NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase.
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.
Department of Clinical Microbiology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand. mtvpr@mucc.mahidol.ac.th
Virapong
V
Prachayasittikul
Sarah
S
Ljung
Chartchalerm
C
Isarankura-Na-Ayudhya
Leif
L
Bülow
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005691:Galactose Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D009243:NAD / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011551:Pseudomonas fluorescens / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology*
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D005691:Galactose Dehydrogenases; D007770:L-Lactate Dehydrogenase; C047959:galactose dehydrogenase