NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase

Virapong Prachayasittikul, Sarah Ljung, Chartchalerm Isarankura-Na-Ayudhya, Leif Bülow

Research output: Contribution to journalArticlepeer-review

15 Citations (SciVal)

Abstract

A chimeric bifunctional enzyme composing of galactose dehydrogenase (galDH; from Pseudomonas fluorescens) and lactate dehydrogenase (LDH; from Bacillus stearothermophilus) was successfully constructed. The chimeric galDH/LDH possessed dual characteristics of both galactose dehydrogenase and lactate dehydrogenase activities while exhibiting hexameric rearrangement with a molecular weight of approximately 400 kDa. In vitro observations showed that the chimeric enzyme was able to recycle NAD with a continuous production of lactate without any externally added NADH. Two fold higher recycling rate (0.3 mM/h) than that of the native enzyme was observed at pH values above 8.5. Proximity effects became especially pronounced during the recycling assay when diffusion hindrance was induced by polyethylene glycol. All these findings open up a high feasibility to apply the NAD(H) recycling system for metabolic engineering purposes e.g. as a model to gain a better understanding on the molecular proximity process and as the routes for synthesizing of numerous high-value-added compounds.

Original languageEnglish
Pages (from-to)10-6
Number of pages7
JournalInternational Journal of Biological Sciences
Volume2
Issue number1
DOIs
Publication statusPublished - 2006

Subject classification (UKÄ)

  • Biochemistry and Molecular Biology

Keywords

  • Bacterial Proteins
  • Enzyme Stability
  • Escherichia coli
  • Galactose Dehydrogenases
  • Geobacillus stearothermophilus
  • Hydrogen-Ion Concentration
  • L-Lactate Dehydrogenase
  • NAD
  • Protein Engineering
  • Pseudomonas fluorescens
  • Recombinant Fusion Proteins

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