In recent years, an increasing number of studies have underscored the significance of breast milk as the optimal nutritional source for infant growth and development [1]. These studies have highlighted the unique triglyceride structure of breast milk, characterized by long-chain fatty acyl groups at sn-1, 3 and medium-chain fatty acyl groups at sn-2, which facilitate enhanced digestions and absorption in the intestines of infants [2], [3]. As the main component of breast milk, enhancing the quality of milk powder in infant formula is achievable by incorporating 1, 3-dioleoyl-2-palmitoylglycerol (OPO). OPO synthesis is possible via both chemical and enzymatic routes. When compared to chemical approaches, enzymatic synthesis of OPO is distinguished by its mild reaction conditions, high efficiency, strong specificity and a more extensive range of potential applications [3].

Rhizomucor miehei lipase (RML), as a sn-1, 3-specific biocatalyst, exhibited high activity during the OPO synthesis process [4], [5], [6]. However, exposure to harsh conditions such as extreme pH, high temperatures and organic solvents renders free RML susceptible to inactivation and limits its reusability, thereby constraining its practical application. Addressing these limitations is achievable through the immobilization of RML onto a solid carrier [7], [8]. Wang et al. fixed lipase in glycerol halogen and halogenated hydrocarbon modified BSA-15. The results showed that the immobilized enzyme had interfacial activation, which provided greater glycerol decomposition activity and also showed good reusability [9]. Chen et al. encapsulated lipase in nucleotide / metal ion coordination polymers and used it to evaluate its catalytic performance in glycerol decomposition and esterification reactions. In the CALB @ CPs studied, it showed good performance in the esterification of fatty acids and glycerides to synthesize TAGs [10]. It shows that the immobilization of enzyme can improve the shortcomings of free enzyme. Although existing commercial immobilized lipase (Lipozyme RM IM) have been employed for OPO synthesis [11], the substrate high viscosity has a significant negative impact on its catalytic efficiency. Furthermore the inability to adjust existing commercial immobilized lipase by changing immobilized carrier and method poses limitations. Hence the effective combination of novel immobilized carriers and methods is the most important for optimizing biocatalyst activity and longevity [12]. He et al. immobilized Thermomyces lanuginosa lipase (TLL) on C18H37-SBA-15 to obtain TLL @ C18H37-SBA-15 immobilized enzyme, which was used as a catalyst for the synthesis of OPO structure by enzymatic acidolysis. The results showed that the PPP conversion rate reached 99.07%, the OPO content was 73.15%, and the sn-2 palmitic acid content was 90.09% [13]. Ghide et al. immobilized RML lipase on magnetic multi-walled carbon nanotubes (mMWCNTs) to obtain immobilized enzyme RML-mMWCNTs. Using tripalmitin (PPP) / oleic acid (OA) as substrates, OPO structured lipids were synthesized by two-step enzymatic acidolysis, and an effective biocatalyst for the synthesis of OPO-rich structured lipids was obtained [14].

Compared to traditional carriers, covalent organic frameworks (COFs) represent a novel class of porous materials with higher specific surface area, tunable pore volume, and superior thermal stability, which can significantly improve enzymatic properties [15]. Currently, the incorporation of COFs materials in lipase immobilization primarily relies on adsorption and covalent methods. Elmerhi et al. studied a new COFs with cationic properties, and employing adsorption to immobilize, horseradish peroxidase (HRP) and thereby enhancing HRP stability [16]. Similarly, Wang et al. achieved notable improvements in the loading capacity and reusability of natural trypsin by covalently immobilizing it on magnetic COFs [17]. However, due to the limited pore dimensions within COFs, enzymes are typically adsorbed or covalently attached to the surface rather than within the pore channel of the material. As a result, enhancements in organic solvent tolerance and thermal stability remain insufficient.

Generally, COFs synthesis involves the implementation of harsh conditions, such as elevated temperatures, acid-base solutions and organic solvents, rendering the enzyme unable to survive the in-situ embedding process. In this study, immobilized lipase RML@COF-1 was synthesized by an in-situ aqueous phase method at room temperature (Scheme 1). RML was directly encapsulated within the pore channels of the material, simplifying the immobilization process. Simultaneously, this method prevented the activity loss causing by covalent method. Various characterization techniques such as FT-IR, BET, CLSM and XRD were used to prove the successful immobilization of RML. The performance of RML@COF-1 in OPO synthesis was evaluated with respect to pH, temperature, organic solvent, storage stability and reusability.