Immobilization of grain-sorghum (Sorghum bicolor, var. CSH-14) leaf oxalate oxidase on to a modified mica chip and its application in the determination of urinary oxalate.
A new method is described for immobilization of oxalate oxidase purified from grain sorghum (Sorghum bicolor, var. CSH-14) leaves on to mica chip through MnCl(2) x 4H(2)O coupling with a conjugation yield of 0.36 mg of protein/cm(2) and 65.3% retention of the initial activity of the free enzyme. The immobilized enzyme showed maximum activity at pH 6.5 when incubated at 40 degrees C for 15 min. K(m) for oxalate was decreased from 0.7 to 0.53 mM after immobilization. The immobilized enzyme lost 12% of its initial activity after storage at 4 degrees C in reaction buffer. The oxalate in urine was analysed using this immobilized enzyme. The minimum detection limit was 2.5 mg/l of urine. Analytical recovery of added oxalate (10, 20 and 30 mg/l) was 89.2, 91 and 92.1% respectively. Within- and between-batch coefficients of variation were <1.3% and <4.1% respectively. A good correlation (r=0.92) was found between oxalate values obtained using a commercial enzymic colorimetric kit (Oxalate Urinalysis Diagnostic kit; procedure no. 591-C; Sigma) based on free barley oxalate oxidase and the present method.
Bhambi
Manu
M
Biochemistry Research Laboratory, Department of Biochemistry and Genetics, Maharshi Dayanad University, Rohtak, 124001 Haryana, India.
Pundir
Chandra Shekhar
CS
D000327:Adsorption; D000538:Aluminum Silicates / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D020099:Coated Materials, Biocompatible / Q000737:chemistry; D003124:Colorimetry / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D019815:Oxalic Acid / Q000737:chemistry / Q000652:urine*; D010088:Oxidoreductases / Q000737:chemistry*; D018515:Plant Leaves / Q000201:enzymology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D045868:Sorghum / Q000201:enzymology*; D016482:Urinalysis / Q000379:methods*
D000538:Aluminum Silicates; D020099:Coated Materials, Biocompatible; D004800:Enzymes, Immobilized; D019815:Oxalic Acid; D010088:Oxidoreductases; C020598:oxalate oxidase; C011934:mica