Effects of the Water-Miscible Organic Solvents on Lactoperoxidase Purified from Creek-Water Buffalo Milk

H. Özdemir^*, H. Ý. Hacibeyoðlu, and Ö. Ý. Küfrevioðlu

Faculty of Arts and Sciences, Department of Chemistry, Ataturk University, 25240 Erzurum, Turkey; E-mail: hozdemir@atauni.edu.tr

^* To whom correspondence should be addressed.

Received February 26, 2002; Revision received May 27, 2002

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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 SDS-PAGE. The R_z (A₄₁₂/A₂₈₀) 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 25°C was 0.13 mM. The V_max value at optimum pH and 25°C was 5.3 µmol/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 I₅₀ 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.

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KEY WORDS: lactoperoxidase, organic solvents, kinetics

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