Diamonsil c18 2

The cultured ApsA-expressing cells were harvested by centrifugation at 12,000 rpm for 3 min. The pellets were washed three times with PBS and then resuspended in 50 mM Tris-HCl (pH 8.0) at an OD₆₀₀ of 3.0. The enzyme-catalyzed reaction solution was 1 mL, including 500 μL substrate of 200 mM fumaric acid (dissolved in ammonium hydroxide solution, with the pH adjusted to 8.0 with HCl) and 500 μL cell suspension. The reaction was carried out in a metal bath at 37°C and terminated by thermal inactivation at 100°C for 10 min. The product l-aspartate was derivatized with phenyl isothiocyanate, and the concentration was determined using a C₁₈ column [Diamonsil C₁₈(2), 5 μm, 250 by 4.6 mm; DIKMA, China] by high-performance liquid chromatography (HPLC) as described previously (53 (link)). One unit of AspA activity was defined as the amount of enzyme needed for the production of 1 μmol of l-aspartate per minute.

The cultured ApsA-expressing cells were harvested by centrifugation at 12,000 rpm for 3 min. The pellets were washed three times with PBS and then resuspended in 50 mM Tris-HCl (pH 8.0) at an OD₆₀₀ of 3.0. The enzyme-catalyzed reaction solution was 1 mL, including 500 μL substrate of 200 mM fumaric acid (dissolved in ammonium hydroxide solution, with the pH adjusted to 8.0 with HCl) and 500 μL cell suspension. The reaction was carried out in a metal bath at 37°C and terminated by thermal inactivation at 100°C for 10 min. The product l-aspartate was derivatized with phenyl isothiocyanate, and the concentration was determined using a C₁₈ column [Diamonsil C₁₈(2), 5 μm, 250 by 4.6 mm; DIKMA, China] by high-performance liquid chromatography (HPLC) as described previously (53 (link)). One unit of AspA activity was defined as the amount of enzyme needed for the production of 1 μmol of l-aspartate per minute.