چكيده لاتين
In this thesis, Candida rugosa lipase (CRL) was immobilized on graphene oxide nanosheets (GON) and its four chemically modified forms through adsorption and covalent attachment. In this regard, GON was synthesized via modified Hummers method. In order to activate the support surface for covalent immobilization, GON was then chemically modified by various reagents such as thiosulfonate (TS), glutaraldehyde (GLA) and trichlorotriazine (TCT) via two different experimental procedures. CRL was then immobilized on GON and its modified compounds including TS-GON, GLA-APTES-GON, DCT-GON and DCT-APTS-GON at the identical conditions. The success of the synthesis, modification, and immobilization processes were proved using several techniques such as FT-IR, XRD, AFM, FE-SEM, EDS, UV-Vis, and CHNS. For the achievement of the efficient immobilization, the initial concentration of the enzyme and the immobilization time were optimized. The highest loading and enzyme specific activity belong to GON and CRL@DCT-APTS-GON, respectively.After enzyme immobilization, despite the decrease of the enzyme activity, the operational stability of the enzyme amplified significantly. While the free enzyme lost 93.20% of its optimum activity after exposure to pH 10, CRL @ DCT-APTS-GON showed the highest stability compared to other samples under these conditions Moreover, after the thermal stress, the thermal stability of the immobilized enzyme on all supports was higher than that of the free enzyme, so that the free enzyme totally lost its activity at 70°C while the residual activity of CRL @ DCT-APTS-GON was 58.85% of its initialvalue. Also, after 50 days, CRL @ DCT-APTS-GON retained 79.74% of its initial activity. After 5 re-uses, it was found the highest activity belong to CRL @ DCT-APTS-GON.
In the second part of this study, due to the swift separation of the immobilized enzyme from the reaction mixture and obtain more purified products, the magnetic graphene oxide (MGON) nanosheets were synthesized. According to the results of the first part, DCT-APTS-GON showed the best performance for CRL immobilization, so, MGON was chemically modified by DCT-APTS and then applied as support. The processes of synthesis, modification and enzyme immobilization were confirmed using different characterization methods. Subsequently, the initial amount of CRL and immobilization time were optimized. The results showed that the loading capacity and immobilization efficiency decreased and the specific activity, activity recovery, storage stability, pH and thermal stability, and reusability of the immobilized enzyme increased after the MGON modification.
In the third part of this study, for the fabrication of the enzymatic hybrid membrane reactor (EHMR), the facile separation of the product from the reaction mixture and the improvement of the reaction efficiency, the hybrid membranes were prepared through two different methods. In the first approach, GON was coated on the surface of the commercial polymeric membrane through the dip-coating and low temperature hydrothermal (LTH) process. The decreasing of the immobilized enzyme activity on these hybrid membranes in comparison with the pure commercial membrane and total deactivation of the immobilized enzyme in the second cycle of use, indicated the unsuitability of this method for EHMR preparation. In the second approach, the hybrid membranes were prepared by blending the different percentages of GON in polyethersulfone (PES) matrix via phase inversion method and applied for CRL immobilization. The chemical composition, surface morphology, and performance of hybrid membrane and EHMR were assessed through various methods such as ATR-IR, XRD, AFM, contact angle goniometry, surface free energy, pure water flux, and p-NP flux...
