Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications.
Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4 degrees C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Tris-acetate buffer, pH 7.3, at 4 degrees C had a t(1/2) of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77 degrees C, compared with 47 degrees C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.
Kayastha
A M
AM
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India. kayastha@indiatimes.com
Srivastava
P K
PK
D000818:Animals; D001669:Biochemical Phenomena; D001671:Biochemistry; D003386:Brachyura; D002686:Chitin / Q000031:analogs & derivatives / Q000737:chemistry*; D048271:Chitosan; D005976:Glutaral / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D018532:Pisum sativum / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors; D014508:Urea / Q000097:blood / Q000737:chemistry*; D014510:Urease / Q000737:chemistry* / Q000378:metabolism
D002686:Chitin; D014508:Urea; D048271:Chitosan; D014510:Urease; D005976:Glutaral