Response of Free and Immobilized Uricase from Fenugreek Leaves to various treatments and Digestive Enzymes

Abstract

Uricase (Urate: oxygen oxidoreductase (EC 1.7.3.3) was isolated from 15-day old Trigonella foenum-graecum L. seedlings. Kinetin, gibberellin (GA3) and benzylaminopurine induced the enzyme and kinetin was the best inducer. The polyols including mannitol, sorbitol, glycerol,erythritol and xylitol expressed appreciable thermostability for uricase at 60 ºC. Xylitol was better than trehalose as stabilizer at 60 ºC. The enzyme was then purified using 80% ammonium sulfate, DEAE-cellulose, and Sephadex G-200. The final specific activity was 80 units mg- 1 protein. The purified uricase was immobilized on alginate (entrapped) and alginate with glutaraldehyde activity (cross-linked). The cross-linked method was better than entrapped method. The Vmax values were 15.6, 13.8 and 15.2 units mg- 1 protein for the free, entrapped and cross-linked enzyme, respectively. The Km values were 0.072, 0.092 and 0.084 mM, respectively. The optimal pH was 8.5for the free, entrapped, and cross-linked enzyme. The optimal temperature was 35 ºC , for free and entrapped but it was 45 ºC for cross-linked uricase. Uricase exhibited appreciable resistant digestion by pepsin and trypsin.