BIOPROSPECTING OF BACTERIA FOR PRODUCTION AND PURIFICATION OF LACCASE ENZYME
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Date
2013
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Abstract
ABSTRACT
Laccase enzyme has acquired the status of ‘green catalyst’ as it possesses remarkable bioremediation potential
along with numerous applications in effluent detoxification, degradation of textile dyes, herbicide and insecticide
degradation, wine clarification, enzymatic conversion of chemical intermediates, biosensors and organic synthesis. In the
present study, significant high diversity of laccase producing bacteria from rhizosphere of rice plants from paddy fields of H.
P. was assessed whereas medium diversity was obtained from the samples of paper mills of H.P. A total of 449 bacterial
isolates were obtained from 198 samples using M162 and TY media containing 5mM guaiacol and 40 mg/l CuSO4. These
were rescreened on the basis of their ability to oxidise tannic acid and dimethoxyphenol leading to selection of 67 bacterial
isolates which were characterized both morphologically and biochemically alongwith the laccase activity and 14 bacterial
isolates exhibiting maximum laccase activity of 10-19 U/l were selected. Molecular characterization of the selected isolates
was carried out using RAPD-PCR and 16S rrna gene technology and in silico analysis of 16S rrna gene sequences lead to
identification of these bacterial isolates as Pseudomonas putida strain LUA15.1 and LHB7.1, Pseudomonas umsongenesis
strain LHB9.1., Pseudomonas mohnii strain LHN12.2, Pseudomonas chlororaphis strain LUD7.1, Pseudomonas jessenii
strain LHN9.1, Pseudomonas lurida strain LB6.2, Pseudomonas graminis strain LHN8.1, Pseudomonas veronii strain
LUA14.1, Pseudomonas fulva strain LR5.1, Lysnibacillus fusiformis strain LKM7.1, Lysnibacillus sphaericus strain LH3.4
and Bacillus subtilis strain LB6.1 and LR6.3 .
On the basis of maximum laccase enzyme activity Pseudomonas putida strain LUA15.1 was selected for
production and purification of the laccase enzyme. Maximum extracellular enzyme production was achieved at 28°C, pH 7
(24 hrs incubation) with 5mM guaiacol, 50 mg/l CuSO4, 5% tryptone and 3% yeast extract in combination as nitrogen source
in Tryptone Yeast medium. The laccase crude extracellular enzyme preparation was purified by ammonium salt precipitation
(50-90%) followed by gel filtration and ion exchange chromatography which showed 10.74 yield and 61.36 fold purification.
The purified enzyme had optimal activity at pH 7.0 and 40°C and 0.80 mM Km value. The molecular weight of laccase in
the present study was found to be 42.5 kDa. The activity was inhibited by sodium azide and DTT. Strain LUA15.1 as well as
its enzyme preparations were studied for their ability to decolourize dyes which are the potential contributors of water
pollution. All six different synthetic dyes were decolourized RBBR (48%), congo red (35%), indigo carmine (80%), brilliant
green (97%), bromophenol blue (78%) and aniline blue (23%) when treated with the culture of Pseudomonas putida strain
LUA15.1. However, the crude as well as partially purified enzyme preparation of Pseudomonas putida strain LUA15.1
showed greater decolourization of dyes comparatively congo red (98%), indigo carmine (99%), RBBR ( 96%), aniline blue
(37%) bromophenol blue (70%) and brilliant green (60%). The purified enzyme was successfully immobilized using
encapsulation method in calcium alginate beads with 76% immobilization percentage and immobilized laccase enzyme
beads were studied for their ability to degrade dyes. The stability and reusability of the immobilized enzyme system has the
potential to make the entire treatment process inexpensive.
An extracellular laccase producing gene has been isolated using degenerate primer based on the copper I and II
conserved site of laccase enzyme, from the rice rhizospheric bacteria, Pseudomonas putida strain LUA15.1 followed by
determination of the nucleotide sequence of this gene and it showed 91% similarity with Pseudomonas putida strain mt-2
Mn(II)-oxidation-associated multicopper oxidase (cumA) gene, partial cds. This nucleotide sequence of laccase was
translated into amino acid and encodes a polypeptide comprised of 113 amino acids which showed 85 % identity with the
amino acid sequences of bacterial laccases i.e. Mn (II)-oxidation-associated multicopper oxidase [Pseudomonas putida].
Further multiple sequence alignment using MULTALIN and structure prediction using Phyre 1 & 2 revealed conserved
histidine residues.
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Keywords
enzymes, bacteria, fungi, productivity, aromatic compounds, proteins, genes, sampling, colourants, selection, Laccase Enzyme