Data-Driven Discovery of a Novel Cutinase OryEst1 for Efficient Ester Synthesis under Aqueous Phase and Enhancement of Activity through Channel Engineering

Fatty acid esters are usually synthesized in the organic phase, and the presence of water drives the reaction toward hydrolysis, limiting it as a green solvent for esterification. This study identified a novel cutinase OryEst1 from <i>Aspergillus oryzae</i> using a data-driven strategy. It exhibited a broad substrate spectrum and great stability and efficiently catalyzed the synthesis of ethyl octanoate (con. 49.6%) at pH 5.0 and 30 °C in the aqueous phase. The catalytic triad Ser170-His235-Asp222 was stabilized by the π stacking formed between Tyr136 and Tyr169. Channel engineering broke the substrate transport bottleneck, raising ethyl octanoate conversion to 64.5% of mutant S95A. Molecular dynamics revealed that the mutation broke hydrogen bonds between the channel-gating site and lid, enhancing structural flexibility to accelerate the transportation of the substrate and product. Hydrophobic interaction in S95A anchors and orients the substrate into an active site, reducing ineffective collisions and boosting activity. The findings provide an important guide for advancing green catalysts for ester synthesis.