| Summary: | Phenylalanine ammonia-lyases (PALs) are attractive biocatalysts for the stereoselective synthesis of non-natural phenylalanines. The rational design of PALs with extended substrate scope, highlighted the substrate specificity-modulator role of residue I460 of <i>Petroselinum crispum</i> PAL. Herein, saturation mutagenesis at key residue I460 was performed in order to identify <i>Pc</i>PAL variants of enhanced activity or to validate the superior catalytic properties of the rationally explored I460V <i>Pc</i>PAL compared with the other possible mutant variants. After optimizations, the saturation mutagenesis employing the NNK-degeneracy generated a high-quality transformant library. For high-throughput enzyme-activity screens of the mutant library, a PAL-activity assay was developed, allowing the identification of hits showing activity in the reaction of non-natural substrate, <i>p</i>-MeO-phenylalanine. Among the hits, besides the known I460V <i>Pc</i>PAL, several mutants were identified, and their increased catalytic efficiency was confirmed by biotransformations using whole-cells or purified PAL-biocatalysts. Variants I460T and I460S were superior to I460V-<i>Pc</i>PAL in terms of catalytic efficiency within the reaction of <i>p</i>-MeO-Phe. Moreover, I460T <i>Pc</i>PAL maintained the high specificity constant of the <i>wild-type</i> enzyme for the natural substrate, <span style="font-variant: small-caps;">l</span>-Phe. Molecular docking supported the favorable substrate orientation of <i>p</i>-MeO-cinnamic acid within the active site of I460T variant, similarly as shown earlier for I460V <i>Pc</i>PAL (PDB ID: 6RGS).
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