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International Journal of Advances in Science and Technology,
Vol. 1, No. 5, 2010
Dye Decolorization Studies on Laccase produced by
Streptomyces gresius MTCC 4734
M.V. Sampoorna Laxmi, Md. Mazharuddin Khan
P. G Department of Microbiology, Mumtaz Degree & P.G College, Malakpet, Hyderabad-500036.
sampoorna83@gmail.com
Abstract
The present paper studies the applicability of the enzyme laccase produced by Streptomyces gresius
MTCC 4734 for industrial purposes. Dye decolorization studies were performed and analyzed. The
enzyme produced was found to be efficient in decolorizing the azo dye Acid red 26 which was used for
the study. Decolorization was observed up to 31/2 hrs and then a decreasing trend was observed in the
activity.
Keywords: Acid red 26, laccase, decolorization, Streptomyces gresius.
1. Introduction
Laccases (EC 1.10.3.2 benzenediol: oxygen oxidoreductases) are multicopper oxidases that catalyze the
1 electron oxidation of several aromatic substrates with the simultaneous reduction of dioxygen to two
molecules of water (Piontek et al., 2002). These enzymes oxidize a group of phenolic compounds,
including monophenols, polyphenols and methoxy substituted phenols as well as aromatic amines and
metallic ions (Schlosser D et al., 1999; Larrondo LF et al., 2003). Laccases are widely distributed in nature
and have been described in fungi (Baldrian P, 2006), plants (Mayer AM, 1987), insects (Dittmer NT et al.,
2004) and in bacteria (Arias ME, 2003).
Currently the catalytic properties of laccases are being exploited for a range of biotechnological
applications. Use of redox mediators to expand the degree of oxidation, offers considerable
biotechnological potential. The application of laccases to textile industry is particularly important. Infact,
90% of reactive textile dyes entering activated sludge sewage treatment plants pass through unchanged and
are discharged in to rivers (Abadulla E et al., 2000). Although several combined oxic and anoxic treatments
have been reported to enhance the degradation of textile dyes, the generation of carcinogenic amines from
azo dyes through anoxic processes poses a serious health hazard. In addition, the colored industrial
effluents significantly reduce oxygen solubility in receiving waters and are thus an important environmental
hazard. Laccases combined with synthetic or natural mediators have been reported to decolorize several
groups of textile dyes (Camarero S et al., 2005). However, the suitability of these enzymatic systems to
decolorize and detoxify azo-type dyes has yet to be fully evaluated.
This work describes the production, partial purification of laccase by Streptomyces gresius MTCC
4734 together with its effectiveness in decolorizing an azo dye.
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International Journal of Advances in Science and Technology,
Vol. 1, No. 5, 2010
2. Materials and Methods
Microorganism and its maintenance
Streptomyces griseus MTCC 4734 used in the present study is an aerobic, filamentous
actinomycete procured from Microbial Type Cell Culture (MTCC), IMTECH, Chandigarh, India. It was
isolated from soil (Tripathi, CDRI, Lucknow). The culture was grown and maintained on medium
containing (g L-1): glucose – 4.0, yeast extract- 4.0, malt extract – 10.0, CaCO3 – 2.0, agar – 12.0, dis. H2O
– 1L, pH = 7.2 (adjusted with KOH) and temperature 30oC.
Enzyme production
Enzyme production was done by submerged fermentation in liquid media under shaking
conditions (120rpm) for 8days at 30oC. After the incubation, the enzyme was extracted by centrifuging the
broth contents at 10,000rpm for 10min at 4 oC in a refrigerated centrifuge. The supernatant was collected
and used for enzyme assay, partial purification and dye decolorization studies.
Enzyme assay
The reaction mixture used to determine laccase activity consisted of 66.6mM sodium malonate at
pH = 4.5, 1.3mM 2, 6- dimethoxy phenol and 500 lt of sample. Absorbance changes at 465nm and 30oC
were monitored for 5min (Molar absorption coefficient = 10,000 M-1 cm-1) (Font X et al., 1997).One
activity unit was defined as the amount of enzyme that oxidizes 1mole of the substrate (DMP) per minute.
Partial purification
The supernatant collected after centrifugation was used for fractionated precipitation by
ammonium sulphate between 30% to 50% saturation. The precipitate was suspended in 100mM phosphate
buffer, pH = 6.8 and centrifuged at 10,000rpm for 15min at 4oC; many times (Khammuang S et al., 2007).
An attempt was previously made to enhance the laccase production using natural inducers and the enzyme
was characterized to an extent (M.V.Sampoorna Laxmi et al., 2010). The enzyme was found to be stable at
relatively high pH (8.0) and temperatures (80 oC).
Dye decolorization
To study the applicability of the enzyme laccase that has been extracted, dye decolorization
studies were performed. For this an azo dye acid red 26 has been chosen. Dye decolorization capability of
the partially purified laccase from S.griseus was monitored with 5 mg/lt dye concentration. Broth for
decolorization was prepared containing the dye and 2, 6-DMP. Reaction was started by the addition of
0.05U/ml of the enzyme and incubating at 30 oC. The reaction mixtures were monitored by following the
decrease in absorbance of acid red 26 (λmax; 510nm) using spectrophotometer. Experiments were
monitored immediately after enzyme addition and after every 30min. Dye decolorization is expressed in
terms of percentage calculated according to equation.
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International Journal of Advances in Science and Technology,
Vol. 1, No. 5, 2010
Where Ao is the absorbance at 510nm immediately measured after adding the enzyme solution and A t is
absorbance at 510nm after the incubation time.
3. Results and Discussion
From the table given below, it can be emphasized that with increase in time interval, the
percentage decolorization is also increasing. But after 3 ½ hrs, there is no significant decreasing trend
observed in the decolorization activity.
Table 1: % Decolorization activity with respect to time
S.No Time interval in Absorbance at % decolorization
min 510nm
1 30 0.48 11.4
2 60 0.452 16.4
3 90 0.44 19.5
4 150 0.42 24
5 210 0.41 25.8
The observation of dye decolorization till 3 ½ hrs only suggests a slow reaction rate or a sigmoidal
behavior. The acid red decolorization ability of this laccase might be improved if the enzyme is used in
higher amounts or in more pure form. The effect of pH on dye decolorization also needs to be studied. A
small molecular weight redox mediator was found to be effective in decolorizing several synthetic
recalcitrant dyes. For example, Soares et al; (2001) reported that the decolorization by laccase from
Aspergillus showed a sharp increase in the presence of either 1-hydroxybenzotriazole (HOBT) or violuric
acid (VA), whereas no decolorization took place when the laccase alone was used. Since, the enzyme
showed decolorization, the use of mediators might improve the activity of the enzyme produced by this
species. The enzyme was found to be thermo stable which improves its use in industrial and environmental
applications.
References
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Vol. 1, No. 5, 2010
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