Abstract
Enzyme uricase has wide application in diagnostics and other industrial sectors. Here we report isolation, purification and characterization of microbial uricase from Penicillium purpurescens cultured on firm media such as solid biological waste. Purifications of the enzyme from the firm media included stepwise fractionation with ammonium sulfate and further purification with ion exchange columns subsequently through gel filtration chromatography. We obtained a final purified fraction of about 51.7% with specific enzymatic activity of 3920 U/mg protein. Electrophoretic purity revealed a single band of molecular weight 42 KDa. Kinetic studies on the purified enzyme by Line Weaver-Burk plot assessment showed a K-M value of 0.5 mM and V-max of 909 IU. The purified enzyme's stability was over a wide range of pH between 6-10 and tolerated temperature up to 70 degrees C, however maximum enzyme activity was at pH 9 and 30 degrees C. The enzyme activity was stimulated greatly in presence Fe++, Na+, Cat, and Cu+ ions, chelated by addition of EDTA and inhibited by KCN. Amino acid analysis proved the enzyme to be rich in methionine, glutamic acid and glycine.