Zhang_2026_Int.J.Biol.Macromol_350_150986

Although numerous hydrolases have shown promiscuous acyltransferase activity in aqueous media, none have been reported to efficiently synthesize feruloyl esters. Here, we demonstrate that a ferulic acid esterase (AmCE1) from Anaeromyces mucronatus functions as a promiscuous hydrolase/acyltransferase, exhibiting unprecedented acyltransferase activity in water by efficiently catalyzing the transfer of bulky aromatic acyl donors and acceptors. This function was rapidly identified using a novel precipitation-based screening method, the Ferulate-acyltransferase (Ferulate-AcT) assay. We subsequently characterized key enzymatic properties of AmCE1, including its pH and temperature profiles, as well as its dependence on substrate and enzyme concentration. Under optimized conditions (35 degreesC, pH 8.5, 240 mM methyl ferulate (MFA), 810 mM phenethyl alcohol), the wild type AmCE1 (WT) achieved up to 34.7% conversion within 4 h. Rational engineering yielded the mutant Y68T, which showed 49.0% conversion under the same conditions, a 40% improvement over the WT. The enzyme displays strict specificity for phenylpropenoid ester scaffolds, particularly methyl ferulate, coupled with broad acceptor promiscuity that includes regioselective acylation of glycosides. We further illustrate its application in the one-step, aqueous-phase synthesis of phenethyl ferulate, a high-value anti-inflammatory natural product. This study establishes a sustainable and efficient enzymatic platform for the industrial production of cinnamate derivatives.