AbstractBackgroundsGlycerolysis, with its advantages of readily available raw materials, simple operation, and mild reaction conditions, is a primary method for producing diacylglycerol (DAG). However, enzymatic glycerolysis faces challenges such as high enzyme costs, low reuse efficiency, and poor stability. The study aims to develop a cost‐effective immobilized enzyme by covalently binding lipase to pre‐activated carriers through the selection of suitable lipases, carriers, and activating agents. The optimization is intended to improve the glycerolysis reaction for efficient DAG production.ResultsLipase CN‐TL (from Thermomyces lanuginosus) was selected through glycerolysis reaction and molecular docking to catalyze the glycerolysis reaction. Optimizing the immobilization method by covalently binding CN‐TL to poly(ethylene glycol) diglycidyl ether (PEGDGE)‐preactivated resin LX‐201A resulted in the preparation of the immobilized enzyme TL‐PEGDGE‐LX. The immobilized enzyme retained over 90% of its initial activity after five consecutive reactions, demonstrating excellent reusability. The DAG content in the product remained at 84.8% of its initial level, further highlighting the enzyme's potential for reusability and its promising applications in the food and oil industries.ConclusionsThe immobilized lipase TL‐PEGDGE‐LX, created by covalently immobilizing lipase CN‐TL on PEGDGE‐preactivated carriers, demonstrated broad applicability and excellent reusability. This approach offers an economical and convenient immobilization strategy for the enzymatic glycerolysis production of DAG. © 2024 Society of Chemical Industry.