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

Abstract

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.