反应详情

The substrate specificity of the purified amino acid dehydrogenase was investigated. The enzyme activity was measured by using each substrate in place of the phenylpyruvic acid substrate in the procedure previously described for measuring the enzyme activity. In the case of the enzyme activity for naphthylpyruvic acid, 100 μL of the appropriately diluted enzyme solution was added to 100 μL of 0.1 M sodium carbonate buffer (pH 9.0) that contained 1.0 mg naphthylpyruvic acid, 12.3 mg ammonium sulfate, and 6.1 mg NADH and the increment in the 2-naphthylalanine produced in 30 minutes at 30° C. was determined by quantitation by HPLC. The HPLC analysis was run using the following: column: COSMOSIL 5C18-ARII (4.6 mm×250 mm, Nacalai Tesque, Inc.), eluent: 10 mM potassium phosphate buffer (pH 2.0)/acetonitrile=5/1, flow rate: 1.0 mL/min, column temperature: 40° C., detection: 210 nm. Here, 1 unit is defined as the quantity of enzyme that produces 1 μmol 2-naphthylalanine in 1 minute. In the case of the enzyme activity for indolepyruvic acid, 100 μL of the appropriately diluted enzyme solution was added to 100 μL of 0.1 M sodium carbonate buffer (pH 9.0) that contained 2.0 mg indolepyruvic acid, 26.0 mg ammonium sulfate, and 7.6 mg NADH and the increment in the L-tryptophan produced in 30 minutes at 30° C. was determined by quantitation by HPLC. The HPLC analysis was run using the following: column: CROWNPAK CR—(4.6 mm×150 mm, Daicel Chemical Industries, Ltd.), eluent: aqueous HClO4 (pH 2.0), flow rate: 1.0 mL/min, column temperature: 30° C., detection: 210 nm. Here, 1 unit is defined as the quantity of enzyme that produces 1 μmol L-tryptophan in 1 minute. The relative activity values, assigning 100% to the activity for phenylpyruvic acid, are shown in Table 1.