Philippine E-Journals

Isolation, Purification, and Characterization of Urease From Pigeon Pea (Cajanus Cajan)

Gracia Fe Yu | Angelita Reyes

Discipline: veterinary sciences

 

Abstract:

Urease was isolated and purified by a series of citrate buffer extractions and chromatography through Sephadex Fine G-200. The molecular weight of the enzyme, obtained through Sephadex G-200 chromatography, was estimated at 540,000 daltons. SDS polyacrylamide gel electrophoresis set the molecular weights of the sub-units at 90,000, 46,000 and 31,000 daltons respectively. The isoelectric point, determined by isoelectric focusing, was about 5.8. Thiosemicarbazide was slightly acted upon, while urea was completely hydrolyzed by this enzyme. Th Km value obtained from the Lineweaver-Burk plot was 9.9 x 103 mM and V value of 189 units/mg protein. The Eadie Hofstee diagnostic plot rendered values of 10.4 x 10-3 mM and 193 units/ mg protein for Km and V respectively. The low Km value of this enzyme for urea indicates its high affinity for the said substrate. The optimum conditions for the assay, with urea as substrate, were at pH 7.0 and temperature at 40°C. The crude enzyme was stable when suspended in 50% glycerol and stored at 0°C for 6 months. These properties are comparable to the commercially available Sigma jackbean urease.