Overexpression, purification and properties of ldhL gene from Lactobacillus casei in Escherichia coli

Abstract:

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 expression 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 40 kD. The biochemical properties of L-LDH showed that the specific activity were up to 1 722 U/mg with optimum catalysis temperature of 40 °C?45 °C and pH of 6.6?6.8. Fructose-1,6-bisphosphate (FBP) is a positive allosteric modifier of the enzyme, the addition of Mn2+ to the assay in the presence of FBP broadens the pH profile, particularly towards neutral pH values. Mn2+, Ca2+ and Mg2+ increases the activity of L-LDH, but Zn2+ decreases its activity.