Major Role of NAD-Dependent Lactate Dehydrogenases in the Production of l-Lactic Acid with High Optical Purity by the Thermophile Bacillus coagulans

Abstract

Bacillus coagulans 2-6 is an excellent producer of optically pure l-lactic acid. However, little is known about the mechanism of synthesis of the highly optically pure l-lactic acid produced by this strain. Three enzymes responsible for lactic acid production-NAD-dependent l-lactate dehydrogenase (l-nLDH; encoded by ldhL), NAD-dependent d-lactate dehydrogenase (d-nLDH; encoded by ldhD), and glycolate oxidase (GOX)-were systematically investigated in order to study the relationship between these enzymes and the optical purity of lactic acid. Lactobacillus delbrueckii subsp. bulgaricus DSM 20081 (a d-lactic acid producer) and Lactobacillus plantarum subsp. plantarum DSM 20174 (a dl-lactic acid producer) were also examined in this study as comparative strains, in addition to B. coagulans. The specific activities of key enzymes for lactic acid production in the three strains were characterized in vivo and in vitro, and the levels of transcription of the ldhL, ldhD, and GOX genes during fermentation were also analyzed. The catalytic activities of l-nLDH and d-nLDH were different in l-, d-, and dl-lactic acid producers. Only l-nLDH activity was detected in B. coagulans 2-6 under native conditions, and the level of transcription of ldhL in B. coagulans 2-6 was much higher than that of ldhD or the GOX gene at all growth phases. However, for the two Lactobacillus strains used in this study, ldhD transcription levels were higher than those of ldhL. The high catalytic efficiency of l-nLDH toward pyruvate and the high transcription ratios of ldhL to ldhD and ldhL to the GOX gene provide the key explanations for the high optical purity of l-lactic acid produced by B. coagulans 2-6.

FIG 1

Active staining of nLDHs after native PAGE. Extracts of B. coagulans 2-6 (lanes 1, 4, and 7), L. plantarum subsp. plantarum DSM 20174 (lanes 2, 5, and 8), and L. delbrueckii subsp. bulgaricus DSM 20081 (lanes 3, 6, and 9) cells were used for native PAGE. dl-Lactate (lanes 1, 2, and 3), d-lactate (lanes 4, 5, and 6), and l-lactate (lanes 7, 8, and 9) were used as substrates for active staining. M, molecular size marker.

FIG 2

Changes in the optical purity of lactic acid during the fermentation process. Shown are the time courses of the optical purity values of l-lactic acid produced by B. coagulans 2-6 (a), d-lactic acid produced by L. delbrueckii subsp. bulgaricus DSM 20081 (b), d-lactic acid produced by L. plantarum subsp. plantarum DSM 20174 (c), and l-lactic acid produced by L. plantarum subsp. plantarum DSM 20174 (d).

FIG 3

Determination of the relative transcription levels of ldhD, ldhL, and the GOX gene by RT-PCR analyses. (a and b) Transcript ratios of ldhL to ldhD (a) and ldhL to the GOX gene (b) at different growth phases in B. coagulans 2-6. (c and d) Transcript ratios of ldhD to ldhL at different growth phases in L. delbrueckii subsp. bulgaricus DSM 20081 (c) and L. plantarum subsp. plantarum DSM 20174 (d). Error bars represent the standard deviations of the means for three independent experiments.