Water Melon (Citrullus Vulgaris) Urease Immobilization in Alginate Beads and Characterization

Abstract

Immobilization of urease has been carried out on various matrices for clinical analytical applications. Effective enzyme immobilization can be achieved using several techniques including adsorption to insoluble materials, entrapment in polymeric matrix encapsulation, cross linking with a bifunctional reagent, or covalent linking to an insoluble carrier. However, no single method and support is best for all enzymes and their applications. Cross linking of alginate with divalent ions (like Ca2+) and glutaraldehyde improves the stability of enzymes. Immobilization on a conducting matrix (e.g., a metal electrode) is generally difficult but is desirable in biosensor application. Several enzyme biosensors have been designed and constructed using various kinds of immobilization techniques. Calibration curve of NH4Cl v/s Absorbance found a straight line and 0.1 optical density at around 80μg. From steady state kinetics investigation, optimum temperature for urease is 800C for both soluble and immobilized urease. The optimum pH for 7-7.1 for both soluble and immobilized enzyme. Effect of substrate on urease activity, we made a Lineweaver Burk plot and calculated Km for soluble and immobilized urease is 26.31mM and 33.33mM respectively. There was a decrease in Vmax after immobilization and the values calculated were 13.33 and 12.5 μmole-1NH3min-1mg-1 for soluble and immobilized urease respectively. From Reusability studies it is clear that after eight rounds of use, the immobilized enzyme loose original activity more than 50% may be due to the continuous washing with buffer. Storage stability studies showed that the lose of activity of enzyme at both the temperatures (40C and room temperature), per cent relative activity was 45% for soluble urease and 37% for immobilized urease. Thermal inactivation studies showed that per cent relative activity increases first from (10-30) min upto the 100% and then decrease from (40-70)min to 55.5% at constant temperature (700C).