Preparation and characterization of molecularly imprinted hydrogel for recognition of lipase enzyme

In the thesis study, using the molecular imprinting technique, lipase imprinted (MIP) and non-imprinted (NIP) hydrogels were prepared using monomers with different functions such as hydroxyethylmethacrylate (HEMA), 1-vinylimidazole (1-VI), methyl acrylate (MA), acrylamide (AAm), 2-dimethyl aminoethyl methacrylate (2-DMAEMA). Lipase rebinding studies were performed in different pH environments for these prepared hydrogels. The amount of protein adsorped on MIP and NIP hydrogels and imprinting efficiency (IF) values were calculated and compared. Imprinting efficiencies found to be high for the hydrogels prepared with AAm, AAm-1-VI, AAm-MA monomers so further studies were performed with these hydrogels. Determination of functional groups and surface morphology analysis of molecularly ımprinted hydrogels for recognition of lipase enzyme were done by Fourier Transform Infrared Spectroscopy (FTIR) scanning electron microscopy (SEM). For optimization of rebinding conditions of lipase enzyme on MIP and NIP hydrogels, effect of pH, contact time, temperature and enzyme concentration were investigated. Maximum rebing of lipase enzyme on MIP and NIP hydrogels were occurred at 25 °C, pH 3.0 and 90 min. When the initial enzyme concentration reached to 5 mg/mL, the adsorption reached to saturation. Adsorption isotherms and adsorption kinetics were studied under optimized conditions. In order to investigate the selectivity of lipase imprinted hydrogel, rebinding studies were performed with α-amylase, β-glucosidase and BSA. Selectivity of lipase imprinted hydrogels was found to be low towards these enzymes and protein. Swelling test was performed to investigate swelling properties of MIP and NIP hydrogels. The lipase imprinted hydrogel prepared with the acrylamide monomer was used to selectively separate lipase from the medium containing different pancreatic enzymes. The purity control of the samples was done by SDS-PAGE method.