Immobilization of watermelon (Citrullus vulgaris) urease in agarose gel for urea estimation

Urease from dehusked seeds of watermelon was immobilized in 1.5% agarose gel with 53.9% entrapment. There was negligible leaching (< 10% at 4 degrees C) and the same gel membrane could repeatedly be used for seven days. The immobilization exhibited no apparent change in the optimum pH but there was a significant decrease in the optimum temperature (50 degrees C as compared to 65 degrees C for soluble urease). The immobilized urease revealed an apparent K-m of 9.3 +/- 0.3 mM; 1.2 times lower than the soluble enzyme (11.4 +/- 0.2 mM). Unlike soluble enzyme which was inhibited at 200 mM urea, the immobilized urease was inhibited at 600 mM of urea and above, and about 47% activity was retained at 2 M urea. The time-dependent thermal inactivation kinetics at 48 and 52 degrees C was found to be biphasic, in which half of the initial activity was destroyed more rapidly than the remaining half. These gel membranes were also used for estimating the urea content of the blood samples from the University hospital. The results obtained matched well with those obtained by the usual method employed in the clinical pathology laboratory. The significance of these observations is discussed. (c) KSBB.