Characterization and application of a l-specific amino acid oxidase from Rhodococcus sp. AIU LAB-3

An l-specific amino acid oxidase (l-AAO) suitable for assay of N-acyl-l-amino acid amidohydrolase (l-aminoacylase) activity was purified from Rhodococcus sp. AIU LAB-3. The enzyme exhibited broad substrate specificity and catalyzed an oxidative deamination of the α-amino group of l-amino acids. The optimal enzyme activities for l-amino acids tested were observed in the pH range from 6.0 to 8.5, and more than 80% of the maximum activity was obtained at pH 7.5. The enzyme was stable in the pH range from 7.0 to 8.5, and the apparent Km values for those l-amino acids were small. We, therefore, developed a new enzymatic method for assay of l-aminoacylase activity using the l-AAO at pH 7.5. The new enzymatic method had advantages that the l-aminoacylase reaction was spectrophotometrically followed by measuring absorbance at 555 nm. The l-aminoacylase activity was assayed within 10 min using a small reaction volume. Thus, the new enzymatic method was simple and sensitive compared to the ninhydrin method.