Purification Profiling and Kinetic Properties of Tri –Responsive Enzymes from Hydrocarbonoclastic Bacteria

Purification is though a cost implicative aspect in production and application of enzymes. Lipase, urease and peroxidase were produced from strains of Pseudomonas isolated from a crude oil polluted soil using standard microbiology and biochemical techniques. The stress responsive enzymes were produced through submerged fermentation technique. Crude extracts were precipitated at physiologic pH show the crude extract precipitation of lipase with maximum protein precipitation at 70% saturation of the ammonium salt, precipitation of the crude extracts of urease with maximum protein precipitation at 60% saturationof the ammonium salt while precipitation of the crude extracts of peroxidase with maximum protein precipitation at 50% saturation of the ammonium salt. Crude protein of lipase was purified upto 2.32 folds after gel filtration (sephadex G-100) with percentage yield of 2.00%.The specific activity of lipase increased from 211.81 to 490.55 U/m g after gel filtration. Crude urease was purified up to 3.20 folds after gel filtration (sephadex G-100) with percentage yield of 2.30%. The specific activity of urease increased from 170.56 to 546.88U/mg after gel filtration. Crude protein extract of peroxidase was purified upto 2.8 folds after gel filtration (sephadex G-100) with percentage yield of 2.20%. The specific activity of peroxidase increased from 215.38 to 602.20U/mg after gel filtration. lipase, urease and peroxidase activity showed optimum activity at pH of 6.0; 7.0 and 4.5 respectively while 50°C;60°C and 40°C were optimum activity of lipase, urease and peroxidase respectively. Michaelis-menten constant (KM) and maximal velocity of catalysis (Vmax) of 0.32mM and 200 μmol/min were obtained at different concentrations of p-NPP for lipase; whereas KM of 0.370mM and velocity maximal of 197.27μmol/min were obtainedat different concentrations of standard urea for urease and Michaelis-Menten constant (Km)and maximal velocity(Vmax) of 3.45mM and 270μmol/min were obtained at different concentrations of o-dianisidine hydrogen peroxide standard substrates for peroxidases. The purification and kinetic properties of enzymes defines their much physiologic stability and efficient catalytic profile for wide application.