Characterization and application of aminoamide-oxidizing enzyme from Aspergillus carbonarius AIU 205

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We isolated Aspergillus carbonarius AIU 205 as a new producer of an enzyme catalyzing oxidative deamination of 4-aminobutanamide (4-ABAD) to 4-oxobutanamide with the subsequent release of ammonia and hydrogen peroxide. Since the strain produced three enzymes with different Km values for 4-ABAD, the enzyme with lowest Km value (0.31 mM) was purified and revealed certain remarkable properties. The enzyme also oxidized aliphatic monoamines, aromatic amines and aliphatic aminoalcohols, but did not oxidize l-amino acids and aliphatic diamines. The Vmax/Km values for aliphatic monoamines were higher than that for 4-ABAD, and the enzyme activity was strongly inhibited by inhibitors of copper-containing amine oxidases. Thus, it was concluded that the enzyme might belong to a group of copper-containing amine oxidase. The 4-ABAD oxidase activity of this enzyme was optimum at pH 7.0, and the enzyme activity at pH 6.0 was 65% of that at pH 7.0. The enzyme was useful for increasing the sensitivity of l-lysine assay using l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813.

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Chemicals

l-Lysine, l-ornithine, aliphatic amines and diamines were purchased from Wako Pure Chemicals (Osaka, Japan). 4-ABAD and plasma amine oxidase were from Toronto Research Chemicals (Toronto, Canada) and Worthington Biochemical Co. (Lakewood, NJ, USA), respectively. Peroxidase was gift from Amano Enzyme (Nagoya, Japan). All other chemicals used were of analytical grade and commercially available.

Screening of microorganism

Fungal strains were incubated in a 500-ml shaker flask containing 100 ml of a n-butylamine medium, pH

Screening of microorganisms

We isolated 12 fungal strains as producers of an enzyme exhibiting oxidase activity for 4-ABAD. The crude enzyme solution from each isolated strain also exhibited oxidase activity for n-butylamine (Table 1). Since it was known that amine oxidase from Aspergillus niger AKU 3302 catalyzed oxidative deamination of aliphatic monoamines, aromatic monoamines and aliphatic diamines 4, 5, we purified the amine oxidase, and assayed the 4-ABAD oxidase activity. The amine oxidase also oxidized 4-ABAD, but

Discussion

Here, we demonstrated a new result that the copper-containing amine oxidase from A. niger AKU 3302 oxidized 4-ABAD as well as amines. However, the amine oxidase was not applicable for l-lysine assay coupling with l-AAO/MOG from Pseudomonas sp. AIU 813, because the Km value for 4-ABAD was high. Since, enzymes with low Km values are generally suitable for coupling enzymes, we selected A. carbonarius AIU 205 as a new producer of enzymes exhibiting oxidase activity for 4-ABAD, and revealed certain

Acknowledgment

This work was financially supported by the Hokuriku Innovation Cluster for Health Science, Ministry of Education, Culture, Sports, Science, and Technology.

References (11)

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    The N-terminal sequence of intact enzyme III was found to be APHPLAILSEEETNLARDVV, which was similar to those of copper-containing amine oxidase from A. oryzae (4), an copper-containing amine oxidase I from A. niger AKU 3302 (2), a putative copper amine oxidase from A. kawachii IFO 4308 (8), and a putative copper amine oxidase from Asperillus nidulans (11) (Fig. 4B). We have reported that A. carbonarius AIU 205 inductively produced three enzymes in the mycelia that catalyze the oxidative deamination of 4-ABAD and release hydrogen peroxide and ammonia (5). In these three enzymes, the characteristics of enzyme I have been revealed, but those of enzymes II and III have not.

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