Kinetic characterisation of recombinant Corynebacterium glutamicum NAD+-dependent LDH over-expressed in E. coli and its rescue of an lldD- phenotype in C. glutamicum: the issue of reversibility re-examined

The ldh gene of Corynebacterium glutamicum ATCC 13032 (gene symbol cg3219, encoding a 314 residue NAD⁺-dependent l-(+)-lactate dehydrogenase, EC 1.1.1.27) was cloned into the expression vector pKK388-1 and over-expressed in an ldhA-null E. coli TG1 strain upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The recombinant protein (referred to here as CgLDH) was purified by a combination of dye-ligand and ion-exchange chromatography. Though active in its absence, CgLDH activity is enhanced 17- to 20-fold in the presence of the allosteric activator d-fructose-1,6-bisphosphate (Fru-1,6-P₂). Contrary to a previous report, CgLDH has readily measurable reaction rates in both directions, with V _max for the reduction of pyruvate being approximately tenfold that of the value for l-lactate oxidation at pH 7.5. No deviation from Michaelis–Menten kinetics was observed in the presence of Fru-1,6-P₂, while a sigmoidal response (indicative of positive cooperativity) was seen towards l-lactate without Fru-1,6-P₂. Strikingly, when introduced into an lldD ⁻ strain of C. glutamicum, constitutively expressed CgLDH enables the organism to grow on l-lactate as the sole carbon source.