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 center dot 4H(2)O coupling with a conjugation yield of 0.36 mg of protein/cm(2) and 6S.3 % retention of the initial activity of the free enzyme. The immobilized enzyme showed maximum activity at pH 6.S when incubated at 40 degrees C for IS min. K-m for oxalate was decreased from 0.7 to O.S3 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.S 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.