Effects of the water-miscible organic solvents on lactoperoxidase purified from creek-water buffalo milk

Water buffalo lactoperoxidase (WBLPO) was purified with Amberlite CG-50 (NH4+ form) resin, CM-Sephadex C-50 ion-exchange chromatography. and Sephadex G-100 gel-filtration chromatography from skimmed buffalo milk. The purity of the WBLPO was shown with SIDS-PAGE. The R-z (A(412)/A(280)) value for the WBLPO was 0.9. The optimum pH for the WBLPO was at 6.0. The K-m value at optimum pH and 25degreesC was 0.13 mM. The V-max value at optimum pH and 25degreesC was 5.3 mumol/min per ml. The K-i values for methanol, ethanol, dimethyl sulfoxide (DMSO), acetonitrile, isopropanol, tetrahydrofuran (THF), N,N'-dimethylformamide (DMF), and ethylene glycol were 1.087, 0.364, 0.302, 0.459, 0.330, 0.126, 0.093, and 2.125 M, respectively. All the solvents showed competitive inhibition. The I-50 values of methanol, ethanol, dimethyl sulfoxide, acetonitrile, isopropanol, tetrahydrofuran, N,N'-dimethylformamide, and ethylene glycol were 2.910, 0.942, 0.537, 1.320, 0.875, 0.470, 0.405, and 3.920 M, respectively. Ethylene glycol, methanol, acetonitrile, and ethanol have been found to be very promising solvents for performing biocatalytic reactions with LPO in organic media.