Isolation, purification and characterization of urease from pigeon pea (Cajanus cajan).
1992
Yu G.F.B.
Urease (Urea amidohydrolase, E.C. 3.5.1.5) was isolated and purified from pigeon pea (Cajanus cajan) 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 subunits 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 fully hydrolyzed by this enzyme. The Km value obtained from the Lineweaver-Burk plot was 9.9 x 10 to the negative third power M and Vmax value of 189 units/mq protein. The Eadie Hofstee diagnostic plot rendered values of 10.4 x 10 to the negative third power M and 193 units/mg protein for Km and Vmax, respectively. The optimum conditions for the assay, with urea as substrate, were at pH 7.0 and temperature at 40 deg C. Although Cu2+ activated the enzyme at high metal concentration (8.16 ppm), a 0.163 ppm inhibits the enzyme activity, thus the order of inhibition at metal concentration of 0.163 ppm was Ni2+ > Cu2+ > Ca2+ > Mg2+. The crude enzyme was stable when suspended in 50% glycerol and stored at 0 deg C for 6 months.
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