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. - Archive ouverte HAL Accéder directement au contenu
Article Dans Une Revue Archives of Microbiology Année : 2011

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.

Michael A Sharkey
  • Fonction : Auteur
Marcus A Maher
  • Fonction : Auteur
Paul C Engel
  • Fonction : Auteur

Résumé

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 Vmax 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.

Dates et versions

hal-00639213 , version 1 (08-11-2011)

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Citer

Michael A Sharkey, Marcus A Maher, Armel Guyonvarch, Paul C Engel. 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.. Archives of Microbiology, 2011, 193 (10), pp.731-40. ⟨10.1007/s00203-011-0711-z⟩. ⟨hal-00639213⟩
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