Overexpression and Characterization of L-Lactate Dehydrogenase from Lacticaseibacillus casei HY2782 to Control Post-Acidification in Yogurt

Post-acidification is a common phenomenon in fermented milk that can cause adverse effects, in which lactic acid bacteria used as starter cultures continue the lactic acid fermentation during storage. Lactate dehydrogenase (LDH) is a key enzyme for lactic acid fermentation. Therefore, the control of LDH could be one of the solutions to prevent post-acidification. In the present study, four genes (<i>ldhL</i>1, <i>ldhL</i>2, <i>ldhL</i>3, and <i>ldhL</i>4) encoding _L-LDH were identified by whole genome sequencing of the <i>Lacticaseibacillus casei</i> HY2782, cloned into expression vector, pET22b (+), transformed into <i>Escherichia</i> <i>coli</i> BL21 (DE3), and expressed by IPTG induction. Four recombinant _L-LDHs were purified using Ni-NTA agarose column and characterized. The molecular weights of purified _L-LDHs were 30-37 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Among the four _L-LDHs, _L-LDH3 exhibited the highest enzyme activity. _L-LDH3 exhibited maximal activity of 302,343.16 U/mg at 43°C and pH 4.0 in the presence of 8 mM fructose 1,6-bisphosphate. In acidic condition, _L-LDH3 was activated by 2 mM of Mg²⁺, Ca²⁺ and Mn²⁺ ions, inhibited by Zn²⁺ and Cu²⁺ ions. In addition, activity of _L-LDH3 was gradually decreased as KH₂PO₄ concentration increased. Consequently, the _L-LDH3 seems to be the major enzyme among the _L-LDHs of <i>L. casei</i> HY2782 and may play an important role in lactic acid fermentation of this strain. Therefore, the inhibition of _L-LDH3 activity using Zn²⁺, Cu²⁺and KH₂PO₄ could be one of the solutions to mitigate the post-acidification caused by <i>L. casei</i> HY2782.