Purification and Properties of Membrane Bound Oxalate Oxidase from Leaves of Bougainvillea glabra

摘要

A plastid membrane bound oxalate oxidase from leaves of Bougainvillea glabra has been solubilized by taurodeoxycholate and purified by 80% ammonium sulphate fractionation, gel filtration on Sephadex G-100 and ion exchange chromatography on DEAE-Sephacel. The enzyme was purified by 60.47-fold with 31%) yield and exhibited a single band in simple PAGE and SDS-PAGE with a M_r of 133 kDa and two identical subunit of 66.5 kDa each. The enzyme showed maximum activity at an optimum pH 5.5, when incubated at40degC for 4 min. A hyperbolic relationship between enzyme activity and oxalate concentration was obtained. K_m and V_(max) as calculated from LB plot were found to be 1.5 mM and 121 nmoles H_2O_2/ml/min respectively. Enzyme was stimulated by Cu~(+2) ions but inhibited by NaCl, KC1 and HgCl_2. UV-visible spectra of enzyme did not match with that of flavoprotein. A positive response of enzyme to H_2SO_4-orcinol indicated its glycoprotein nature. The analytic use of enzyme for oxalate determination in urine is demonstrated.