2.7. Enzyme activity assay
The activities of DAADHs were determined in
NH4Cl-NH3·H2O buffer
(200 mM, pH 9.5) with phenylpyruvate and NADPH. Enzyme activity was
measured by detecting the NADPH concentration at 340 nm by Infinite 200
PRO spectramicroplate reader (TECAN). The initial velocities was
calculated as consumption rate of NADPH when the conversion rate was
below 5%. The amount of enzyme consumed for generation or consumption
of 1 μmol of NADPH per minute was defined as one unit of enzyme
activity. Specific activity (U/mg) of DAADH is defined as the ratio of
enzyme activity (U/mL) to enzyme concentration (mg/mL). Each data was
obtained by five parallel experiments.
D-phenylalanine was analyzed by HPLC. HPLC analysis with standard
solution of L-phenylalanine and D-phenylalanine showed that the peak
time of L-phenylalanine was 25 min, and the peak time of D-phenylalanine
was in 33 min. HPLC detection condition for D/L phenylalanine was as
follow: Chromatographic column: Philomon Chirex 3126 (D)-Penicillamine
250 nm*4.6 nm, 5 μm; mobile phase, 5% (v/v) acetonitrile and 95% (v/v)
2.0 mM, CuSO4 aqueous solution; flow rate, 0.8 mL/min;
detection wavelength, 254 nm[22].