A novel two-step enzymatic esterification-hydrolysis method that generates high-purity conjugated linoleic acid (CLA) isomers was developed. CLA was first partially purified by enzymatic esterification and then further purified by efficient, selective enzymatic hydrolysis in a three-liquid-phase system (TLPS). Compared with traditional two-step selective enzymatic esterification, this novel method produced highly pure cis-9, trans-11 (c9,t11)-CLA (96%) with high conversion (approx. 36%) and avoided complicated rehydrolysis and reesterification steps. The catalytic efficiency and selectivity of CLA ester enzymatic hydrolysis was greatly improved with TLPSs, as high-speed stirring provided a larger interface area for the reaction and product inhibition was effectively reduced by extraction of the product into other phases. Furthermore, the enzyme-enriched phase (liquid immobilization support) was effectively and economically reused more than 8 times because it contained more than 90% of the concentrated enzyme. Therefore, this novel enzymatic esterification-hydrolysis method can be considered ideal to produce high-purity fatty acid monomers.
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