Preparation And Catalytic Properties Of Biomimetic Immobilized Enzyme

The key problem for enzyme immobilization is the rational design and preparation of carrier. In this study, a core-shell structured capsule was designed by mimicking the structure, component, function, and formation process of multi-layered cell.β-D-glucuronidase (GUS) encapsulated in this biomimetic capsule was utilized for the enzymatic conversion of baicalin to baicalein. The preparation conditions of capsule core, shell membrane and shell wall were systematically investigated, and their effects on the activity and stability of encapsulated GUS were extensively studied. Finally, the guideline for efficiently preparing core-shell structured capsule carrier was tentatively proposed.The cationic, neutral and anionic polysaccharides were separately acted as the liquid core of biomimetic capsule by mimicking the component and function of cell sap. It was found that the electrostatic repulsion interaction between the polysaccharide core and the enzyme enhanced the enzyme activity and stability. The shell membrane of biomimetic capsule was then fabricated by mimicking the structure and function of cell membrane. Under the optimum preparation conditions, the porous Ca-alginate membrane of the capsule could not only confine the enzyme inside but also permit the substrate and product to freely diffuse in and out. Finally, mimicking the biosilicification process and function of diatom cell wall, protamine was for the first time utilized to mediate the formation of intact protamine/silica shell wall on the surface of capsule. The rigid silica shell wall dramatically inhibited the swelling of the capsule and significantly enhanced the recycling stability of capsule. In addition, the silica-precipitating and templating roles of protamine were elucidated, and the mediation mechanism of organic molecules in biomimetic silicification was also tentatively analyzed.Baicalin was converted into baicalein by GUS encapsulated in biomimetic capsules. The sodium carboxymethyl cellulose (CMC) core created a biomimetic anionic microenvironment, and thus enhanced the activity of GUS. Meanwhile, the alginate/protamine shell membrane and the protamine/silica shell wall prevented the swelling of capsule and the leakage of GUS, and thus enhanced the recycling stability of GUS. The encapsulated GUS exhibited up to 125% of its free-form activity with an encapsulation efficiency of 69%. Moreover, the relative activity of encapsulated GUS under extreme pH conditions was up to 85%. The encapsulated GUS showed no appreciable loss in activity after 10 repeated cycles, and 90% of its initial activity remained after 26-day storage at 4 oC. Under the optimum conversion conditions (37℃, pH 7), a baicalein productivity as high as 73% was obtained.