Purification and Characterization of Thermostable H2O2-forming NADH Oxidase From 2-phenylethanol-assimilating Brevibacterium Sp. KU1309

Appl Microbiol Biotechnol. 2008 Aug;80(1):71-8. doi: 10.1007/s00253-008-1535-x. Epub 2008 Jun 3.


A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification*
  • Bacterial Proteins / metabolism
  • Brevibacterium / chemistry
  • Brevibacterium / enzymology*
  • Brevibacterium / genetics
  • Brevibacterium / metabolism
  • Cholesterol / metabolism
  • Enzyme Stability
  • Hot Temperature
  • Hydrogen Peroxide / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Multienzyme Complexes / chemistry*
  • Multienzyme Complexes / genetics
  • Multienzyme Complexes / isolation & purification*
  • Multienzyme Complexes / metabolism
  • NADH, NADPH Oxidoreductases / chemistry*
  • NADH, NADPH Oxidoreductases / genetics
  • NADH, NADPH Oxidoreductases / isolation & purification*
  • NADH, NADPH Oxidoreductases / metabolism
  • Oxidation-Reduction
  • Phenylethyl Alcohol / metabolism*
  • Sequence Alignment
  • Substrate Specificity


  • Bacterial Proteins
  • Multienzyme Complexes
  • Cholesterol
  • Hydrogen Peroxide
  • NADH oxidase
  • NADH, NADPH Oxidoreductases
  • Phenylethyl Alcohol