Purification and characterization of lactate dehydrogenase from Geotrichum candidum

Abstract

An appreciable quantity of lactate dehydrogenase (EC 1.1.2.3) was present in the crude extract of Geotrichum candidum grown previously in sauerkraut processing waste effluents. The enzyme catalyses the oxidation of lactate to pyruvate with potassium ferricyanide and 2,6-dichlorophenol indophenol as electron acceptors. The enzyme was purified 64-fold by ammonium sulfate fractionation, gel filtration, and ion exchange chromatography with a yield of more than 5%. The specific activity of the purified enzyme was more than 6 units per mg protein. pH and temperature optima were 7·5 and 30°C, respectively. The enzyme was stable at pH values between 7·0 and 8·0, but lost most of its activity at 40°C in 10 min. The enzyme was specific for l(+)-lactate and its K m value with potassium ferricyanide as an electron acceptor was 0·71 mm. The enzyme had a molecular weight of > 200 000 as determined by gel filtration, and its pl was estimated to be 7·5. The enzyme activity was inhibited by heavy metals, and EDTA could be used to prevent heavy metal inhibition.