反应详情

The standard PAM activity assay mixture contains 5 mM L-α-phenylalanine, 1 to 10 μg enzyme in 50 mM Tris-HCl buffer (pH 7.5) in a final volume of 0.5 ml. Enzyme reactions were carried out at 28° C., 150 rpm for 18 hours and terminated by addition of 50 μl ethanol to precipitate proteins. After centrifugation for 5 minutes, the supernatants were analyzed by a reverse phase HPLC method. The formation of β-phenylalanine was quantified based on peak area compared to the β-phenylalanine standard curve. Enzyme activity is expressed as μg β-phenylalanine formed per hour per mg protein. HPLC analysis used a YMC Ph column (4.6×150 mm; Waters, Milford, Mass.), and a flow rate of 1.2 ml/minutes. Absorbance was monitored at 210 nm. The method employed two solvents A, water; and B, CH3CN and a gradient consisting 0% to 12% B for 6 minutes, 12% to 90% in 1 minute and returning to 0% in 1 minute. Retention times of L-α-phenylalanine and β-phenylalanine are 4.3 minutes and 3.5 minutes, respectively.

1. customwere carried out at 28° C.
2. customterminated by addition of 50 μl ethanol
3. customto precipitate proteins
4. waitAfter centrifugation for 5 minutes
5. customformed per hour per mg protein
6. customfor 6 minutes
7. wait12% to 90% in 1 minute
8. waitreturning to 0% in 1 minute