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

Prachayasittikul, Virapong; Ljung, Sarah; Isarankura-Na-Ayudhya, Chartchalerm and Bülow, Leif LU (2006) In International Journal of Biological Sciences 2(1). p.6-10
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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
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
in
International Journal of Biological Sciences
volume
2
issue
1
pages
7 pages
publisher
Ivyspring International Publisher
external identifiers
  • scopus:33646535853
ISSN
1449-2288
DOI
10.7150/ijbs.2.10
language
English
LU publication?
yes
id
aafefe18-d01e-4f62-aa08-4748cc76fcbb
date added to LUP
2016-04-18 15:53:18
date last changed
2017-01-01 08:23:11
@article{aafefe18-d01e-4f62-aa08-4748cc76fcbb,
  abstract     = {<p>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.</p>},
  author       = {Prachayasittikul, Virapong and Ljung, Sarah and Isarankura-Na-Ayudhya, Chartchalerm and Bülow, Leif},
  issn         = {1449-2288},
  keyword      = {Bacterial Proteins,Enzyme Stability,Escherichia coli,Galactose Dehydrogenases,Geobacillus stearothermophilus,Hydrogen-Ion Concentration,L-Lactate Dehydrogenase,NAD,Protein Engineering,Pseudomonas fluorescens,Recombinant Fusion Proteins},
  language     = {eng},
  number       = {1},
  pages        = {6--10},
  publisher    = {Ivyspring International Publisher},
  series       = {International Journal of Biological Sciences},
  title        = {NAD(H) recycling activity of an engineered bifunctional enzyme galactose dehydrogenase/lactate dehydrogenase},
  url          = {http://dx.doi.org/10.7150/ijbs.2.10},
  volume       = {2},
  year         = {2006},
}