Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus

Enzyme Microb Technol. 2012 Apr 5;50(4-5):255-62. doi: 10.1016/j.enzmictec.2012.01.009. Epub 2012 Feb 4.


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 ~50 kDa 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 °C, and k(cat)/K(m) of 3.77×10(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 °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 H(2)O-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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biotechnology / methods
  • Cloning, Molecular*
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics
  • Hot Temperature
  • Kinetics
  • Lacticaseibacillus rhamnosus / enzymology*
  • Lacticaseibacillus rhamnosus / genetics
  • Models, Molecular
  • Molecular Sequence Data
  • Multienzyme Complexes* / chemistry
  • Multienzyme Complexes* / genetics
  • Multienzyme Complexes* / metabolism
  • NAD / metabolism
  • NADH, NADPH Oxidoreductases* / chemistry
  • NADH, NADPH Oxidoreductases* / genetics
  • NADH, NADPH Oxidoreductases* / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Analysis, DNA
  • Water / metabolism


  • Multienzyme Complexes
  • Recombinant Proteins
  • Water
  • NAD
  • NADH oxidase
  • NADH, NADPH Oxidoreductases