Investigating purification and activity analysis of urease enzyme extracted from jack bean source: A green chemistry approach

Abstract

Urease is an enzyme of historical importance in the field of biochemistry, generally microbial and plant urease is the primary sources of urease. The significant applications of urease enzyme are found to be foremost in food industry, medical equipment's and biosensors. In this work, urease has been extracted from Jack bean meal using ammonium sulphate and acetone precipitation. A significant amount of urease was precipitated and concentrated at 60% saturated solution of ammonium sulphate. The obtained precipitates were dissolved in 50 mM phosphate buffer (pH 8) after centrifugation, and subjected to sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the molecular weight of urease. Results obtained from the SDS-PAGE were validated using Zymography. Anion exchange chromatography was used to separate the desired protein at different pH (7.0, 7.5 and 8.0). The eluted fractions were assessed for urease activity using phenol-nitroprusside dependent ammonia release assay. Under these assay conditions, one unit of urease activity was calibrated as the amount of enzyme liberating 1 μM of NH3 from urea per unit time. The eluted fraction and Zymography analysis show the purified urease observed at 90 kDa and activity of purified urease, respectively. The obtained results for specific activity (173.67Units mg) and % purification (99.71%) for urease has been compared with the available literature, which are found to be in close relation with existing results. The proposed method is a novel approach which has recorded highest % purification and specific activity. Furthermore, it can be suitable for extracting urease from jack bean source for various industrial applications.