Purification .and Properties of NAD-Dependent L-Lactate Dehydrogenase from a Rhizopus Oryzae Mutant

Abstract

NAD-dependent L-lactate dehydrogenase from Rhizopus oryzae mutant 3Nf3 was purified39-fold with an 11% yield by ammonium sulfate fractionation, DEAE-Sephadex chromatography,hydroxy apatite chromatography, and chromatofocusing. The enzyme showed optimal pH and temperature of 7.0 to 7.5 and 25 to 30°C. respectively. It was stable over a pH range of 7.0 to 8.0, but lost its activity above 30°C. The Km for sodium pyruvate and NADH were 0.61 and 0.10 n-&I. respectively. The Vmax for sodium pyruvate and NADH were 23.5 and 34.5 mM permg protein per min. respectively. The molecular weight was 38 kDa as determined by SDSPAGE and 150 kDa by non-denaturing PAGE. The enzyme had an isoelectric point (~1) of 5.2 as determined by chromatofocusing.