Characterization and Purification of Urate Oxidase in Highly Active Condition from Bacillus Subtilis for Biomedical Applications

Abstract

Objective: This research aimed to extract and purify urate oxidase (uox) for use in the pharmaceutical industry. 

Methodology: The experimental study was carried out at Govt. College University of Faisalabad  with the collaboration of Peoples University of Medical and Health Sciences for Women Nawabshah, Sindh.For the purification of Bacillus subtilis derived uox, different techniques were used i.e ammonium sulfate precipitation (ASP), gel filtration chromatography (GFC), and ion exchange chromatography (IEC). The purity of this enzyme as well as molecular weight was assessed by using SDS-PAGE. At every step of the purification process, the specific activity and purification fold was increased which showed that the enzyme was purified after the use of every purification technique.

Result: There was initial purification step involving ASP, eliminating impurities. This stage yielded 22.95 units of total activity, with a specific activity of 0.166 units/mg and 138.43 mg of protein. The enzyme was purified 1.34 folds as compared to crude cell lysate. When the enzyme was purified in the next step by using IEC, the specific activity was significantly increased to 2.40U/mg with 19.34 fold increase in purification as compared to crud. In the last step of purification (GFC) the enzyme was 42.96 fold increase purification with 5.33 U/mg specific activity.

Conclusion: This research successfully extracted highly pure uox from B. subtilis using various purification techniques. The final enzyme sample has higher specific activity and purity, which bodes well for future industrial applications and research uses.