Purification and characterization of thermostable H2O 2-forming NADH oxidase from 2-phenylethanol-assimilating Brevibacterium sp. KU1309

Jun Ichiro Hirano, Kenji Miyamoto, Hiromichi Ohta

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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°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.

Original languageEnglish
Pages (from-to)71-78
Number of pages8
JournalApplied Microbiology and Biotechnology
Volume80
Issue number1
DOIs
Publication statusPublished - 2008 Aug

Fingerprint

Brevibacterium
Phenylethyl Alcohol
Purification
Enzymes
NAD
Oxidation
Biocatalysts
Enzyme activity
NADH oxidase
Oxidoreductases
Recycling
Phenylalanine
NADP
Ammonium Compounds
Bacteria
Gels
Molecular weight
Salts
Carbon
Gel Chromatography

Keywords

  • Brevibacterium sp.
  • Cofactor regeneration
  • Hydrogen peroxide
  • Molecular oxygen
  • NADH oxidase

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology
  • Bioengineering
  • Microbiology (medical)

Cite this

Purification and characterization of thermostable H2O 2-forming NADH oxidase from 2-phenylethanol-assimilating Brevibacterium sp. KU1309. / Hirano, Jun Ichiro; Miyamoto, Kenji; Ohta, Hiromichi.

In: Applied Microbiology and Biotechnology, Vol. 80, No. 1, 08.2008, p. 71-78.

Research output: Contribution to journalArticle

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