Syncephalastrum racemosum amine oxidase with high catalytic efficiency toward ethanolamine and its application in ethanolamine determination

Yoshitaka Hirano, Keisuke Chonan, Kazutaka Murayama, Shin ich Sakasegawa, Hideyuki Matsumoto, Daisuke Sugimori

Research output: Contribution to journalArticlepeer-review

2 Citations (Scopus)

Abstract

Our screening study yielded a copper amine oxidase (SrAOX) from Syncephalastrum racemosum, which showed much higher affinity and catalytic efficiency toward ethanolamine (EA) than any other amine oxidase (AOX). Following purification of the enzyme to electrophoretic homogeneity from a cell-free extract, the maximum activity toward EA was detected at pH 7.2–7.5 and 45 °C. The SrAOX complementary DNA (cDNA) was composed of a 2052-bp open reading frame encoding a 683-amino acid protein with a molecular mass of 77,162 Da. The enzyme functions as a homodimer. The deduced amino acid sequence of SrAOX showed 55.3 % identity to Rhizopus delemar AOX and contains two consensus sequences of Cu-AOX, NYDY, and HHQH, suggesting SrAOX is a type 1 Cu-AOX (i.e., a topaquinone enzyme). Structural homology modeling showed that residues 112ML113, 141FADTWG146 M158, and N318 are unique, and T144 possibly characterizes the substrate specificity of SrAOX. The recombinant enzyme (rSrAOX) was produced using Escherichia coli. Steady-state kinetic analysis of rSrAOX activity toward EA (pH 7.5 and 45 °C) gave Km and kcat values of 0.848 ± 0.009 mM and 9.11 ± 0.13 s−1, respectively. The standard curves were linear between 0.1 and 2 mM EA, and 10 μg mL−1–2.5 mg mL−1 (15 μM–3.6 mM) phosphatidylethanolamine using Streptomyces chromofuscus phospholipase D, respectively, was sufficiently sensitive for clinical use.

Original languageEnglish
Pages (from-to)3999-4013
Number of pages15
JournalApplied Microbiology and Biotechnology
Volume100
Issue number9
DOIs
Publication statusPublished - 2016 May 1

Keywords

  • Characterization
  • Cloning
  • Cu amine oxidase
  • Enzymatic determination of ethanolamine
  • Heterologous expression
  • Syncephalastrum racemosum

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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