BIOPROSPECTING OF THERMOPHILIC BACTERIA FROM HOT WATER SPRINGS OF HIMACHAL PRADESH FOR LACCASE ENZYME PRODUCTION

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dc.contributor.advisorSHIRKOT, POONAM
dc.contributor.authorSHARMA, RUCHIKA
dc.date.accessioned2016-06-14T15:23:55Z
dc.date.available2016-06-14T15:23:55Z
dc.date.issued2015
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, significantly high diversity of laccase producing bacteria from hot water springs of Himachal Pradesh was assessed. A total of 118 laccase producing thermophilic bacterial isolates were obtained from 200 hot water spring samples using TYM containing 5 mM guaiacol which were morphologically characterized. These were rescreened on the basis of their ability to oxidise tannic acid, dimethoxyphenol and syringaldazine leading to selection of 50 laccase producing thermophilic bacterial isolates, which were characterized biochemically. Eighteen thermophilic bacterial isolates exhibiting maximum laccase activity of 0.0007-0.0038 U/l were selected for further, molecular characterization using RAPD-PCR and 16S rrna gene technology. In silico analysis of 16S rrna gene sequences led to identification of these bacterial isolates and they were found to belong to genus Bacillus, Aneurinibacillus and Pseudomonas, as Bacillus licheniformis strain RSV20, Bacillus licheniformis strain RSM8, Bacillus licheniformis strain RSV10, Bacillus sonerensis strain RSM17, Bacillus sonerensis strain RSV8, Bacillus licheniformis strain RSP1, Bacillus licheniformis strain RSP2, Bacillus licheniformis strain RSP3, Bacillus licheniformis strain RSP7, Bacillus sonerensis strain RSP5, Bacillus sonerensis strain RSP11, Aneurinibacillus thermophilus strain RSP13, Bacillus aerius strain RSP4, Bacillus aerius strain RSP9, Bacillus subtilis strain RSP8, Bacillus amyloliquifacience strain RSP10, Bacillus pumilis strain RSP12 and Pseudomonas taiwanensis strain RSP6. On the basis of maximum laccase enzyme activity Bacillus licheniformis strain RSM8 was selected for production and purification of the laccase enzyme. Maximum extracellular enzyme production was achieved at 60°C, pH 9.0 and 24 hrs incubation with 5 mM guaiacol, 5 % tryptone and 3 % yeast extract in combination with nitrogen source. Crude extracellular thermolaccase enzyme preparation was purified by ammonium salt precipitation (50-90%) followed by gel filtration and ion exchange chromatography which showed 15.21 yield and 10.5 fold purification. The purified enzyme had optimal activity at pH 9.0 and 60 °C, and 16.22 μM Km value. The molecular weight of thermolaccase in the present study was found to be 72.5 kDa. However activity was inhibited by sodium azide and DTT. Bacillus licheniformis strain RSM8 as well as its enzyme preparations were investigated for their ability to decolourize dyes which are the potential contributors of water pollution. Six different synthetic dyes were decolourized RBBR (68 %), congo red (86 %), indigo carmine (73 %), brilliant blue (40 %), bromophenol blue (51 %) and aniline blue (54 %) when treated with the crude enzyme preparation of Bacillus licheniformis strain RSM8. And partially purified enzyme preparation of Bacillus licheniformis strain RSM8 showed greater decolourization of dyes comparatively RBBR (74 %), congo red (91 %), indigo carmine (80 %), brilliant blue (60 %), bromophenol blue (64 %) and aniline blue (67 %). The purified enzyme was successfully immobolized using adsorption method in calcium alginate beads with 76% immobolization percentage and immobolized 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. Bacillus licheniformis strain RSM8 enzyme preparations was investigated for phytotoxicity evaluation of three dyes viz., Congo red, RBBR and Indigo carmine and each of enzyme treated dyes for Phaseolus mungo and Calendula officinalis and Tagetes patula plant species respectively, under in vitro conditions and Phaseolus mungo with Congo red dye under in vivo conditions. Significant germination inhibition, a slower rate of plumule and radicle seedlings growth was observed for Congo red, RBBR and Indigo carmine dyes as compared to enzyme treated dyes. 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 hot water spring bacteria, Bacillus licheniformis strain RSM8 followed by determination of the amino acid which were translated from nucleotide sequence and encodes a polypeptide comprised of 50 amino acids showeng 97 % identity with the amino acid sequences of bacterial laccases i.e. copper oxidase [Bacillus licheniformis]. Further multiple sequence alignment using MULTALIN and structure prediction using Phyre1 & 2 revealed conserved histidine residues.en_US
dc.identifier.urihttp://krishikosh.egranth.ac.in/handle/1/67377
dc.language.isoenen_US
dc.subMolecular Biology and Biotechnology
dc.subjectenzymes, bacteria, fungi, colourants, productivity, genes, aromatic compounds, proteins, irrigation, surface wateren_US
dc.subjectLaccase Enzyme , bioremediationen_US
dc.these.typePh.D
dc.titleBIOPROSPECTING OF THERMOPHILIC BACTERIA FROM HOT WATER SPRINGS OF HIMACHAL PRADESH FOR LACCASE ENZYME PRODUCTIONen_US
dc.typeThesisen_US
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