Improved Substrate Specificity of Fatty Acid Hydratases Enables Efficient and Selective Linoleic Acid Isolation

Isolation of linoleic acid (LA, C18:2) from oleic acid (OA, C18:1) remains challenging due to their structure and property similarity. Conventional methods such as molecular distillation and silver-ion chromatography face energy inefficiency or complexity limitations. Enzymatic hydration using fatty acid hydratases (FAHs) selectively converts OA to hydroxy fatty acid (HFA) in favor of further isolation. However, natural FAHs show inadequate OA specificity. Herein, an FAH from Bifidobacterium breve (BbMCRA2) was engineered by optimizing its substrate tunnel geometry through semirational design. Mutants I206 V and I202 V/I209 V enhanced OA selectivity by 4.7- and 5.5-fold. Following optimization of the OA/LA reaction kinetics, LA isolation achieved 94.3% purity and 81.7% recovery in a 5 L system. Compared with traditional LA isolation strategies, the combined enzymatic hydration process simultaneously reduced both energy consumption and processing time. This strategy, by improving the substrate specificity of HFAs, provides a green and efficient approach for industrial fatty acid purification, enabling selective linoleic acid isolation with superior cost-effectiveness compared with conventional methods.