Phenylpyruvate

Phenylpyruvate, phenylethanol and 2-ethylbutyric acid were purchased from Sigma-Aldrich (China). NADPH was obtained from Roche Products (China). The Bacterial DNA Kit and the Plasmid Mega Kit were bought from OMEGA. The Competent Cell Preparation Kit was from TaKaRa (Dalian, China). The other reagents were all analytical grades.

10 μL of enzymes (200 μM) were mixed with 90 μL of one of the twenty native L-amino acids (10 mM) with or without E. coli membrane (2 mg/mL). Reactions were incubated at 25 °C for 60 min, and then 100 μL trichloroacetic acid (20%) were added to stop the reactions. After that, 40 μL 2,4-dinitrophenylhydrazine (DNP, 20 mM) was added to produce the corresponding 2,4-dinitrophenylhydrazone derivatives. After incubating at 37 °C for 15 min, 800 μL of NaOH (0.8 M) was added to stop the reactions. Additional 5 min incubation was applied at room temperature. Finally, the reactions were transferred into the plastic cuvettes and absorbance at 520 nm were measured. The concentrations of α-keto acid were calculated according to the standard curve generated by different concentrations (25, 100, 250, 500, 1000 and 2000 μM) of phenylpyruvate (Sigma-Aldrich).