Characterization of gelatin-immobilized pigeonpea urease and preparation of a new urea biosensor.
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.
Srivastava
P K
PK
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi 221 005, India.
Kayastha
A M
AM
Srinivasan
D015374:Biosensing Techniques; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005780:Gelatin / Q000737:chemistry; D005976:Glutaral / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D012639:Seeds / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D014508:Urea / Q000097:blood / Q000494:pharmacology; D014510:Urease / Q000737:chemistry*
D003432:Cross-Linking Reagents; D014508:Urea; D005780:Gelatin; D014510:Urease; D005976:Glutaral