Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/2165
Title: Diagnostic potential of indigenously hyperproduced urate oxidase
Authors: Meraj, Munazzah
Keywords: Natural Sciences
Chemistry & allied sciences
Chemistry
Biochemistry
Issue Date: 2012
Publisher: University of Agriculture, Faisalabad, Pakistan.
Abstract: The main purpose of this research work was to optimize the production of urate oxidase through mutagensis of Bacillus subtilis. The organism was subjected to ultra violet irradiation and chemical mutagenesis. Ethyle methane sulfonate treated B. subtilis (180 minutes) was proved to be the best for optimum production of urate oxidase by 3 log kill/survival curve. Fermentation medium was also optimized, it was found that substrate concentration (0.5%), fermentation period (36 h), pH (8.5), temperature (35 oC), yeast extract (0.3%) and sucrose (2%) enhanced the activity of the parent and mutant derived enzyme. The enzyme was purified by adopting different techniques i.e ammonium sulfate precipitation, ion exchange and gel filtration chromatography. It was observed that mutated enzyme exhibited 97.56 U/mg specific activity with 256.73 fold improvement. The purified urate oxidase was run on SDS-PAGE which determined a single band with molecular weight of 34 kDa. The purified BSM-2 possessed Km and V max value 0.067 M and 133.3 IU mg-1 min-1 respectively. The optimum pH and temperature for catalytic activity were 7.5 and 35oC respectively. The activation energy for formation of ES complex was 43.4 kJ/mol. Enthalpy (ΔH*), entropy of activation and Gibbs free energy demands for urate oxidase inactivation were 30.26 kJ/mol, -106.27 J mol-1 K-1, 62.99 kJ/mol respectively. Barium chloride, potassium cyanide and zinc sulfate decreased the activity of the enzyme. Whereas, sodium chloride (0.6M), potassium chloride (0.4M) and calcium chloride (0.4M) enhanced the enzymatic activity 123%, 117% and 119% respectively.
URI:  http://prr.hec.gov.pk/jspui/handle/123456789//2165
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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