Vitamin E is a vital nutrient for human health that has been widely exploited in the food and pharmaceutical industries [1]. Due to the inherent instability of the phenol group at position 6, it is highly susceptible to oxidation, which in turn leads to a reduction in activity [2]. At the same time, the fat-soluble of VE affects the effective absorption of the human body, so it is necessary to modify the molecular structure of VE [3]. Vitamin E succinate(VES) is the most commonly utilized of the major VE derivatives owing to its exceptional water solubility and stability [4]. Moreover, Vitamin E succinate exhibits strong biological activity and has potential applications in field of food nutrition [5].

Currently, both chemical and enzymatic methods are widely employed for the synthesis of Vitamin E succinate [6]. However, chemical approaches tend to have a negative impact on the environment, thereby hindering sustainable development. Consequently, the enzymatic method is consistent with the trend of green development and merits further investigation [7]

Yin [8] reported that Novozym 435 lipase catalyzed the enzymatic synthesis of Vitamin E succinate using 8.6 % concentration of VE and succinic anhydride at 1:5 molar ratio. After 48 h of reaction, the esterification yield of Vitamin E succinate reached 78.1 % in dimethyl sulfoxide (DMSO) and 53.3 % in N, N-dimethylformamide (DMF), respectively. Subsequently, Tao [9] described the synthesis of VES catalyzed by Candida rugosa lipase (CRL) in DMSO for 24 h, achieving an esterification yield of 70 %, whereas the yield in DMF for the same reaction time was only 39.8 %. Xia [10] examined the use of immobilized CRL by nanogels with ionic liquid modified to catalyze the synthesis of Vitamin E succinate with 8.6 % concentration of VE and succinic anhydride at 1:4 molar ratio, resulting in an esterification yield of 62.6 % after 15 h of reaction.

The studies described above indicate that the enzymatic synthesis of Vitamin E succinate yields suboptimal esterification results. Dissolving succinic anhydride requires the use of polar organic solvents, which adversely affect the catalytic activity of lipases. Therefore, to improve the reaction efficiency of enzymatic VES synthesis, it is crucial to enhance both the esterification efficiency of lipase and their catalytic stability in polar organic solvents.

Recently, bio-imprinting by substrate analogs has emerged as a promising approach to enhance lipase catalytic efficiency. Accordingly, Kahveci [11] compared the effects of fatty acids and triacylglycerols as bio-imprinting molecules and found that bio-imprinting with oleic acid could significantly improve the transesterification activity of lipase in organic solvents. Wang [12] reported that bio-imprinted Thermomyces lanuginosus lipase with palmitic acid as the imprint molecule, immobilized with polyacrylamide gel matrix, exhibited 2.5–4.7 times for activity in organic solvents. Gao [13] employed fatty acid imprinting on Burkholderia cepacia lipase (BCL) and immobilized the imprinted lipase via cross-linking, resulting in 53.8 % enhancement its activity. Brandão [14] further investigated the bio-imprinting effects of four fatty acids with different chain lengths on BCL and found that the esterification efficiency of BCL bio-imprinted by fatty acids was significantly improved, with the imprinting effect being closely related to the carbon chain length of the fatty acids. The findings from these studies indicate that bio-imprinted lipase with fatty acids as substrate analogs in esterification reactions can effectively enhance esterification activity. This approach holds significant potential for further exploration in the enzymatic synthesis of Vitamin E succinate.

Sampath [15] investigated oleic acid bio-imprinted lipase, which was further immobilized through cross-linking. The resulting lipase exhibited a hydrolytic activity 10.4 times higher than that of the free lipase and significantly improved reusability. Li [16] also utilized lecithin to bio-imprint phospholipase A1, and the activity of the immobilized imprinted phospholipase A1 increased by 30.9 folds in hexane solution, accompanied by a considerable enhancement in substrate selectivity. The mechanism is which substrate analog bio-imprinting enhances the catalytic esterification efficiency of lipase lies in inducing the exposure of the catalytic active site, thereby improving catalytic performance. Therefore, immobilization is required to maintain the conformational stability of the imprinted lipase protein, stabilize the enhanced catalytic activity conferred by bio-imprinting, and enable its application in catalytic reactions.

Cross-linked enzyme aggregates have been recognized as an ideal method for enzyme immobilization [17]. Through precipitation, lipase can form stable tertiary structures, thereby enhancing its catalytic performance. Thus, cross-linked enzyme aggregate technology can serve as an immobilization strategy for bio-imprinted lipase to maintain its structural conformation [18]. Muley and Alves [19], [20] prepared cross-linked lipase aggregates using ammonium sulfate as the precipitating agent and glutaraldehyde as the cross-linker, achieving higher enzyme activity recovery. Furthermore, the stability of the lipase was significantly enhanced, and its reusability in catalytic reactions was improved. Therefore, applying the cross-linked enzyme aggregate method for immobilizing bio-imprinted lipase is worthy of exploration.

In this study, fatty acids, as substrate analogs of succinic acid, were used as imprint molecules to bio-imprint Aspergillus niger lipase, aiming to enhance its catalytic efficiency in the esterification reaction of Vitamin E succinate. The cross-linked enzyme aggregate method was employed to immobilize the imprinted lipase, which improved the reaction efficiency and catalytic stability. Fluorescence spectroscopy characterized the variations in catalytic activity during the bio-imprinting process, which can provide deeper insights into the mechanism of substrate analog bio-imprinting for enhancing lipase catalytic efficiency.