Sodium ions activated phosphofructokinase leading to enhanced D-lactic acid production by Sporolactobacillus inulinus using sodium hydroxide as a neutralizing agent

Abstract

Sporolactobacillus inulinus is a superior D-lactic acid-producing bacterium and proposed species for industrial production. The major pathway for D-lactic acid biosynthesis, glycolysis, is mainly regulated via the two irreversible steps catalyzed by the allosteric enzymes, phosphofructokinase (PFK) and pyruvate kinase. The activity level of PFK was significantly consistent with the cell growth and D-lactic acid production, indicating its vital role in control and regulation of glycolysis. In this study, the ATP-dependent PFK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. The PFK was allosterically activated by both GDP and ADP and inhibited by phosphoenolpyruvate; the addition of activators could partly relieve the inhibition by phosphoenolpyruvate. Furthermore, monovalent cations could enhance the activity, and Na⁺ was the most efficient one. Considering this kind activation, NaOH was investigated as the neutralizer instead of the traditional neutralizer CaCO₃. In the early growth stage, the significant accelerated glucose consumption was achieved in the NaOH case probably for the enhanced activity of Na⁺-activated PFK. Using NaOH as the neutralizer at pH 6.5, the fermentation time was greatly shortened about 22 h; simultaneously, the glucose consumption rate and the D-lactic acid productivity were increased by 34 and 17%, respectively. This probably contributed to the increased pH and Na⁺-promoted activity of PFK. Thus, fermentations by S. inulinus using the NaOH neutralizer provide a green and highly efficient D-lactic acid production with easy subsequent purification.

Keywords: Allosteric regulation; D-Lactic acid production; Neutralizer; Phosphofructokinase; Sodium ions; Sporolactobacillus inulinus.