Abstract
NADH oxidase (Nox) catalyzes the conversion of NADH to NAD(+). A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 453 amino acid residues. The molecular mass of the purified LrNox enzyme was estimated to be similar to 50kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 100 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had optimal activity at pH 5.6 and temperature 65 degrees C, and k(cat)/K-m of 3.77 x 10(7) s(-1) M-1, the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 degrees C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of LrNox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H2O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications such as NAD(+) regeneration. (C) 2012 Elsevier Inc. All rights reserved.
| Original language | English |
|---|---|
| Journal | Enzyme and Microbial Technology |
| Volume | 50 |
| Issue number | 4-5 |
| Pages (from-to) | 255-262 |
| ISSN | 0141-0229 |
| DOIs | |
| Publication status | Published - 2012 |
| Externally published | Yes |
Keywords
- BIOTECHNOLOGY
- XYLITOL DEHYDROGENASE
- COFACTOR REGENERATION
- STREPTOCOCCUS-MUTANS
- LEUCONOSTOC-MESENTEROIDES
- MOLECULAR-CLONING
- PURIFICATION
- REDUCTASE
- PROTEIN
- OXYGEN
- SANFRANCISCENSIS
- Characterization
- Cofactor regeneration
- H2O-forming NADH oxidase
- Lactobacillus rhamnosus
- Thermal stability
- enzyme activity
- thermostability
- Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Regular Nonsporing Gram-Positive Rods [07830] Lactobacillus rhamnosus species
- Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli species expression system strain-BL21(DE3)
- Lactobacillus rhamnosus Nox gene [Regular Nonsporing Gram-Positive Rods] open reading frame
- NAD+
- NADH oxidase 9032-21-7 EC 1.6.99.3 homoclimer
- polypeptide amino acid residue
- 03502, Genetics - General
- 10802, Enzymes - General and comparative studies: coenzymes
- 31000, Physiology and biochemistry of bacteria
- 31500, Genetics of bacteria and viruses
- 39008, Food microbiology - General and miscellaneous
- Biochemistry and Molecular Biophysics
- cloning laboratory techniques, genetic techniques
- gel filtration chromatography laboratory techniques, chromatographic techniques
- heat inactivation laboratory techniques
- molecular dynamics simulation mathematical and computer techniques
- sequence analysis laboratory techniques, genetic techniques
- sodium dodecyl sulfate-polyacrylamide gel electrophoresis electrophoretic techniques, laboratory techniques
- Bioprocess Engineering
- Enzymology
- Molecular Genetics
- Amino Acid Sequence
- Biotechnology
- Cloning, Molecular
- Electrophoresis, Polyacrylamide Gel
- Enzyme Stability
- Escherichia coli
- Hot Temperature
- Kinetics
- Models, Molecular
- Molecular Sequence Data
- Multienzyme Complexes
- NAD
- NADH, NADPH Oxidoreductases
- Recombinant Proteins
- Sequence Analysis, DNA
- Water
- 059QF0KO0R Water
- 0U46U6E8UK NAD
- EC 1.6.- NADH oxidase
- EC 1.6.- NADH, NADPH Oxidoreductases
- Amino acids
- Bacilli
- Cloning
- Electrophoresis
- Gel permeation chromatography
- Genes
- Industrial applications
- Molecular dynamics
- Sodium
- Sodium sulfate
- Thermodynamic stability
- Enzyme activity
- amino acid
- dodecyl sulfate sodium
- homodimer
- metal ion
- polypeptide
- reduced nicotinamide adenine dinucleotide dehydrogenase
- water
- article
- bacterial gene
- catalysis
- cloning
- concentration response
- controlled study
- enzyme analysis
- enzyme inactivation
- enzyme kinetics
- enzyme metabolism
- enzyme purification
- enzyme stability
- gel filtration chromatography
- gene overexpression
- genetic code
- heat
- molecular docking
- molecular dynamics
- molecular weight
- nonhuman
- open reading frame
- pH
- physical chemistry
- polyacrylamide gel electrophoresis
- protein quaternary structure
- sequence analysis
- site directed mutagenesis
- temperature
- Amino acid residues
- Gel-filtration chromatography
- Heat inactivation
- High activity
- Homodimers
- Molecular dynamics simulations
- NADH oxidase
- Open reading frame
- Optimal activity
- Sequence analysis
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- H 2O-forming NADH oxidase