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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 3; Year 2015; Page: 182 - 185

Research Article

PRODUCTION, PARTIAL PURIFICATION AND INDUSRIAL

APPLICATION OF LACCASE ENZYME
C. Janaki* and G. Sarojini
PG and Research Department of Microbiology, Sri Akilandeswari Womens College, Wandiwash,
Thiruvannamalai, Tamil Nadu, India
_______________________________________________________________________________________
Abstract
Human and industrial activities result in the discharge of various pollutants in to the aquatic
environment threatening the health of the population and damaging the quality of the environment by
rendering water bodies unsuitable. The textile mills daily discharge millions of liters of untreated effluents in
the form of wastewater into public drains that eventually empty into rivers. Most of them are recalcitrant in
nature, especially azo dyes. In this study, decolourization of dye was carried out by using Laccase enzyme
from white rot fungi. At first, white rot fungi was sub cultured on PDA plates by incubating for 5 days. The
selected species was inoculated in agro waste substrate for mass production. After incubation for 12 days the
enzyme was extracted from the grown culture using sodium acetate buffer. The extracted enzyme was
partially purified by using two techniques such as salting out technique and Dialysis. The substrate wheat
bran showed more protein concentration 0.373 mg/ml for crude enzyme. Wheat Bran showed more enzyme
activity 3.0215 IU/ml after salting out. Protein was separated by SDS-PAGE. The partially purified enzyme
was used for textile dye decolourization. According to our work the enzyme laccase produced by white rot
fungi effectively decolourize textile dye. Thus, it was found that the dye degradation can be done using
laccase enzyme.
Key words: Textile mills, Recalcitrant azo dyes,
White rot fungi, Decolourization and Laccase
enzyme.

Article History
Received : 05.04.2015
Revised : 12.05.2015
Accepted : 18.05.2015
1. Introduction

Human and industrial activities result in

the discharge of various pollutants in to the
aquatic environment threatening the health of the
population and damaging the quality of the
environment by rendering water bodies unsuitable
(Abowei and Sikoki, 2005). Discharge from
tannery, food, pharma and textile industries has
raised the threats of water pollution in developed
as well as developing countries. The textile mills
daily discharge millions of liters of untreated
* Corresponding author: C. Janaki, PG and Research

Department of Microbiology, Sri Akilandeswari

Womens College, Wandiwash.

effluents in the form of wastewater into public

drains that eventually empty into rivers. Most of
them are recalcitrant in nature, especially azo
dyes. The stability and their xenobiotic nature of
reactive azo dyes makes them recalcitrant hence
they are not totally degraded by conventional
wastewater treatment processes that involve light,
chemicals or activated sludge (Chung et al., 1992).
WRF are key regulators of the global C-cycle.
Their LME, i.e., manganese peroxidases (MnP),
lignin peroxidases (LiP), and laccases (Lac), are
directly involved not only in the degradation of
lignin in their natural lignocellulosic substrates
(Becker and Sinitsyn, 1993; Hatakka, 1994) but

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C. Janaki / Life Science Archives (LSA), Volume 1, Issue 3, Page 182 - 185, 2015
also in the degradation of various xenobiotic
compounds (Barr and Aust, 1994 Pointing, 2001)
including dyes (Glenn and Gold, 1983). Some
white rot fungi produce all three LME while
others produceonly one or two of them (Hatakka,
1994).
Laccase belongs to the blue multicopper
oxidases and participates in cross-linking of
monomers, degradation of polymers, and ring
cleavage of aromatic compounds. It is widely
distributed in higher plants and fungi. It is present
in
Ascomycetes,
Deuteromycetes
and
Basidiomycetes and abundant in lignin-degrading
white-rot fungi. It is also used in the synthesis of
organic substance, where typical substrates are
amines and phenols, the reaction products are
dimers and oligomers derived from the coupling
of reactive radical intermediates. In the present
study we are using white rot fungi as a source of
laccase enzyme for decolourizing textile industry
dye.
2. Materials and Methods
Textile Dyes
The textile dyes Black B, Crimson, Yellow
F3R, Rhodamin and Merrun V was obtained
from Armats Biotek (P) Ltd, located at Chennai.
Culture media
PDA was used to cultivate white rot fungi
Pleurotus ostreatus
Enzyme production in solid-state fermentation:
Lignocellulosic substrate namely wheat
bran were used for laccase production under Solid
state fermentation condition.
Extraction of Enzyme
A volume of 0.2 M Sodium acetate buffer
(pH - 5.8) was added to 12 days old mass culture
and the mycelium was broken completely. Then, it
was kept in the shaker for overnight. Next day the
culture was filtered and centrifuged at 5000 rpm
for 20 minutes. Supernatant was collected in
conical flask. The enzyme was precipitated using
ammonium sulphate. The precipitated solution
was centrifuged at 5000 rpm for 20 minutes.

183

Partial purification of enzyme

Partial purification was done by Salting out
by using 19.02g of ammonium sulphate and
dialysis by using 5 mM sodium acetate buffer.
Estimation of Protein
The protein was determined according to
the method of Bradford (1976) for both the crude
as well as salted out and partially purified enzyme.
Purification and Characterization of Laccase
Preparation of enzyme for purification
The proteins in the supernatant were
precipitated with 75% ammonium sulphate
saturation. All the subsequent purification steps
were carried out at 4C. The protein was dissolved
in 0.1 M sodium acetate buffer, pH 5.8 and
dialyzed against the same buffer with
concentration 5 mM.
Sodium Dodecyl Sulphate- Polyacrylamide gel
electrophoresis (SDS-PAGE)
SDS-Polyacrylamide gel electrophoresis
was performed on slab gel with separating and
stacking gels (10 & 5 % w/v) by the method of
(Laemmli,1970). The molecular mass of the
purified laccase was determined on SDS-PAGE.
Purified protein samples were run on SDS-PAGE
with concurrent run of standard protein markers
consist of 97.4, 66.0, 43.0, 29.0, 20.1, and 14.3
kDa purchased from Genie. After separation, the
gels were stained with silver nitrate.
Decolourisation of Textile dye using Laccase
enzyme

Water agar medium was prepared,

autoclaved and was poured into sterile petriplates
and was allowed to get solidified. Then, 4 wells (8
mm diameter) were made by using a sterile cork
borer. The dyes of different concentrations (10 g,
50 g, 75 g and 100 g) were prepared and
loaded in each of the well. Distilled water served
as control. After 24 hours of incubation the zone
of decolourization should be measured.
3. Results and Discussion
Mycelia discs (6 mm) from 5 days old
culture were grown on PDA (Potato dextrose agar)
plates and were used as inoculum for sub-

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C. Janaki / Life Science Archives (LSA), Volume 1, Issue 3, Page 182 - 185, 2015
culturing. The inoculated plates were incubated
for 10 days and the white rot fungi were grown
without any contamination.
Table 1: Estimation of protein
S.
No

Substrates

1.

Wheat Bran

Protein concentration (mg/ml)

Crude Salting out Dialyzed
enzyme
sample
sample
0.373

0.778

1.408

Decolourization of textile dye using laccase

enzyme
The dye decolourizing activity was
observed in rhodamin dye and decolourization was
high at 1000g/ml. The decolourization activity
was not observed in any of the other dyes.
Table 2: Dye decolourization
Dye
Rhodamin

Zone of inhibition (mm)

100
500
750
Control
g/ml g/ml g/ml
16
22
mm
mm

1000
g/ml
24
mm

Worldwide, dye wastewater has become

one of the main sources of severe pollution
problems due to the greater demand for textile
products and the proportional increase in
production and applications of synthetic dyes
(Santos et al., 2007). Unfortunately, most of these
dyes escape conventional wastewater treatment
processes and persist in the environment as a
result of their high stability against light,
temperature, water, detergents, chemicals, and
microbial
attack
(Coutoet
al.,
2009).
Notwithstanding, industries are required to
eliminate color from their effluents containing
dyes, before disposal into water bodies, due to
environmental legislation (Neill et al., 1999).
Various types of oxidative enzymes are
produced by white rot fungi in order to make use
of lignocellulosic substrates for nutrition (Martin
Ruhl et al., 2008; Leonowicz et al., 1999). SSF
has been considered as an efficient method for
enzyme production in biotechnological process
due its potential advantages and high yield. The
high level of laccase production could be
attributed due to the presence of ferulic acid in

184

wheat bran (Hedge et al., 2006). The optimal

temperature range for fungal laccase activity is 30
to 60 C (Munoz et al., 1997). The laccase
examined in this study had a temperature optimum
70 C and was stable at higher temperatures than
the laccases purified from mesophillic fungi, such
as Pyncoporus cinnabarinus (Eggert et al., 1996).
Fungal laccases are generally active at low pH
values (pH 3 to 5) (Bollag et al., 1984). The
treatment system based on fungi, especially the
white-rot, have not been applied extensively due
to many factors such as, high costs, longer fungal
growth periods (Wesenberg et al., 2003) and an
early inactivation of enzymes (Cing et al., 2004).
4. Conclusion
It can be concluded that textile effluents
containing dye should undergo decolourization
before being released into the water bodies in
order to reduce its harmful effects to the living
beings. According to our work the enzyme laccase
produced by white rot fungi effectively
decolourize the textile dye. Thus it was found that
the dye degradation can be done using laccase
enzyme.
5. References
1) Barr DP,Aust SD (1994), Mechanisms whiterot fungi use to degrade pollutants,
Environmental Science Technology, 28: 78
87.
2) Becker HG, Sinitsyn AP (1993), Mnperoxidase from Pleurotusostreatus: the action
on the lignin, Biotechnol Lett., 15: 289 294.
3) Chung, K. T., Cerniglia, C.E., (1992),
Mutagenicity of azo dyes: structure activity
relationship, Mutational Research, 277: 201
220.
4) Couto, Jos Luis Toca Herrera (2006),
Industrial and biotechnological applications of
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5) Glenn JK, Gold MH (1983), Decolorization of
several polymeric dyes by the lignin-degrading
basidiomycete Phanerochaete chrysosporium,
Applied Environmental Microbiology, 45(1):
741 748.
6) Hatakka, A (1994), Lignin-modifying enzymes
from selected white-rot fungi production and

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C. Janaki / Life Science Archives (LSA), Volume 1, Issue 3, Page 182 - 185, 2015

185

role in lignin degradation, FEMS Microbiology

Review, 13: 125 135.
7) Osma J F,1 Jose L. Toca-Herrera, and Susana
Rodrguez-Couto (2010), Uses of Laccases in
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8) Pasti-Grigsby MB, Paszczynski A, Gosczynski
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