[Catalytic synthesis of dihydroavenanthramide D by lipase RWL]

Dihydroavenanthramide D (DHAvD) is an aromatic amide with anti-inflammatory, antipruritic, and antioxidant properties. Its production currently relies on chemical synthesis. Lipases can catalyze the aminolysis of esters or acids to synthesize a series of amide compounds. However, the enzymatic synthesis of DHAvD is particularly challenging due to the ortho-substituted carboxyl group on its amine donor, 2-aminobenzoic acid (2-AA). Addressing this challenge, this study aims to identify a novel lipase capable of tolerating ortho-substituted anilines and to establish a suitable DHAvD-catalyzed synthetic system. Through genomic mining, this study successfully identified lipase RWL from Rhizorhabdus wittichii, which utilizes 2-AA as the amine donor and either methyl 3-(4-hydroxyphenyl)propionate (MHPP) or p-hydroxyphenylpropionic acid (PHBPA)as the acyl donor to synthesize DHAvD in aqueous phase. To enhance reaction efficiency, we established the optimal organic-phase reaction systems for both carboxylic acid and ester substrates: hexane for MHPP and methyl tert-butyl ether for PHPA. This switch from aqueous phase increased the DHAvD yield from 0.1% to 3.9%. Building on this foundation, molecular docking-guided alanine scanning and saturation mutagenesis were employed to modify the active pocket, resulting in the optimal mutant H245S with 1.55-fold enhanced synthetic activity raising the DHAvD yield to 6.1%. This study achieved the first enzymatic synthesis of DHAvD and characterized its enzymatic properties. The work not only lays a foundation for subsequent development but also provides theoretical and technical insights for research on other lipases.