






893.203







4.12







3.3






4.12
I. Dialysis
LCC1

LCC2

LCC3

190

183

187

4.0

3.0

4.0

47.5

61.0

46.75

950

1220

935

4

3

4
J. Superdex-75
Gelfiltration
chromatograp
hy (tube 384)


468.5


1.64


285.6707


5713


1.64


Electrophoresis and Molar mass determination
300mM KCl elute (pI 3.0) from Isoelectric-focussing on Toyopearl DEAE-650 column containing LCC 1 and 2 was
applied to Superdex-75 gel filtration column and the native molar mass was estimated to be 130kDa however
bands of 65 and 60kDa were obtained in 6% SDS-PAGE suggesting that LCC 1 and 2 form heterodimer. 200mM
Glucose elute (pI 3.0), 300mM glucose elute (pI 3.0) and 300mM glucose elute (pI 4.7) from ConA-sepharose 4B
showed 65kDa, 60kDa and 80kDa bands respectively for LCC1,2 and 3. So, LCC 3 is always monomeric but LCC 1
and 2 can form a heterodimer.


fig. 300mM KCl in 50mM Tris-HCl (pH 2.5) elute from Iso electric focussing was loaded in lane 2 of 6% SDS-
PAGEmwith Phosphorylase b (97.4KD), regulatory subunit of serine/ threonine protein phosphatase 2A(65KD),
Mitochondrial Heat shock chaperon300mM KCl in 50mM Tris-HCl (pH 2.5) elute from Isoelectric focusing was
loaded in Lane2 of 6% SDS-PAGE with Phosphorylae (Hsp60) (60 KD) and Ovalbumin (45KD) as MW markers in
Lane 1 (from left). MW markers phospharylase b (97.4 KD), cytochrome oxidase of Pseudomonas aeruginosa
(89.8 KD), Human tyrosine kinase (80 KD), Regulatory subunit of serine/ threonine protein phosphatase 2A (65
KD) and Mitochondrial Heat shock chaperone (Hsp 60) (60 KD) were loaded in lane 3. Staining was done with
Coomassie Brilliant Blue G-250. Two bands of molecular mass 65 KD and 60 KD were obtained. The 65KD band
was named as laccase isozyme-1 (LCC 1) (pI- 3.0) and 60 KD band as laccase isozume-2 (LCC 2) (pI 3.0).

Fig. 300mM Glucose elute of ConA-sepharose 4B affinity column was loaded in lane 2 & 3 of 6% SDS-PAGE with
cytochrome oxidase of P. aeruginosa (89.8 KD), Bovine Serum Albumin (66.2 KD) and carbonic anhydrase (31 KD)
in lane 1 (from left). 200mM Glucose elute from ConA-Sepharose 4B was loaded in lane 4 of 6% SDS-PAGE (from
left). Staining with Coomassie Brilliant Blue G-250. 200mM Glucose elute from ConA-Sepharose 4B was loaded in
lane 4 of 6% SDS-PAGE (from left). Staining with Coomassie Brilliant Blue G-250. 65KD bands in lane 2 is of LCC 1
(pI 3.0) and 60KD band in lane 3 of LCC2 (pI 3.0).A band of 80KD was observed in lane 4 of LCC3 .


Fig. 300mM KCl in 50mM Tris-HCl (pH 4.5) elute from Iso-electric focusing was loaded in lane 2 (from left) of a
separate 6% SDS-PAGE. MW marker Human Tyrosine Kinase Ltk (80 KD) was loaded in lane 1, 3 and 4 (from left).
Staining with Coomasie Brilliant Blue G-250. Band of mol.mass 80KD was named as laccase isozyme 3 (LCC 3).


Western Blotting of 60kDa LCC 2(pI 3.0)
Purified 60kDa LCC2 protein was transferred to polyvinyl idenefluoride (PVDF) membrane after transblotting at
40V (300mA) for 2 hrs at room temperature. The protein was detected by monoclonal Lcc Cbr2 rabbit antibodt
against copper binding region II of 60kDa laccase from Agaricus bisporus (Abbiotech LLC) as primary antibody
and anti IgG conjugated to horse radish peroxidase (Abazyme) as secondary Ab. 0.05% (w/v) 3,3,5,5-
tetramethyl benzidine and 0.01% H
2
O
2
were used as substrates for HRP.

Fig. Detection of a 60KD LCC 2 band by western blotting with monoclonal Lcc Cbr 2 rabbit antibody against
copper binding region 2 of laccase from Agaricus bisporus.

Peptide sequencing
Concentration of SH groups in purified LCC1 was determined using Ellmans reagent [5,5-dithiobis(2-
nitrobenzoic acid)] (DTNB) to be 0.422mM and number of SH groups to be 4.68. It suggests presence of 2
disulphide bonds with 4 cysteine and 1 free SH group. The sample was denatured partially with 6(M) urea for
30mins and disulphide bonds were cleaved with 0.1%(v/v) Dithiothreiotol (DTT) at 37C for 2 hrs. Free cysteine
residues were alkylated using 10mM Iodoacetamide for 1hr. Desalting of the sample was done by passing it
through a PD 10 desalting column (G.E Healthcare, Lifesciences) and elution with 0.4% Ammonium bicarbonate
(pH 8.5). The eluted fraction was subjected to cleavage by peptidase like Trypsin (100U/ml) (sigma-Aldrich)
(cleaves at C-terminus of lysine and arginie), Endo proteinase Glu-C (4g/ml) (sigma-Aldrich) (cleaves at C-
terminus of Glutamate) and Endoproteinase Lys-C (7.2g/ml) (sigma-Aldrich) (cleaves at C-terminus of lysine) by
incubation at 37C for overnight. The tryptic digested fraction was denatured with 1X Glycoprotein denaturing
buffer at 100C for 10mins (New England Biolabs). The fraction was deglycosylated with N-glycosidase F
(PNGaseF) (0.15U/ml) with 10% Tergitol type NP_40 for breaking cell membranes at 37C for overnight
digestion. PNGaseF is an amidase that cleaves between the innermost N-acetyl D-glucosamine and Asparagine
residues of high mannose hybrid and complex oligosaccharides from N-linked Glycoproteins. 20l of each
digested fraction (conc. 200pmol) was separately loaded on HP1090A HPLC fitted with Vydac C-18 Reverse
phase (2.1mm 25cm) (Grace Vydac) and eluted with a linear gradient of 5-50% acetonitrile containing 0.1%
Trifluoroacetic acid. The C-terminal digestion fractions of endoproteinase LysC and GluC were filtered through
Zitex
R
-G-filter membrane (pore size 5-6m, Saint Gobain Performance plastic) and applied to HPLC. All the
eluted fractions were analysed by Perkin-Elmer Applied Biosystems 477A pulsed-liquid protein sequencer
equipped with model 120A phenylthiohydantoin analyser. The 533 amino acid peptide sequence of LCC1 was
deposited in Uniprot public protein database with Acc. No. Q12739 and entry name LAC1_PLEOS.
280
for the
protein was calculated to be 57005 M
-1
cm
-1
.

PEPTIDE SEQUENCE OF Pleurotus ostreatus STRAIN V-184 LACCASE ISOZYME (LCC 1)
Analysed by Perkin-Elmer Applied Biosystems 477A pulsed liquid protein sequencer equipped with
Model 120A Phenylthiohydantoin analyser. [Performed at Bone Narrow Transplant (BMT) and Leukemia
unit of Apollo Cancer Research Center, Kolkata)
UniProt Accession no. Q12739
Entry name- LAC 1- PLEOS
Total length of amino acid- 533
10 20 30 40 50 60
M F P G A R I L A T L T L A L H L L H G A H A A I G P A G N M Y I V N E D V S P D G F
A R S A V V A R S V P A T D P T P

70 80 90 100 110 120
A T A S I P G V L V Q G N K G D N F Q L N V V N Q L S D T T M L K T T S I H W H G F
F Q A G S S W A D G P A F V T Q C P

130 140 150 160 170 180
V A S G D S F L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D
P S D P H L S L Y D I D N A D T V I

190 200 210 220 230 240
T L E D W Y H I V A P Q N A A L P T P D S T L I N G K G R Y A G G P T S P L A I I N V E
S N K R Y R F R L V S M S C D P

250 260 270 280 290 300
N F T F S I D G H S L L V I E A D A V N I V P I T V D S I Q I F A G Q R Y S F V L T A N Q
A V D N Y W I R A N F N L G S

310 320 330 340 350 360
T G P V G G I N S A I L R Y A G A T E D O P T T T S S S T S T P L L E T N L V P L E N P G
A P G P P V P G G A D I N I N L

370 380 390 400 410 420
A M A F D F T T F E L T I N G V P F L P P T A P V L L Q L L S G A S T A A S L L P S G S I Y
E E E A N K V V E I S M P A

430 440 450 460 470 480
L A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G T G A
N D N Y T I R F V T D N P G P W
490 500 510 520 530
F L H C H I D W H L E I G L A V V F A E D V T S I S A P P A W D D L C P I Y N A L S D N D
K G G L V P S

No. of Y = 12 No. of W = 7 No. of C = 5

280
( M
-1
cm
-1
) = n (Trp) 5500 + n (Tyr)1490 + n (Cys) 125
= 7 5500 + 121490 + 5125
= 38500 + 17880 + 625
= 57005
Signal peptide sequence (1 23) M F P G A R I L A T L T L A L H L L H G A H A


Identification of conserved domains and results of homology searches with public protein databases
Reverse position specific BLAST (RPS-BLAST) of LCC1 peptide sequence with conserved domain databases has
identified Plastocyanin like 1 domain (25-171), Plastocyanin like 2 domain (382-501). Homology based searches
(BLAST) with public protein databases and translated nucleotide databases have shown 100% homology of LCC1
from Pleurotus ostreatus V-184 strain with phenol oxidase 2 of Pleurotus ostreatus (Acc. No. B7XGB7), 96% with
laccase 2 of P. pulmonarius (Acc. No. Q2VT18), 94% with laccase 4 of P. ostreatus (Acc. No. Q7Z853), 90.6% with
P. eryngii (Boletus of Steppes) ery3 protein (Acc. No. BOJDP8), 94% with P. sapidus laccase (Acc. No. Q6A1A1),
97% with P. ostreatus Bilirubin oxidase (Acc. No. Q9UVYA) and 5.6% with Trametes versicolor laccase 2 (Acc. No.
Q12718).
Identification of conserved domains in LCC 1 from Conserved domain database (CDD) using Reverse
Position specific BLAST (RPS-BLAST)
I G P A G N M Y I V N E D V S P D G F A R S A V V A R S V P A T D P T P A T A S I P G V L V Q G N K G D N F Q
L N V V N Q L S D T T M L K T T S S I H W H G F F Q A G S S W A D G P A F V T Q C P V A S G D S F L V N F N
V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D P S D P H L S L V (25-171) Plastocyanin-like 1
domain.

I D N A D T V I T L E D W Y H L V A P Q N A A I P T P D S T L I N G K G R Y A G G P T S P L A I I N V E S N K R Y
R F R L V S M S C D P N F T F S I D G H SL L V I E A D A V N I V P I T V D S I Q I F A G Q R V S F V L T A N Q A
V D N Y W I R A N P N L G S T G F V G G I N S A I L R Y A G A T E D D P T T T S S S T S T P L L E T (173 336)
Plastocyanin like 2 domain.

A P V L L Q I L S G A S T A A S L L P S G S I Y E L E A N K V V E I S M P A L A V G G P H P F H L H G H T F D V I
R S A G S T T Y N F D T R A R R D V V N T G T G A N D N V T I R F V T D N P G P W F L H C H I D W H L E I G L
A V V F A E D (382 501) Plastocyanin like 3 domain.

Homology based searches (BLAST) of LCC1 peptide sequence (ACC no. Q12739) showed 100% sequence
homology with phenol oxidase-2 of Pleurotus ostreatus (Acc no. B7XGB7)

BLASTp results of Laccase 1 ofPleurotus ostreatus (Acc no. Q12739) with laccase 2 of Pleurotus
pulmonarius (Acc no. Q2VT18) (Length 532)
10 20 30 40 50 60
M R P G A R I L A T L T L A L H L L H G A H A A I G P A G N M Y I V N E D V S P D G F A R S A V V A R S V P A
T D P T F M R P G A R I L A T L T L A L H L L H G A L A A I G P V G D M Y I V N E D V S P D G F S R S A V V A R
S V P I T G P T P

70 80 90 100 110 120
A T A S I P G V L V Q G N K G D N F Q L N V V N Q L S D T T M L K T T S I H W H G F F Q A G S S W A D G P A
F V T Q C P A T A S I P G V L V Q G N K G D N F Q L N V V N Q L S D T T M L K T T S I H W H G F F Q A G S S
W A D G P A F V T Q C P

130 140 150 160 170 180

A S G D S F L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D L R G P F V V Y D P S D P H L S L Y D I D N A D
T V I A S G D S F L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D L R G P F V V Y D P T D P H L S L Y D I D N
A D T V I
190 200 210 220 230 240
L E D W Y H I V A P Q N A A I P T P D S T L I N G K G R Y A G G P T S P L A I I N V E S N K R Y R F R L V S M S
C D P L E D W Y H I V A P Q N A A K S H P D S T L I N G K G R Y A G G P T S P L A I I N V E S T K R Y R F R L V
S M S C D P

250 260 270 280 290 300
F T F S I D G H S L L V I E A D A V N I V P I T V D S I Q I F A G Q R Y S F V L T A N Q A V D N Y W I R A N P N L
G S F T F S I D G H S L L V I E A D A V N I V P I T V D S I Q I F A G Q R Y S F V L T A D Q A V D N Y W I R A N P
N L G S

310 320 330 340 350 360
T G F V G G I N S A I L R Y A G A T E D D P T T S S T S T P L L E T N L V P L E N P G A P G P P Y P G G A D I N I
N L T G F A G G I N S A I L R Y V G A A D A D P T T S S T S T P L L E T N L V P L E N P G A P G P A V P G G A D
I N I N L



370 380 390 400 410 420
A M A F D F T T F E L T I N G V P F L P P T A P V L L Q I L S G A S T A A S L L P S G S I Y E L E A N K V V E I S M
P A
A M A F D F T T F E L T I N G V P F H P P T A P V L L Q I L S G A S S A A S L L P S G S I Y E L A P N K V V E I S
M P A

430 440 450 460 470 480
L A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G T G A N D N V T I R F V T D
N P G P W
L A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G T G A N D N V T I R F V T D
N P G P W

490 500 510 520 530
F L H C H I D W H L E I G L A V V F A E D V T S I S A P P A A W D D L C P I Y N A L S D N D K G G I V P S
F L H C H I D W H L E I G L A V V F A E D V T S I S A P P A A W D A L C P I Y D A L S D T D K G G I I P

Score = 1095
E value = 0.0
Identities = 96%

BLASTp results of laccase 1 of Pleurotus ostreatus (Acc. no. Q12739) with laccase 4 of Pleurotus
ostreatus (Acc no. Q)

10 20 30 40 50 60
M F P G A R I L A T L T L A L H L L H G A H A A I G P A G N M Y I V N E D V S P D G F A R S A V V A R S V P A
T D P T P
M F P G A R I L A T L A L A L H L L H G A L A A I G P V G D M Y I V N E D V S P D G F S R S A V V A R S V P I T
G P T P

70 80 90 100 110 120
A T A S I P G V L V Q G N K G D N F Q L N V V N Q L S D T T M L K T T S I H W H G F F Q A G S S W A D G P A
F V T Q C P
A T A S I P G V L V Q G N K G D N F Q L N V V N Q L S D T T M L K T T S I H W H G F F Q A G S S W A D G P A
F V T Q C P

130 140 150 160 170 180
V A S G D S F L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D P S D P S D P H L S L Y D
I D W A D T V I
V A P G D S L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D P T D P H L S L Y D I D N A
D T V

190 200 210 220 230 240
T L E D W Y H I V A P Q N A A I P T P D S T L I N G K G R Y A G G P T S P L A I I N V E S N K R Y R F R L V S M S
C D P
T L E D W Y H I V A P Q N A A I P T P D S T L I N G K G R Y A G G P T S P L A V I N V E S T K R Y R F R L V S M
S C D P

250 260 270 280 290 300
N F T F S I D G H S L L V I E A D A V N I V P I T V D S I Q I F A G Q R Y S F V L T A N Q A V D N Y W I R A N P N
L G S
N F T F S I D G H S L L V I E A D A V N I V P I T V D S I Q I F A G Q R Y S F V L T A D Q T P D N Y W I R A N P N
L G S

310 320 330 340 350 360
T G F V G G I N S A I L R Y A G A T E D D P T T T S S T S T P L L E T N L V P L E N P G A P G P P Y P G G A D I N
I N I
T G F A G G I N S A I L R Y V G A A D A D P T T T S S T S T P L L E T N L V P L E N P G A P G P P Y P G G A D I N
I N I

370 380 390 400 410 420
A M A F D F T T F E L T I N G V P F L P P T A P V L L Q I L S G A S T A A S L L P S G I Y E L E A N K V V E I S M P
A
A M A F D F T T F E L T I N G V P F H P P T A P V L L Q I L S G A S S A A S L L P S G S I Y E A P N K V V E I S M
P A

430 440 450 460 470 480
A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G T G A N D N Y T I R F V T D N
P G P W
A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G T G A N D N Y T I R F V T D N
P G P W

490 500 510 520 530
L H C H I D W H L E I G L A V V F A E D V T S I S A P P A A W D D L C P I Y N A L S D N D K G G I V P S
L H C H I D W H L E I G L A V V F A E D V T S I S A P P A A W D A L C P I Y D A L S D A D K G G I I P S
E value = 0.0
Identities = 94%
Score = 1045

Multiple sequence allingment between Pleurotus ostreatus LCC 1 (Acc no. Q12739) (1); Pleurotus eryngii
(Boletus of the steppes) ery3 protein (Acc no. BOJDP8) (2); Pleurotus sapidus laccase (Acc no. Q6A1A1)
(3); Pleurotus ostreatus bilirubin oxidase (Acc no. Q9UVY4) (4); Trametes versicolor laccase 2 (Acc no.
Q12718) (5)
10 20 30 40 50 60
1. M F P G A R I L A T L T L A L H L L H G A H A A I G P A G N M Y I V N E D V S P D G F A R S A V V A R S V
P A T D P T P
2. M F P G A R I L A T L T L A L H L L H G T H A A I G P I A D M Y I V N E D V S P G D F A R S A V V A R S V P
A T D P T P
3. M F P G A R I L A T L T L A L H L L R G T H A A I G P I D D M Y I V N E D V S P D G F T R S A V V A R S V P
A T G P A P
4. M F P G A R I L A T L T I A L H L L G T H A A I G P T G N M Y I V N E D V S P D G F A R S A V V A R S V P A
T D P T P
5. M G L Q R F S F F V T L A L V A R S L A I G P V A S L V V A N A P V S P D G F L R D A I V V G V V P S P L I T
G K K
70 80 90 100 110 120
1. A T A S I P G V L V Q G N K G D N F Q L N V V Q L S D T T M L K T T S I H W H G F F Q A G S S W A D G P A F
V T Q C P
2. A S V S V P G V L V Q G N K G D N F Q L N V R Q L S D S T M L K T T S I H W H G F F Q S G S T W A D G P A F
V N Q C P
3. A T V S I P G V L V Q G N K G D N F Q L N V V Q L S D T T M L K T T S I H W H G F F Q F G T S W A D G P A F
V T Q C P
4. A T V S I P G V L V Q G N K G D N F Q L N V V N Q L S D T T M L K T T S I H W H G F F Q A G S S W A D G P A
F V T Q C P
5. G D R F Q L N V D D T L T N H S M L K S T S I H W H G F F Q A G T N W A D G P A F V N Q C P I A S G H S F L
Y D F H V P

130 140 150 160 170 180
1. V A S G D S F L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D P S D P H L S L Y D I D N
A D T V I
2. I A S G N S F L Y D F N V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D P S D P H L S L Y D V D N
A D T V I
3. I A S G D S F L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D P S D P H L S L Y D I D N A
D T V I
4. V A S G D S F L Y N F N V P D Q A G T F W Y H S H L S T Q Y C D G L R G P F V V Y D P S D P H L S L Y D I D N
A D T V I
5. D Q A G T F W Y H S H T Q Y C D G L R G P F V V Y D P K D P H A S R Y D V D N E S T V I T L T D W Y H T A A
R L G P

190 200 210 220 230 240
1. T L E D W Y H I V A P Q N A A I P T P D S T L I N G K G R Y A G G P T S P L A I I N V E S N K R Y R F R L V S M S
C D P
2. T L E D W Y H V A A P Q G A V L P T P D S T L I N G K G R F A G G P T S P L A I I N V ES N K R Y R F R L I S M S
C D P
3. T L E D W Y H V V A P Q N A V L P T P D S T L I N G K S R Y A G G P T S P L A I I N V E S T K R Y R F R L V S M S
C D P
4. T L E D W Y H I V A P Q N A A I P T P D S T I L N G K G R Y A G G P T S P L S I I N V E S N K R Y R F R L V S M S
C D P
5. R F P L G A D A T L I N G L G R S A S T P T A A L A V I N V Q S G K R Y R F R L V S I S C D P N Y T F S I D G H N
L T V

250 260 270 280 290 300
1. N F T F S I D G H S L L V I E A D A V N I V P I T N D S I Q I F A G Q R Y S F V L T A N Q A V D N Y W L R A N P
N L G S
2. N P T F S I D G H S L Q V I E A D A V N I V P L V V D S I Q I F A G Q R Y S F V L N A N Q A V D N Y W I R A N P
N L G S
3. N F M F S I D G H S L Q V I E A D A V N I V P I P V D S I Q I F A G Q R Y S F V L T A D Q T V G N Y W I R A N P
N L G S
4. N F T F S I D G H S L L V I E A D A V N I V P I T V P S I Q I F A G Q R Y S A V L T A D Q T V G N Y W I R A N P N
L G S
5. I E V D G I N S Q P L L V D S I Q I F A A Q R Y S A V L N A N Q T V G N Y W V R A N P N F G T V G F A G G I N
S A I L R

310 320 330 340 350 360
1. T G F V G G I N S A I L R Q A G A T E D D P T T S ST S T P L L E T N L V P L E N T V A P G P P V P G G A D I N I
N
2. T G F E G G I N S A I L R Y A G A T E N D P A T T S S T S T P L L E T N L V P L E N P G A P G P A V P G G A D I N
I N
3. T G F A G G I N S A I L R Y A G A T E D D P T T T S S T S T P L L E T N L V P L E N P G A P G P A V P G G A D I
N I N
4. T G F D G G I N S A I L R Y A G A T E D D P T T T SS T S T P L L E T N L V P L E N P G A P G P A V P G G A D I N
I N
5. Y Q G A P V A E P T T Q T S V I P L I E T N L H P L A R M P V P G P T P G G V D K A L N L A F N F N G T N F F I
N

370 380 390 400 410 420
1. A M A F D F T T F E L T I N G V P F L P P T A P V L L Q I L S G A S T A A S L L P S G S I Y E L E A N K V V E I S M
P A
2. A M G F D F T N F E M T I N G S P F K A P T A P V L L Q I L S G A T P A A S L L P S G S I Y A L E A N K V V E I S
I P A
3. A M A F D F T N F E L T I N G A P F H A P T A P V L L Q I L S G A T T A A S L L P S G S I Y E L E A N K V V E I S I
P A
4. A M A F D F T T N F E L T I N G V P F I P P T A P V L L Q I L S G A S S A A S L L P S G S I Y A L E P N K V V E I S
M P A
5. N A T F T P F T V P V L L Q I L S G A Q T A Q D L L P A G S V Y P L P A H S T I E I T L L A T A L A P G A G H P F
H L S

430 440 450 460 470 480
1. L A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T R A R R D V V N T G T G A N D N V T I R F V T D
N P G P W
2. L A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G I D A N D N V T I R F V T D
N P G P W
3. L A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G T G A N D N V T I R F V D N
P G P W
4. L A V G G P H P F H L H G H T F D V I R S A G S T T Y N F D T P A R R D V V N T G T G A N D N V T I R F V T D
N P G P W
5. G H A F A V V R S A G A T T Y N Y N D P I F R D V V S F G T P A A G D N V T I R F Q T D N P G P W F L H C H I
D F H L D

490 500 510 520 530
1. F L H C H I D W H L E I G L A V V F A E D V T S I S A P P A W D D L C P I Y N A L S D N D K G G I V P S
2. F L H C H I D W H L E G L A V V F A E D V A S I K A P P A A W D D L C P I Y D A L S D S D R G G I A
3. F L H C H I D W H L E I G L A V V F A E D V T S I S A P P A A W D D L C P I Y D A L S D S D K G G I V
4. F L H C H I D W H L E I G L A V V F A E D V T S I S A P P A A W D D L C P I Y D A L S D N D K G G I V P S
5. A G F A I V F A E D V A D K A A N P V P K A W S D L C P I Y D G L S E A N Q


Sequence homology of Pleurotus ostreatus LCC 1 (Acc no. Q12739)
A> with Pleurotus eryngii (Boletus of the steppes) ery3 protein (Acc no. BOJDP8)
90.6%
B> with Pleurotus sapidus laccase (Acc no. Q6A1A1) 94%
C> with Pleurotus ostreatus Bilirubin oxidase (Acc no. Q9UVY4) 97%
D> with Trametes versicolor laccase 2 (Acc no. Q12718) 5.6%

Antiproliferative effect of LCC 1 against Hep G2 (human liver carcinoma) cell line and MCF-7 (human brest cancer cell
line):-
Hep G2, hepatocellular carcinoma, human, Abcam, USA, ATCC no. HB-8065 was grown on Dulbeccos Modified
Eagles Medium (DMEM) supplemented with 10% Fetal bovine serum (FBS) for 5 weeks at 37C at concentration
of 110
6
ml cells/ml media. 1ml of 1.6, 2.6, 3.6, 4.6, 5.6 and 6.6M laccase was added to 5 weeks old Hep G2 cell
line and incubated at 37C for 72hrs. The half maximal inhibitory concentration (IC 50) of LCC1 was studied to be
2.8M concentration. However the control set with no laccase showed normal cell growth. Normal cell growth
was observed in negative control where 5 weeks old Hep G2 cell line was incubated with 5.6M LCC1 and
dimethylsulfoxide (DMSO). All the culture slides were visualized under Phase-contrast Microscope (100X
magnitude). However when 5 weeks old Hep G2 grown on DMEM and 10% FBS was incubated with 4.6M LCC1
for 72hrs at 37C and visualized under phase-contrat microscopr, 25 cells were observed/ cm
2
area of
microscopic field. In negative control with no LCC1 but with 5M Menadione, the number of cells observed per
cm
2
microscopic field was 25. But when 5 weeks old Hep G2 cell line was incubated with 4.6M LCC1 and 1, 2, 3,
4, 5M concentration of 2-methyl-1,4-napthiquinone (Menadione), the number of cells/cm
2
microscopic field
decreased from 24 at 1M Menadione to 0 at 5M menadione. All the slides were prepared after 72hrs
incubation and observed under Phase-contrast Microscope. The quinine redox cycle catalyzed by laccase
produces semiquinone intermediate which drives hydroxyl radical (OH
-
) production by Fenton reaction (Victor
Gomez Toribio, Anna B. Garcia, 2009) which killed the Hep G2 cells. However Menadione itself cannot kill the
cells. DMSO is a hydroxyl radical scavenger; so in negative control no cell deaths were observed.
Similarly MCF-7 (human breast cancer cell line Lonza AG, USA) was grown on DMEM media with
10% FBS was incubated with 2.6-7.6M LCC1 for 72hrs and IC
50
was calculated to be 3.3M LCC1. Both in control
set with no LCC1 and in negative control set with 6.6M LCC1 and DMSO; normal cell growth was observed
under Phase-contrast Microscope.

Fig. MCF7 (human breast cancer cell line); 5weeks old cells growing on DMEM supplemented with 10% FBS visualized
under Phase-Contrast microscope (100X magnitude).


fig. 5weeks old Hep G2 (human hepato carcinoma cell line) growing on DMEM media with 10% FBS visualized
under Phase-contrast Microscope (100X magnitude) (National Model 162-PH, Binocular version) (left) and dead 5 weeks
old Hep G2 cells on DMEM media with 10% FBS after addition of 5.6M LCC 1, also visualized under phase-contrast
microscope (100X magnitude).

Fig. 5weeks old Hep G2 cell line on DMEM media with 10% FBS after addition of 4.6M LCC1 and 38M
Menadione (fig-a) and with 1M Menadione with 4.6M LCC1 (fig-b) visualized under Phase-contrast
Microscope (magnitude 100X).
Control set:
No. of cells/cm
2
area of microscopic field 25

Negative control set:
No. of cells/cm
2
area of microscopic field 25

Experimental set:
Conc. of 2-methyl-1,4-napthoquinone Conc. of LCC 1 in each set No. of cells/cm
2
area of microscopic
field
1M 4.6M 24
2M 4.6M 14
3M 4.6M 8
4M 4.6M 3
5M 4.6M 0





Biochemical properties
The temperature optima of LCC1, 2 and 3 was 30C and all the isozymes were stable between 7C to 57C for
10mins. The pH optima of LCC1, 2 and 3 was determined to be pH 5.0. Both isozymes LCC2 and 3 were stable
between pH 4-7.0 but LCC1 was stable between pH 3.9-7.0.
Spectral wavelength scan of LCC1 between wavelengths 240-690 showed absorption bands at
278nm (with a shoulder at 290nm); corresponding to aromatic amino acids and peaks at 330 and 340nm
corresponding to Type 3 Cu (II) and peaks at 612nm corresponding to Type 1 Cu(II) (Reinhammer and Oda;
1985). When LCC1 was incubated with 1mM CuSO
4
, absorbance at 612nm decreased from 0.35 to 0.15
suggesting reduction of Type 1 copper during breakdown of Guaiacol; suggesting that Type 1 copper is involved
in catalytic mechanism (B. G. Malmstrom, A. Finazzi Agro, E. Antonni; 1969). The K
M
for purified LCC1, 2 and 3
was determined to be 84.034M, 74.074M and 158.73M while V
max
was found to be 714.25 U/min,
204.081633 U/min, 714.29 U/min for LCC 1, 2 and 3 respectively.




Discussion
Purification, characterization of isozymes, peptide sequencing and study of antiproliferative activities of laccase
isozymes of Pleurotus ostreatus strain V-184 has been described against Hep G2 and MCF-7 cell lines. However isozymes
of blue laccase and their gene and protein structure has been preciously described (Paola Giardina, Gianna Palmieri,
0 0 0 0
35
65
102
188
73
38
8
0 0 0 0 0 0
60
181
62
32
4
0 0 0 0 0 0
98
52
32
2
0
105
184
60
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
1 2 3 3.5 3.9 4 4.5 5 6 6.5 7 10
LCC 1
LCC 2
LCC 3
0
5
35
65
135
155
175
188
183
180
73
68
33
0
7
28
50
95
145
181
175
172
78
62
45
0
6
30
40
165
180
183
178
78
62
48
165
184
178
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
200
5 7 15 19 25 27 29 30 33 35 41 45 47
LCC 1
LCC 2
LCC 3
Andrea Sealoni, Bianea Fontanella, Vincenza Forace, Gioranna Cennamo, Gicovanni Sannia; 1999). They have also
sequenced the N-terminal peptide sequence and C-terminal peptide sequences of laccase isozymes LCC1, 2 and 3
however the entire peptide sequencing of laccase isozymes have never been reported. However laccase have been
purified from other fungases like Aspergillus niger (Scherer and Fischer, 1998); Myceliophthora thermophila (Xu et. al.,
1998), Rhizoctonia solani (Wahleithner et. al., 1996), Fusarium proliferatum (Kwon and Anderson, 2001), Botrytis cinerea
(Marbech et. al., 1984), Aspergillus oryzae (Ducros et. al.,2001), Agrocype cylindracea (J. H. Wong, Y. Jiang, F. Liu, S.C.
Sze; 2010). While laccase from Pleurotus ostreatus (Gianna Palmieri et. al., 1997), extracellular laccase from P. sajor-caju
(R. Sahay, R.S. Yadav, K.D. Yadav;2008), P. florida laccase 1 (N. Das, T. K. Chakraborty, M. Mukherjee; 1997), P. eryngii (F.
Guillen et. al., 1994), extracellular laccase from Hortaea acidophila (Larusa Tetsch, Jutta Bend, Udo Holker; 2006) and
Trametes versicolor (Wu-Xiang-bo and Xie Yi min, 2008). Antiproliferative activity of laccase from edible mushroom
white common Agrocype cylindracea on Hep G2, MCF 7 and cell lines and its inhibitory effect on HIV-1 reverse
transcriptase was studied previously (J. H. Wong, Y. Jiang et. al., 2010). And induction of extracellular hydroxyl radical
production by white-rot fungi through Quinone redox cycling was previously studied (Victor Gomez Toribio et. al., 2009),
however, Antiproliferative effect of LCC 1 laccase isozyme on Hep G2 and MCF 7 cell lines have never been described
before. Quinone redox cycle xatalysed by laccase produces semiquinone intermediate which drives hydroxyl radical
production by Fenton reaction. Increasing the conc. of 2-methyl-1,4-napthoquinone from 1 to 5M with 4.6M LCC 1
showed progressive decrease in no. of Hep G2 cells. Similar reports have been observed with other quinines like 1,4-
benzoquinone (BQ), 2-methoxy-1,4-benzoquinone (MBQ), 2,6-dimethoxy-1,4-benzoquinone (DBQ) by others (Victor
Gomez Toribio; 2009).
However, highest rate of OH
-
radical formation was obtained with Menadione followed by DBQ, MBQ and
BQ, In negative controls addition of hydroxyl radical scavenger DMSO prevented killing of Hep G2 cells.But 4.6M
laccase LCC1 isozyme conc. couldnot kill Hep G2 cells in absence of Menadione suggesting that 4.6M laccase alone
cannot kill Hep G2 cells. Similar reports have been observed by addition of hydroxyl radical chelators Mannitol and
Dimethyl sulfoxide (DMSO) by others in Crytococcus neoformans (Liu Lide, Ram P. Tewari, Peter R. Williamson et. al.,
1999). However 5.6M LCC1 was sufficient to inhibit growth of 5 weeks old Hep G2 cell line on DMEM media probably
by using Menadione (K
3
) (a parent compound of Vit. K series present in media), Menaquinones (K
2
), Menaquinone-7 or
any other quinine produced by animal tissue. So, lower conc. of LCC1 (4.6M) requires externally added quinines to kill
Hep G2 cells but slightly higher conc. of LCC1 (5.6M) can kill Hep G2 cells by using internally synthesized quinones. The
mode of action of laccase in Antiproliferative action against Hep G2 cell line has been registered as a patent, application
no. 3299/ DELNP/2011A on 18/02/2011 at Controller General of patents Designs and Trademarks (CGPDTM), intellectual
property registration section, Boudhik Sampada Bhavan, CP2, Sector 5, Sech Bhawan, Kolkata, West Bengal- 700091.
IC50 for Antiproliferative activity of LCC 1 against Hep G2 cells and MCF 7 cells were found to be 2.8 and 3.3M
respectively. However, IC50 of laccase from Agrocype cylindracea against Hep G2 and MCF-7 cells was found to be much
higher 5.6 and 6.5M respectively by others (j.H.Wong et. al., 2010) and in P. cornucopiae (K.Y.Zhang et. al., 2010).
Peptide sequencing of 533 amino acid LCC1 from Pleurotus ostreatus stain -184 using Perkin-Elmer Applied Biosystemics
977A pulse-liquid protein sequencer has identified an N-terminal signal peptide of 23 amino acids and 3 plastocyanin
domains (25- 171), (173-336) and (382-501). The N-terminal signal peptide was found to be highly conserved in other
laccase peptide sequences from P. eryngii, P. sapidus, P. ostreatus laccase 4 and P. pulmonarius. The peptide sequence
of LCC 1 from P. ostreatus strain V-184 was deposited in Public protein database Uniprot under accession no. Q12739.
The peptide sequence show high homology with laccase 2 of P. pulmonarius (96%) (Acc. no. Q2VT18), laccase 4 of P.
ostreatus (94%) (Acc.no. Q7Z8S3). P. eryngii ery3 protein (Acc. no. BOJDP8) (90.6%), P. sapidus laccase (94%) (Acc. no.
Q6A1A1) and Bilirubin oxidase of P. ostreatus (97%) but showed little homology with Trametes versicolor laccase 2
(5.6%) (Acc. no. Q12718). Homology with laccases of other Pleurotus species suggests that the peptide sequence of
laccase is highly conserved. Bilirubin: oxygen oxidoreductase (Bilirubin oxidase) (EC 1.3.3.5) catalyses the reaction
between Bilirubin and O
2
to produce biliverdin and water and it acts specifically on CH.CH group of donor oxygen as
acceptor. It participates in porphyrin and chlorophyll metabolism (S. Murao and N. Tanaka; 1981, Masuda-Nishimura, K.
Ichikawa;1999). High homology with Bilirubin oxidase suggests that laccase also belongs to oxidoreductase family and
their mode of catalysis is similar. Ery3 encodes a mature laccase isozyme of S31 aa from P. eryngii (G. Bleve, C. Lezzi, G.
Mita et. al., 2008). Similar reports were observed by others (Paola Giardiana et.al., 1999).
Plastocyanin like domain is distorted trigonal pyramidal. The trigonal plane of the pyramidal base is composed of two N
atoms (N1 and N2) from separate histidines and sulphur (S1) from a cysteine sulphur (S2) from an axial methionine
forms the apex. This distortion occurs in the bond lengths between the copper and sulphur ligands. The Cu-S1 contact is
shorter (207pm) than Cu-S2 (282pm). The elongated Cu-S2 bonding destabilizes the Cu(II) form and increases the redox
potencial of the protein. (Victor Gomez et. al., 2009). Number of cysteine residue has been calculated to 4.68 with
reaction of the thiol group with 5,5-dithiobis (2-nitrobenzoate acid) (DTNB). Similar reports have been observed in P.
ostreatus by others (Paola Giardina et. al., 1999. The optimum temperature of laccase isozymes LCC1,2 and 3 were
found to be 30C. Similar reports have been observed by Miguel Alcade, Thomas Butter in Fungal laccases (2002) and
28C in P. ostreatus strain V-184 by A.M. Mayer and R.C. Staples (2002). However the temperature optima of laccase
from P. sajor-caju strain MTCC141 (37C) (R.Sahay, R.S. Yadav; 2008), P. florida (50C) (N. Das, T.K. Chakraborty,
M.Mukherjee; 1997) and Trametes versicolor (40C) (Wu Xiang-bo et. al.;2008) were found to be higher.
The pH optima of laccase isozymes LCC1,2 and 3 was pH 5.0. Similar reports have been observed in fungal
laccases by Miguel Alcade, Thomas Butter (2002), P. ostreatus strain V-184 by A.M. Mayer and R.C. Staples (2002) and
Trametes versicolor (Wu Xiang-bo, Xie-Yi min et. al.;2008) but the pH optima was slightly lower in P. sajor-caju strain
MTCC141 (pH 4.5) (R. Sahay, R.S. Yadav, K.D. Yadav; 2008), P. florida (pH4.1) (N. Das, T.K. Chakraborty; 1997).
Spectral analysis of LCC1 showed an absorption bands at 278nm (with a shoulder at 290nm); corresponding to aromatic
amino acids and peaks at 330, 340 and 612nm. According to Reinhammar and Oda (1985); absorptions band at 330nm
and 340nm correspond to Type-3 Cu(II) and band at 612nm corresponds to Type-1 Cu(II). Similar reports have been
observed for laccase of Sycamore maple (Acer pseudoplatanus) (Raja Sterjiades, Jeffrey F.D.Dean; 1992). Absorbance at
612nm was found to decrease from 0.35 to 0.16 during Guaiacol catalysis suggesting reduction of Type-1 Cu is involved
in catalysis. Similar reports have been observed by B.G. Malmstrom, A. Finazzi Agro and E. Antonni; 1969).
The molar mass of laccase isozymes LCC1,2 and 3 were observed to be 65, 60 and 80 kDa respectively in P.
ostreatus strain V-184 by SDS-PAGE. Similar reports have been observed by A.M. Mayer, R.C. Staples in P. ostreatus
strain V-184. However they found an additional isozyme LCC4 (82kDa), pI-4.3 which was not detected here. The 60KD
band for LCC2 was confirmed by Western Blotting by using monoclonal LCC Cbr2 rabbit Ab against copper binding region
2 of 60KD laccase from Agaricus bisporus. However it was shown that LCC1 and 2 form a heterodimeric protein of native
molecular mass of 130kDa by gel filtration. However bands of molar mass 61 and 67kDa for P. ostreatus white laccase
(Gianna Palmieri et. al.,1997) 90kDa for P. sajor-caju strain MTC141 laccase (R. Sahay et. al.,2008), bands of 77 and
82kDa for P.florida laccase (N. Das et.al., 1997) and 56.6kDa band for P. eryngii laccase (F. Gullien et. al.,1994). The
native molar mass of laccase from Acer pseudoplatanus was found to be 115 5kDa (Raja Sterjiades, 1992).
The 3 isozymes of laccase from Pleurotus ostreatus strain V-184 were purified by (NH
4
)
2
SO
4
precipitation,
Toyopearl DEAE-650 anion exchange chromatography, isoelectric focusing, ConA-sepharose 4B affinity chromatography,
Superdex 75 gel filtration with 5713 folds of purification and recovery of 1.64%. This is much higher than folds of
purification observed by others in Pleurotus ostreatus strain V-184 (11.8 fold) (A.M. Mayer, R.C. Staples; 2002) in
Magnaporthe cyrisea (282 fold) (Gopal Iyer, B.B. Chattoo; 2003), White rot fungus Polyporus (44.7 fold) (Guo, Li-Qiong,
Shuoxin; 2001), Cerrena unicolor (92 fold) (jerzy Rogalskia, Grzegor Z. Janusza; 2010), Pleurotus sajor-caju (132 fold) (Sze
Chung Lo, Yuen Sze Ho; 2001), P. sajor-caju strain MTCC141 (214 fold) (R. Sahay, R.S. Yadav, K.D. Yadav; 2008) and
Trametes versicolor (209 fold) (Wu Xiang-bo, Xie-Yimin; 2008).
Kinetics of purified isozymes of laccase and K
M
has been calculated to be 84.034M, 74.034M and
158.73M for LCC1, 2 and 3 respectively. It suggests that substrate affinity of LCC2 for Guaiacol is highest (K

=
0.01350M
-1
) followed by LCC1 (K

= 0.0119 M
-1
) and LCC3 (K

= 0.0063 M
-1
). However the K
M
value obtained with
extracellular laccase secreted by P. sajor-caju strain MTCC141 is 30M (R. Sahay, R.S. Yadav, K.D. Yadav; 2008) and 10M
for Botrytis cinerea (D. Siomczynksi, J. P. Nakas, S.W. Tanenbaum; 1995).

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