SHRIKOT, POONAMVERMA, AMBIKA2016-06-222016-06-222016http://krishikosh.egranth.ac.in/handle/1/67661ABSTRACT 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 etc. In present study, fourteen bacterial isolates were characterized morphologically and biochemically followed by screening for laccase activity. All the fourteen bacterial isolates were found to test positive for laccase activity producing black zones in case of tannic acid test, orange colour in case of dimethoxyphenol test and purple coloured pigment in case of syringaldazine. Intracellular and extracellular laccase activities were screened quantitatively by ABTS assay and LUA15.1 bacterial isolate showed maximum extracellular laccase activity after 24 hrs of incubation and was identified as Pseudomonas putida LUA15.1. On the basis of maximum extracellular laccase enzyme activity, Pseudomonas putida LUA15.1 was selected for optimization studies using one variable at a time followed by Plackett Burman and Central composite design for laccase enzyme production and as a result 5.52 fold increase in laccase activity has been achieved.Yield of extracellular laccase enzyme has also been increased 12.94 fold by mutagenesis as compare to the wild type Pseudomonas putida LUA15.1. The crude extracellular extract was produced using optimized conditions and further purified using different purification techniques. The procedure yielded 22.5 mg protein with 48.49 fold purification with a percent yield of 10.08 after ion exchange chromatography. The molecular weight of laccase was found to be 42.5 kDa. An extracellular laccase producing gene was isolated using laccase gene specific primers from Pseudomonas putida LUA15.1 followed by cloning and sequencing. Nucleotide sequence obtained after sequencing showed 99% 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 452 amino acids which showed 97% identity with the amino acid sequence of multicopper oxidase [Pseudomonas putida DOT-T1E]. Further secondary and tertiary structures of laccase enzyme were predicted using web based server, PHYRE 1 and 2. Pseudomonas putida LUA15.1 and its extracellular laccase has a significant potential for use in detoxification of eleven textile dyes and hence can be used for treating textile wastewaters particularly for water recycling. Pseudomonas putida LUA15.1 and its crude and purified laccase enzyme preparations were also found to inhibit various fungal and bacterial pathogens in vitro. Purified laccase enzyme preparation from Pseudomonas putida LUA15.1 also lead to clarification of apple juice, in addition to prevention of browning of apple cubes successfullyenenzymes, bacteria, productivity, genes, fungi, colourants, proteins, additives, poultry equipment, aromatic compoundsThesis