苯丙氨酸解氨酶的分子改造及合成L-邻氯苯丙氨酸的研究

Phenylalanine ammonia lyases (PALs) can catalyze the addition reaction between cinnamic acid and NH4OH. However, due to the high substrate inhibition, the ammonia addition reaction can only be achieved at low substrate concentration. In this study, a novel phenylalanine ammonia lyase IaPAL from Idiomarina abyssalis was obtained through genome mining. IaPAL exhibits high catalytic activity and thermostability. Scanning mutagenesis of IaPAL was conducted based on steric hindrance, hydrophobicity, electrically charged properties and hydrogen bonding properties in order to improve the specific activity and conversion ratios at high substrate concentrations. Mutagenesis revealed that Molecular engineering of phenylalanine ammonia lyase for efficient synthesis of L-o-chlorophenylalaninethe specific activity towards 2-chlorocinnamic acid of L196V was 15.2-fold higher than that of IaPALWT, and the conversion ratio of L196V (81.7%) was 4.3-fold higher than that of IaPALWT (18.8%) at 250 mmol/L 2-chlorocinnamic acid. The H88F mutant reshaped the specific activity of phenylalanine ammonia lyases towards natural substrates cinnamic acid and L-phenylalanine. This study provides interesting guidance for enzymatic synthesis of non-natural L-phenylalanine analogues.