Biochemical Studies of Urease From Watermelon (Citrullus Vulgaris)

Abstract

The urease (urea amidohydrolase; EC 3.5.1 .5) was isolated from the mature seeds of watermelon (Citrullus vulgaris). Urease was stored at two different temperatures for several days in order to check its storage stability with tirne. The plot of percent residual activity versus number of days gave a t 112 of 70 days for urease stored at 4 °C and 16 days for urease that stored at 37 °C. The stability pH of watermelon urease was detennined by storing it at different pHs (6, 7, 8, 9, and I 0). The plot of percent residual activity versus pH gave the stability pH of watermelon urease at 8.0. The optimum temperature and pH was found to be 53 oc and 8.3 respectively. To study the effect of substrate concentration, the experiments were carried out by measuring the amount of product formed at different substrate concentrations. The Km and Vuw.\ of urease was found to be 9 mM and 1 25 J.Lmole/min/mg. Thermal inactivation studies of watermelon urease revealed a variation in kinetic pattern at different temperatures. It is evident that at 50 and 60 °C (low temperatures), there is almost no effect on three dimensional structure of urease and the active site and therefore retaining most of its activity. Time dependent thermal inactivation studies showed biphasic kinetics at 70 °C, 80 °C and 85 °C. The inactivation studies strongly favor the oligomeric nature of urease. Further, urease was investigated for the inhibitory effect of compounds such as HgCb and NaCI. Increased levels of soil microbia] urease have been known to decrease the efficiency of urea fertilizers. Due to increased use of urea as fertilizer this has caused severe environmental and econon1ic problems by continuously releasing high amount of ammonia in the environment. These probletns can be successfully solved by tnixing urease inhibitors with urea based fertilizers. The fw values for Hg2 .. and Na+ inhibition were found to be 6.5 nM and 250 mM, respectively. The results clearly show strong inhibitory nature of Hg21 and poor potency ofNa~ due to its high l5o value. The urease activity strongly inhibited by several inhibitors. Since, the amino acid sequence alignment reveals that all known ureases, isolated from different sources, are highly similar and shares a common phylogenetic relationship and proposed to have common structure and catalytic mechanism. Therefore, urease from any source, tnay be bacterial or plant, can be used as model system for studies and results would be equally applicable for any systetn or field of application.