Enzymatic Synthesis of Bioactive Structured DHA Phospholipids via Stable Immobilized Phospholipase-Catalyzed Esterification in a Solvent-Free Medium

The enzymatic esterification of docosahexaenoic acid (DHA) with glycerophosphocholine (GPC) was investigated to produce bioactive structured DHA phospholipids with DHA esterified at the sn-2 position, which may contribute to the prevention of neurodegenerative diseases such as Alzheimer’s. This reaction is complex due to the low solubility of GPC in anhydrous organic media and the limited stability of enzymes under such conditions. The immobilized phospholipase Quara® LowP (QlowP-C18) proved to be the most effective catalyst, achieving a 58% yield of di-substituted DHA phospholipids (Di-DHA-PC) in just 48 h under optimal conditions (solvent-free media at 60 °C) with 95% purity. Advanced immobilization and post-immobilization techniques significantly improved the stability of QlowP-C18, increasing its longevity threefold and enabling reuse for up to five reaction cycles at 40 °C. The total production reached 120.4 mg of highly pure DHA-di-substituted phospholipid. These findings highlight the effectiveness of stable immobilized enzymes in solvent-free systems and underscore their potential for the efficient and sustainable production of highly pure Di-DHA-PC, which could be used as a functional or nutraceutical ingredient for the prevention of neurodegenerative diseases.