Cloning, Expression And Characterization Of L-Lactate Dehydrogenase In Lactobacillus Casei

L-Lactate Dehydrogenase (L-LDH) is a key enzyme which catalyzes the formation of L-lactate from pyruvate in the Lactobacillus SP. The gene ldhL encoding L-LDH was amplified from genome DNA of Lactobacillus casei using PCR technique. The PCR product was cloned into pUcm-T vector and double digested with restriction endonucleases, and then the DNA fragment of ldhL was inserted into pET-28a(+). The recombinants plasmid pET-ldhL was obtained, and was transformed into E.coli BL21. After it was induced to express L-LDH with IPTG, and purified by affinity chromatography. SDS-PAGE showed that the molecular weight of specific fusion protein was 39 kD. The specific activity were up to 1722 U/mg, the purification multiple is 2.62, the recovery is 59%.The biochemical properties of L-LDH showed that the optimum catalysis temperature of 40℃-45℃and pH of 6.6-6.8. Fructose-1, 6-bisphosphate (FBP) is a positive allosteric modifier of the enzyme, and addition of Mn²⁺ to the assay in the presence of FBP broadens the pH profile, particularly towards neutral pH values. The optimal NADH concentration was 0.174 mmol/L, and the optimal pyruvate concentration was 20 mmol/L. The kinetics curve of substrate [S] to v was sigmoidal. After activator was added, the curve becomes hyperbolic. Mn²⁺, Ca²⁺ and Mg²⁺ increased the activity of L-LDH, but Zn²⁺ decreased its activity. When lactic acid concentration was less than 0.22 mol/L in the reaction system, the enzyme activity kept high. It was dropped significantly when the lactate concentration was higher than 0.44 mol/L.The recombinant plasmid pMG-ldhL was constructed on the base of ldhL gene and E. coli-Lactobacillus shuttle vector pMG36e. After electroporation conditions were optimized, it was transferred into strain L.casei G-02 and the recombinant bacterium was identificated. The target protein was expressed, and there was apparent band at about 37 kD. But the amount of protein was very low in the recombinant strain.The fermentation of recombinant strain was studied in shake flask. It was found that the maximum enzyme activity of Lactate Dehydrogenase was 23 U/mL in the initial stain, while the maximum L-LDH activity was 95 U/mL in the recombinant strain L. casei (pMG-ldhL), which has improved 4-fold higher than that of the initial strain, but there was little change in the amount of L-lactic acid. The activator FBP(4 mmol/L), Mn²⁺(3 mmol/L), nicotinic acid (8 mg/L) were added in fermentation medium, and the results showed that the accumulation of lactic acid was 107 g/L, the conversion rate was 91%, and the lactic acid productivity was 2.67 g/(L·h), which were increased by 14%, 11%, 36%, respectively. The consumption rate of glucose was increased significantly, which was almost exhausted after 40 h fermentation. Hence the fermentation was terminated earlier, and the fermentation time was shortened by 8 h.