| Cell-surface carbohydrates (glycocalyx) play essential roles in living creatures. They generally act as molecular recognition sites in normal physiology and disease processes, including fertilization, development, differentiation, virus invasion, cancer cell metastasis and bacterial infection. With the development of bionics, much attention has been paid on fabricating glycocalyx-like surfaces for further probing the biology functions of glycocalyx. Diverse methods were exploited to fabricate glycocalyx-like surfaces, which fall into two categories:non-covalent immobilization and covalent immobilization. However, it is considerably difficult to synthesize glycoconjugate and the fabricated glycocalyx-like surface is usually unstable in terms of non-covalent immobilization method. Covalent immobilization method, in comparison, offers more stable functional surfaces. But the synthesis is also complicated for these sugar-containing monomers and activated sugar derivatives. And there is no site specificity, region-and stereo-selectivity in these synthesis processes. In addition, most of the reactions need to be carried out in organic solvent, which may cause much environmental pollution. Inspired by the enzymatic glycosylation in vivo, a newly method for fabricating glycocalyx-like surface, namely enzymatic glycosyl transfer (transglycosylation) in vitro, has already been developed. Noticeably, enzymatic transglycosylation on surfaces with immobilized acceptor is becoming increasingly important from both biomimetics (biomimicing the reaction in vivo) and biocatalysis (green, region-and stereo-selective, etc.) standpoints.In this thesis, we are dedicated to further developing an enzymatic method for fabricating glycocalyx-like surfaces by glycosidase and glycotransferase. Furthermore, we apply such enzymatic transglycosylation method to prepare glycosylated affinity membranes. The main results of this work are summarized as below.SPR was first used to study the specific interaction between glycosidase (β-glucosidase from almond (EC3.2.1.21) and P-galactosidase (Gal) from Aspergillus oryzae (EC3.2.1.23)) and their corresponding substrate acceptors-polyhydroxy polymer brushes. It was found that poly (2-hydroxy ethy1methacrylate)(PHEMA) brushes could not only adsorb them specifically but also inhibit their activity after adsorption. Compared with PHEMA brushes, the interaction between poly [(oligo (ethylene glycol) methyl ether methacrylate](POEGMA) brushes and the glycosidase is relatively weaker, so POEGMA is the preferent substrate acceptor for glycosidases. QCM was used to monitor the kinetic process of Gal catalyzed transglycosylation on PEG brushes. The kinetic process includes three typical steps:specific binding between Gal and the substrate acceptor, enzymatic transglycosylation induced by adding sugar donor, and dissociation of Gal form the newly glycosylated surface. Kinetic parameters (km and kcat) indicate that this enzymatic strategy is promising for the construction of biomimetic surfaces with glycocalyx-like structure. However, the amount of introduced galactose pendants is insufficient at this stage, and thus there is much room for further improvement.QCM was then used to monitor dextransucrase (DSase)-catalyzed polymerization on the glucose-and maltose-immoblized self-assembly monolayer (SAM) surfaces, repectively. Kinetic parameters of enzymatic polymerization indicate that maltose is the preferent substrate acceptor of DSase. Furthermore, we fabricate the maltose-attached polymer brushes by surface-initiated atom transfer radical polymerization (SI-ATRP) and BF3EtO2catalyzed glycosidic bond formation reaction. Then the branched dextran-attached polymer brushes were successfully fabricated by enzyme-catalyzed polymerization on the surface. Compared with the maltose-attached polymer brushes, the affinity between the branched dextran-attached brushes and canavalin A (Con A) is much stronger. Because the branched dextran brushes are essentially linear polymers (a,1-6bonds) containing5%of a,1-3-linked branches with shorter side-chains. Thus, more nonreducing residues would be available for lectins to bind. In addition, methyl a-D-mannopyranoside (MM) can be used to affinity dissociate almost all lectins from the studied surfaces. The branched dextran-attached brushes have great potential in separation of Con A as affinity ligand. The effective approach to fabricate glycocalyx-like surface with complex structure is also of significant promise in glycomaterials engineering.Based on the above research, we prepared the glycosylated microporous polypropylene membranes (MPPM) by UV-induced grafting polymerization and enzymatic transglycosylation reaction. Both the UV-induced grafting polymerization and enzymatic transglycosylation do not dramatically alter the membrane surface morphology (porosity and pore structure). The glycosylated MPPM not only have good wettability, but also show specific adsorption to the lectins and would be expected to have wide potential applications in affinity separation of lectins. |
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