| Immobilizing enzyme on or within a matrix can lower the costs of the enzymes by reusing and avoid the enzymes contaminating the substrate. Among the substrates, polymeric materials are considered to be ideal substrates for enzymes immobilization due to their merits of low cost, easy processing and superior mechanical properties. However, because most of the polymeric materials have low surface energy and inert surfaces, it is difficult to modify the polymeric materials' surface for enzymes immobilization. In this work, a mild and simple strategy to immobilize enzyme on the polymeric matrix was first proposed based on the visible light induced controlled/living graft polymerization, and then the structure and the application performance of the immobilized enzymes were characterized. In this strategy, one photo-initiator isopropyl thioxanthone (ITX) was first planted onto the polymeric matrix, and then this matrix can initiate the graft polymerization of poly(ethylene glycol) diacrylate (PEGDA). If enzymes were blended with the monomer solutions before the grafting reaction, enzymes can be in-situ encapsulated into the PEG net-cloth after the polymerization. This work mainly included the following results:1. The mechanism of the visible light induced controlled/living graft cross-linking polymerization was studied. Lipase can be embedded in PEG net-cloth on low density poly(ethylene) (LDPE) film via this polymerization, and then this film was used to catalyze the synthesis of the palmitolyglucose ester in tert-butanol. Due to the "controllable" nature of this strategy, the thickness of the net-cloth can be controlled exactly (10-1-103 ?m)by changes of the visible light irradiation time, the concentration of PEGDA and the feed volume. The activity of the immobilized lipase under the visible light was 5 times higher than under the UV irradiation. And compared with free lipase, the esterafication rate in the system of the immobilized lipase increased from 42% to 67% at 50 ?. What's more, the final results shown that about 89% of enzymes were successfully entrapped in the net-cloth after the graft polymerization, and this catalytic film can be reused for seven batches without losing the activities.2. Polymer brushes were grafted on polymeric matrix to immobilize cellulase using adsorption and covalent binding methods, respectively. And then these catalytic fabrics were used to hydrolyze cellulose. The results shown that the immobilized cellulase exhibited a good residual enzymatic activity (45%) but a poor operational stability (6 cycles) when using acrylic acid (AA,30%, v/v) and PEGDA (20%, v/v) as monomers to adsorb cellulase. On the contrary, when grafting polymer brushes to immobilize cellulase via covalent binding, the cellulase can be reused for 10 batches but the conversion ratio of the cellulose maintained only within 10%-15%.3. ?-Glucosidase (BG) can be entrapped in PEG net-cloth on polypropylene (PP) non-woven fabrics via visible light induced controlled/living graft polymerization. This catalytic fabric was then used to hydrolysis the filter paper (cellulose) along with the free cellulase. The results shown that the best conditions for the hydrolysis of filter paper with BG fabric were 0.25% (v/v) GA for 55 mg BG treatment,50 ? and citric acid buffer (pH=4.8). Compared with the system without adding the BG loaded fabric, adding this fabric can lead to a 40% increase of the cellulose conversion after 48 h. In addition, less than 2% of the BGs leached from the net-cloth after immobilization, and the relative activity of BG was still more than 70% after catalyzing for 15 batches.4. According to the "living" characteristics of this grafting polymerization, there were still many initiator residues on the grafting layer. So other kinds of enzymes can be embedded in the new layer on the former layers. Therefore, different kinds of enzymes can be immobilized separately on the same polymeric matrix. According to this theory, two kinds of mutual inhibition enzymes (trypsin and transglutaminase) were separately immobilized in different net-cloth layers on one matrix to make the multi-enzyme loaded film (Type A and Type B). XPS and AFM were used to characterize the structure of these two types of the film. The results shown that more than 90% of the enzymes can be immobilized in the net-cloth without leaching. Compared with the traditional co-immobilization methods, separately immobilized enzymes represented higher activities, and these films can be reused for 4 batches without losing activities.5. Due to that the optimum reaction conditions of some enzymes varies considerably, immobilization all the enzymes on one substrate may cause some enzymes deactivated during the catalytic reaction. So enzymes which have the similar catalytic reaction conditions were separately immobilized on one substrate, and enzymes which have the different catalytic reaction conditions were immobilized on different substrates. Finally, all these catalytic substrates were used together to establish the "tandem and parallel muti-enzyme immobilization system" to catalyze one multiple reaction. This system was then used to synthesize two new target anti-tumor drugs LTV-Azacytidine and LTV-ara-C. The structure of these new drugs were characterized by MS, FT-IR and NMR. And then CCK-8 assay was used to assess the cytotoxic effects against MCF-7. Compared with the original drugs, the new drugs could enhance the anti-tumor activities. |
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