A novel amine oxidase-encoding gene from Aspergillus oryzae

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Amine oxidases (AOXs) are crucial enzymes involved in amine metabolism.
• Understanding AOX diversity and function in fungi like Aspergillus oryzae is important for biotechnological applications.

Purpose of the Study:

• To clone and characterize a novel amine oxidase gene (aoxA) from Aspergillus oryzae.
• To investigate the functional expression and enzymatic properties of the cloned AOX.

Main Methods:

• Gene cloning from an expressed sequence tag (EST) library.
• Molecular analysis including sequence homology and identification of conserved residues.
• Homologous hyperexpression in Aspergillus oryzae.
• Enzyme purification and characterization (gel filtration, SDS-PAGE).
• Substrate specificity assays.

Main Results:

• A novel gene, aoxA, encoding a 672-amino acid amine oxidase was identified and cloned.
• The deduced amino acid sequence showed 83.5% identity to Aspergillus niger AO-I, with conserved residues for cofactor and copper binding.
• Overexpression of aoxA in A. oryzae resulted in a 75-fold increase in AOX activity.
• The purified enzyme is a homo-dimer with a subunit molecular weight of 75,000 Da.
• The enzyme preferentially oxidized aliphatic monoamines (C2-C6).

Conclusions:

• The cloned aoxA gene encodes a functional amine oxidase in Aspergillus oryzae.
• The homologous hyperexpression system effectively enhanced AOX activity.
• The characterized enzyme exhibits properties consistent with copper/quinine-dependent amine oxidases.