Production of racemic lactic acid in Pediococcus cerevisiae cultures by two lactate dehydrogenases

Abstract

Nicotinamide adenine dinucleotide (NAD)-dependent D(-)-and L(+)-lactate dehydrogenases have been partially purified 89and 70-fold simultaneously from cell-free extracts of Pediococcus cerevisiae. Native molecular weights, as estimated from molecular sieve chromatography and electrophoresis in nondenaturing polyacrylamide gels, are 71,000 to 73,000 for D(-)-lactate dehydrogenase and 136,000 to 139,000 for L(+)-lactate dehydrogenase. Electrophoresis in sodium dodecyl sulfate-containing gels reveals subunits with approximate molecular weights of 37,000 to 39,000 for both enzymes. By lowering the pyruvate concentration from 5.0 to 0.5 mM, the pH optimum for pyruvate reduction by D(-)-lactate dehydrogenase decreases from pH 8.0 to 3.6. However, L(+)-lactate dehydrogenase displays an optimum for pyruvate reduction between pH 4.5 and 6.0 regardless of the pyruvate concentration. The enzymes obey Michaelis-Menten kinetics for both pyruvate and reduced NAD at pH 5.4 and 7.4, with increased affinity for both substrates at the acid pH. a-Ketobutyrate can be used as a reducible substrate, whereas oxamate has no inhibitory effect on lactate oxidation by either enzyme. Adenosine triphosphate causes inhibition of both enzymes by competition with reduced NAD. Adenosine diphosphate is also inhibitory under the same conditions, whereas NAD acts as a product inhibitor. These results are discussed with relation to the lactate isomer production during the growth cycle of P. cerevistae.

Bacterial lactate dehydrogenases.

“…D(-)-fLDH's. Most D(-)-nLDH's are reversible (13,27,34,39,49), but a few which use only pyruvate as substrate are known. In an early study of Lactobacillus casei, a D(-)-nLDH was partially purified.…”

“…L(+)-nLDH's are of two kinds, those which are activated by FDP and those which are not. The DL-lactate-forming lactic acid bacteria have L(+)-nLDH's which do not require FDP, and in many species these enzymes catalyze reversible reactions (13,34,39). Some lactobacilli have L(+)-nLDH's which react with lactate weakly or not at all, so that they can be detected after electrophoresis only with pyruvate.…”

“…Other lactic acid bacteria form DL-lactate, but the ratio of the isomers can change at different times in cultures growing without pH control (26,39,85). Pediococcus pentosaceus (called Pediococcus cerevisae [26,39]) and homofermentative lactobacilli (except Lactobacillus plantarum) have a high proportion of L(+)-lactate early in the growth cycle and form D(-)lactate when the medium has a low pH.…”

“…When P. pentosaceus was grown in glucosecontaining media, L(+)-lactate was produced preferentially while carbohydrate was plentiful (39). When the pH fell to about 5.0, the L(+)lactate production stopped, and D(-)-nLDH became active.…”

“…Further purification has been achieved with column chromatography, and a variety of conditions have been satisfactory (5, 6, 10, 13, 23, 35, 38-40, 45, 46, 48, 49, 52, 64, 67, 70, 72, 87, 90, 97, 100, 104, 111). Diethylaminoethyl-cellulose, diethylaminoethyl-Sephadex, and Sephadex G200 have been used most often, but other materials have also been tried; these other materials have included Sepharose 6B (39), hydroxyapatite (39,49), calcium phosphate gel (64,70,100,102,111), diethylaminoethyl protein (10,48), and Ecteola cellulose (111). Agarose permeation chromatography (5) and preparative electrophoresis (40,49) have also been used successfully.…”