Characterization of gelatin-immobilized pigeonpea urease and preparation of a new urea biosensor

Urease purified from pigeonpea seeds was immobilized on gelatin beads via c ross-linking with glutaraldehyde. The maximum immobilization (75%) was obse rved at 30 mg/ml gelatin, 0.414 mg of enzyme/bead, 1% (v/v) glutaraldehyde and 4 degreesC. Beads stored in 50 mM Tris/acetate buffer (pH 7.3) at 4 deg reesC showed a half-life of 240 days and there was practically no leaching of enzyme (less than 2%) over a period of 30 days. These beads can be reuse d more than 30 times (with 24 h intervals) without much loss of enzyme acti vity (i.e. less than 11%). The immobilized urease showed a shift in its opt imum pH from 7.3 to 6.5 in Tris/acetate buffer. Optimum temperature also sh ifted from 47 to 65 degreesC compared with the soluble enzyme. Gelatin-immo bilized pigeonpea urease had a higher K-m (8.3 mM) than that of the soluble enzyme (3.0 mM). The time-dependent temperature inactivation pattern was a lso found to change from biphasic to monophasic kinetics. The immobilized b eads were used for the preparation of a new urea biosensor with a response time of less than 2 min. At least 14 samples of urea can be measured with t his biosensor within an hour. The beads, as well as the biosensor, were use d to analyse the urea content in clinical samples from the local clinical p athology laboratories. The results obtained with the biosensor were strikin gly similar to those obtained with the various commonly employed biochemica l/autoanalyzer (R) methods used. These immobilization studies also have a p otential role in haemodialysis machines that maintain the urea level in kid ney patients and in the construction of a portable/wearable kidney. The eas y availability of the pigeonpea urease, the ease of its immobilization on g elatin and a significantly lower cost of the urease described in the presen t study makes it a suitable product for future applications in therapeutics and diagnostics.