| Dihydromyricetin(DMY) is a natural aglycone flavonoid and has numerous healthpromoting functions, including antioxidant, anti-inflammatory, analgesic, antitussive, expectorant, antibacterial, anti-thrombotic andanti-tumor activities and the ability to protect liver and alleviate hangover. However, DMY shows poor lipid solubility, which limits its efficient absorption by the liver of human body. The enzymatic acylation of DMY has gained increasing attention due to its high efficiency, green process, mild reaction conditions and high regioselectivity. In this paper, an available cheap lipase(Aspergillus niger lipase, ANL) was screened and this ANL can effectively catalyze the acylation of DMY. However, the free ANL has a number of limitations preventing its further applications such as its easy inactivation, difficulty to recycle and separate from the reaction system. So the immobilization of ANL is used to overcome these shortcomings and will bring significantly economic benefits in industry.In recent years, magnetic iron oxide nanoparticles(MNPs) have attracted interest for its properties of biocompatibility, magnetism, high surface-to-volume ratio and low toxicity. In most cases, the MNPs require a modification or functionalization to introduce the catalyst onto the surface. Polydopamine(PDA), which is a polymer inspired by the composition of the adhesive protein in mussels, is easily synthesized. And the surface of PDA contains many active groups(such as phenol hydroxyl), so further reaction could happen on the surface PDA. Therefore, PDA is increasingly applied in the organic and inorganic material surface modification.In this study, MNPs were synthesized via the conventional co-precipitation method. Then MNPs were further surface-modified by a polydopamine coating. The polydopaminecoated MNPs(PD-MNPs) were structurally characterized in detail. It has been found that the size of MNPs is about 10 nm and the PDA layer is about 4 nm. Besides, PD-MNPs has good thermal tolerance and superparamagnetism. We firstly immobilized ANL onto PD-MNPs by the Michael addition or Schiff base reaction between catechols(a moiety of polydopamine or dopamine) and amines or thiols. So a magnetic nanoscale immobilized enzyme(ANL@PD-MNPs) with high activity, high stability had successfully been obtained. The effects of different preparation conditions on the activity recovery of ANL@ PD-MNPs were optimized. The results indicated that, under the optimal conditions: immobilization time of 12 h, support/enzyme mass ratio of 4.8:1, immobilization p H of 8.0, the ANL was immobilized on the PD-MNPs with a protein loading of 138 mg/g and an activity recovery of 83.6%.The secondary structure and enzymology properties of ANL@ PD-MNPs were systematically investigated. The results revealed that the content of alpha helix and beta folding in secondary structure respectively increased by 2.74% and 0.84% after immobilization, leading to the increase of structural rigidity and stability of ANL@ PD-MNPs. The optimal p H of ANL@ PD-MNPs and free ANL were 8.0 and 7.0, respectively; the optimal temperature of ANL@ PD-MNPs was 40°C, and free ANL was 35°C. Besides, the ANL@ PD-MNPs showed superior thermal stability, solvent tolerance and p H tolerance comparing with its free countpart; Moreover, after 20 days, ANL@ PD-MNPs had remained 92.8% activity compared with initial activity, which significantly higher than that of free ANL(21.5%). The kinetic study showed that the catalytic efficiency(Vmax/Km) of ANL@ PD-MNPs was lower than that of free ANL(3.73 ? 10-4 min-1 vs 4.07 ? 10-4 min-1). These results demonstrated that the ANL immobilized on the surface of PD-MNPs had improved adaption of p H and temperature, higher stability and higher catalytic efficiency.The aclyation of DMY catalyzed by the as-prepared ANL@PD-MNPs was performed in organic solvents. Among all the selected acyl donors, only vinyl acetate can acylate with DMY in the presence of ANL@ PD-MNPs and the obtained product was confirmed to be DMY-16-acetate by MS and NMR analysis. The optimal reaction conditions for ANL@ PDMNPs were as follows: the optimum substrate molar ratio of 10:1(vinyl acetate/DMY), the optimal temperature of 40°C, the optimum adding amount of 60 U. Under the above optimal parameters, the initial reaction rate, the product yield was 7.1 m M/h and 83.1%, respectively. Besides, ANL@PD-MNPs retained over 55% of its initial activity after 10 cycles of reuse, and the regioselectivity ANL@PD-MNPs showed no significant effect by its reuse.In order to further impove the reaction efficiency of ANL@PD-MNPs-catalyzed acylation of DMY, the DES-containing systems were applied as the reaction media for the enzymatic acylation. The optimal reaction conditions for the enzymatic acylation carried out in the DES-based co-solvent system(Ch Cl:Glycerol-DMSO, 1/ 3) were as follows: the optimum substrate molar ratio(vinyl acetate/DMY), the optimal temperature, the optimum adding amount were found to be 10:1, 45°C and 50 U, respectively. While the concentration of DMY was 300 m M, the initial reaction rate was 55.2 m M/h in the co-solvent system of Ch Cl:Glycerol-DMSO and the product yield was 85.3%(24 h). But the initial reaction rate was 34.9 m M/h in DMSO and the product yield was 78.8%(48 h). Obviously, the use of the DES-containing co-solvent system as the reaction medium significantly enhanced the efficiency of the acylation of DMY catalyzed by ANL@PD-MNPs. Additionally, the product DMY-16-acetate was separated from the reaction system and purified, and subsequently was evaluated for its antioxidant ability. After acylation, the fat solubility of DMY-16-acetate reached 0.635 g/100 g oil, larger than that of DMY. For DMY-16-acetate, the IC50 of DPPH scavenging, hydroxyl radical scavenging and chelating Fe2+ were 4.16 mg/m L, 1.79 mg/m L and 0.13 mg/m L, respectively, indicating that the formed DMY-16-acetate had excellent antioxidant activity.In this dissertation, the immobilization of ANL on PD-MNPs and the efficient acylation of DMY catalyzed by the prepared ANL@PD-MNPs were for the first time investigated. Compared with the present immobilization technology, this preparation process exhibited many advantages such as simpleness, high enzyme loading, high enzyme activity, high stability of enzyme and rapid separation of enzyme from the reaction medium. More importantly, ANL@ PD-MNPs could efficiently catalyze the acetylization of DMY. This study is helpful to rich immobilization technology of enzyme and promote the the researchers’ understanding of the intereaction of enzymes and carriers. Additionally, the wellestablished biocatalysis system can be extended to enzymatic modification of other flavonoids. |
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