Preparation and characterization of molecularly imprinted hydrogel for recognition of α-amylase enzyme

Molecular imprinting technology (MIT) is based on the synthesis of polymers containing binding sites that specifically recognize the target molecule. In the synthesis of biomacromolecule imprinted polymers, such as proteins, there may be difficulties dueto their large macromolecule structure and instability. In the first step of the thesis study, imprinted hydrogels were prepared with different monomers and α-amylase was used as a template molecule. For this purpose, monomers with different functions such as acrylamide (AAm), hydroxyethylmethacrylate (HEMA), 1-vinylimidazole (1-VI), 4-vinylpyridine (4-VP) and methacrylicacid (MAA) were tested. Imprinting efficiencies of the prepared hydrogels were calculated after rebinding studies. The highest α-amylase binding capacity was obtained with the hydrogel prepared with AAm monomer and further studies were performed with this hydrogel. Structural analysis of the imprinted hydrogel were done with Fourier Transform İnfrared Spectroscopy (FT-IR) and scanning electron microscopy (SEM) analysis. Maximum α-amylase binding capacity was obtained at rebinding time of 120 minutes, when the temperature was 25 ° C, pH of the medium was 7.0 and at 2.5 mg/mL enzyme concentration. The selectivity of the imprinted hydrogel was tested with different proteins (β-glucosidase, lipase and albumin) and α-amylase imprinting efficiency of the hydrogel was found to be higher than other proteins. Under optimized conditions, adsorption isotherms and adsorption kinetics were studied. Swelling tests were done in order to investigate swelling properties of crosslinked hydrogels. The α-amylase imprinted hydrogel prepared with the acrylamide monomer was used for the selective separation of the α-amylase from the tablet containing different pancreatic enzymes. Activity and protein assays were performed to determine the α-amylase activity recovered from MIP and NIP hydrogels. Purity control of the samples were done with SDS-PAGE.