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

Abstract

Urease purified from pigeonpea seeds was immobilized on gelatin beads via cross-linking with glutaraldehyde. The maximum immobilization (75%) was observed at 30 mg/ml gelatin, 0.414 mg of enzyme/bead, 1% (v/v) glutaraldehyde and 4 degrees C. Beads stored in 50 mM Tris/acetate buffer (pH 7.3) at 4 degrees C 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 reused more than 30 times (with 24 h intervals) without much loss of enzyme activity (i.e. less than 11%). The immobilized urease showed a shift in its optimum pH from 7.3 to 6.5 in Tris/acetate buffer. Optimum temperature also shifted from 47 to 65 degrees C compared with the soluble enzyme. Gelatin-immobilized 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 also found to change from biphasic to monophasic kinetics. The immobilized beads 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 this biosensor within an hour. The beads, as well as the biosensor, were used to analyse the urea content in clinical samples from the local clinical pathology laboratories. The results obtained with the biosensor were strikingly similar to those obtained with the various commonly employed biochemical/autoanalyzer(R) methods used. These immobilization studies also have a potential role in haemodialysis machines that maintain the urea level in kidney patients and in the construction of a portable/wearable kidney. The easy availability of the pigeonpea urease, the ease of its immobilization on gelatin and a significantly lower cost of the urease described in the present study makes it a suitable product for future applications in therapeutics and diagnostics.

Effective Use of Plant-Derived Urease in the Field of Geoenvironmental/ Geotechnical Engineering

“…This result is closely related to those reported by Das et al [13] and Srivastava et al [41] but differs from that stated by El-Shora [42]. Urease activity increases with substrate concentration.…”

Immobilization of Aspergillus oryzae α-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

“…It was speculated that hydrophobic interactions and other secondary interactions of the immobilized enzyme might impair conformational flexibility, necessitating higher temperature for the enzyme molecules to reorganize and attain proper conformation for its functioning and binding of the substrate (Arica et al 1996). A shift of temperature from 20°C to 40°C and 40°C to 65°C for tyrosinase and urease respectively was observed after immobilization in gelatin (Munjal and Sawhney 2002;Srivastava et al 2001). …”

“…After 24 h, soluble enzyme retained 44% and gelatin-immobilized enzyme retained 69% activity. The thermal stability of the enzyme will be stabilized because the immobilization process will make the enzyme more rigid and that the immobilization will change the microenvironment of the enzyme, altering its apparent pH optima (Srivastava et al 2001). …”

Immobilization of Urease from Pigeonpea (Cajanus cajan) on Agar Tablets and Its Application in Urea Assay

“…The apparent increase in K m might be due to changes in the accessibility of substrate to the active site as there are diffusional resistances due to agar gel. The significant changes (increase) in K m upon immobilization have been observed most commonly in case of encapsulation and adsorption methods [12,14,29,30].…”