PRODUCTION OF URICASE A THERAPEUTIC ENZYME FROM Pseudomonas putida ISOLATED FROM POULTRY WASTE

Urate oxidase or uricase is the enzyme that catalyzes specifically the oxidation of uric acid to allantoin and plays an important part in nitrogen metabolism. The soil samples were collected from poultry farms and the uricase producing microorganism were isolated. From the uric acid amended plates 5 strains were selected based on different morphology. They were purified and checked for their potential of uricase production by using well assay method. The potential strain which showed a zone of clearance of 14mm on uric acid agar plate was selected and it was to subjected to biochemical tests. The isolated strain was optimized to parameters like temperature, pH, incubation time, carbon sources and nitrogen sources by adopting one parameter at a time method. The study was conducted for a period of hours where analysis was done for every 6hrs interval. The isolated protein was estimated and its was separated by SDS –PAGE. uricase producing gene was isolated and amplified by PCR.The Purified protein Sample was subjected to FTIR spectroscopy. The isolated strain was identified as Pseudomonas putida. The optimized parameters, tem 37?C, pH-7.0, 36 hrs of incubation time are found suitable for higher production, 1% starch and 0.2% ammonium nitrate were found to be ideal for growth. The highest level of uricase activity attained for the crude enzyme was 38U/ml/min, whereas after purification 67.0U/ml/min. SDS-PAGE analysis revealed the protein profile of partially purification a 66kDa protein was obtained. The amplified uricase gene of Pseudomonas putida showed 240bp length. FTIR results also showed characteristic positive vibrations for uricase. Thus the present study contributes much new information on microbial uricase and proved its potential as a industrial scale producer.