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Site‐specifically Incorporated Non‐Canonical Amino Acids into Pseudomonas alcaligenes Lipase to Hydrolyze L‐menthol Propionate among the Eight Isomers

It remains a challenge to improve the diastereopreference of enzymes when there are multiple chiral centers in the substrate, mainly because the limited understanding of mechanism determining diastereoselectivity. Compared with natural amino acids, non‐canonical amino acids (ncAAs) provide side chains with wider range of functional groups and genetically encoded ncAAs have been applied in probing the complex enzyme mechanisms, improving catalytic activity, and even designing enzymes with new catalytic mechanisms. Here, the ncAAs were site‐specifically incorporated into a lipase (PaL) produced by Pseudomonas alcaligenes to explore its diastereopreference mechanism. Menthol propionate has three chiral centers, eight isomers in total. Molecular dynamics (MD) simulations were first applied to analyze the interactions between the active sites of PaL and the target substrate L‐menthol propionate. Furthermore, the four ncAAs (o‐bromophenylalanine, o‐chlorophenylalanine, p‐cyanophenylalanine and p‐aminophenylalanine) were substituted for 9 amino acids sites that potentially influenced three chiral centers and several variants with significant improvement in the diastereopreference were obtained. The diastereomer selectivity of beat variant at Ala253 was 100 % higher than that of the wild‐type. A linear relationship was found between volume, flexibility of the active center and diastereoselectivity.