Kinetic and Thermodynamic Properties of Purified Wild and Mutant Derived Urate Oxidase
Urate oxidase (uox) has a great significance for diagnosis and treatment of uric acid in body fluids and body tissues respectively. The present research work was designed to hyper produce, purify and characterize the uox for its multiple uses. Uox was produced by mutated Bacillus subtilis in submerged fermentation. The organism was subjected to ultra violet irradiation and chemical mutagenesis. Ethyl methane sulfonate treated B. subtilis (BSM-2) (180 minutes) was proved to be the best for optimum production of uox by 3 log kill/survival curve. The enzyme was purified by adopting different techniques i.e. ammonium sulfate precipitation, ion exchange and gel filtration chromatography. It was observed that uox from BSM-2 exhibited 23.21 and 97.56 U/mg specific activities when subjected to ion exchange and gel filtration chromatography with 156.82 and 256.73-fold improvement respectively. Wild type uox (WT uox) showed 7.42 and 8.42 U/mg specific activity after purification by ion exchange and gel filtration chromatography with 85.37 and 136.57-fold purification respectively. The purified BSM-2 was run on SDS-PAGE which determined a single band with molecular weight of 34 k Da. The purified BSM-2 and WT derived uox possessed Km 0.067 ± 0.004 M & 0.80 ± 0.018 M and V max 133.3 ± 4.5 & 83.3 ± 2.4 IU mg-1 min-1 respectively. The activation energy required for BSM-2 and WT uox to hydrolyze uric acid was 32.8 & 43.4 KJ/mol respectively. The enthalpy (ΔH*) and entropy of activation (ΔS*) for both BSM-2 and WT uox was 30.26 & 40.84kJ/mol and -106.27 & -70.81 J/mol. K respectively.
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