BIOPROSPECTING OF BACTERIA FOR PRODUCTION AND PURIFICATION OF LACCASE ENZYME

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dc.contributor.advisorSHIRKOT, POONAM
dc.contributor.authorDHIMAN, KARUNA
dc.date.accessioned2016-05-31T15:59:17Z
dc.date.available2016-05-31T15:59:17Z
dc.date.issued2013
dc.description.abstractABSTRACT 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.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/66502
dc.language.isoenen_US
dc.subBiotechnology
dc.subjectenzymes, bacteria, fungi, productivity, aromatic compounds, proteins, genes, sampling, colourants, selectionen_US
dc.subjectLaccase Enzymeen_US
dc.these.typePh.D
dc.titleBIOPROSPECTING OF BACTERIA FOR PRODUCTION AND PURIFICATION OF LACCASE ENZYMEen_US
dc.typeThesisen_US
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