Study On Dehydrogenases Immobilization By Alginate-Silica Hybrid Gels

Enzyme encapsulation, as one of the effective immobilization method, has become a popular approach with widely biotechnological and bioengineering applications. It is very important to design the novel carriers for enzyme encapsulation, especially to create the appropriate microenvironment for encapsulated enzymes in the carrier. The microenvironment for enzyme encapsulation in the carriers was affected by many factors, including the hydrophilicity, the structure and morphology, and the reactivity of the carriers. To this objective, in this thesis, the biocompatible and widely used silica gel (SiO₃) and alginate (ALG) gel were used to prepare the novel alginate-silica (ALG-SiO₃) hybrid composites according to the factors affecting the microenvironment. The activity and stability of three dehydrogenases coencapsulated or encapsulated separately in ALG-SiO₃ hybrid composites prepared by different methods were compared.Three types of ALG-SiO₃ hybrid composites were prepared: (1) class I ALG-SiO₃ hybrid composite was prepared by doping of silica gel (SG) or silica nanotubes (SiNTs) into alginate gel to form ALG-SG hybrid composite or ALG-SiNTs hybrid composite; (2) class II ALG-SiO₃ hybrid composite was prepared through the hydrolysis and condensation of tetramethoxysilane (TMOS) in situ in an alginate solution, followed by Ca2+ crosslinking with alginate; (3) class III ALG-SiO₃ hybrid composite was prepared through coating the outer surface of the alginate beads with silica gel ,which was derived from the gelation of TMOS. Chemical and physical properties, structure and morphology of alginate gel and ALG-SiO₃ hybrid composites were detected by FTIR, SEM, TEM, DMA and BET etc.. Diffusion properties of coenzyme NADH in the alginate gel and ALG-SiO₃ hybrid composites were determined according to the unsteady-state model. ALG gel and ALG-SiO₃ hybrid composites were employed for yeast alcohol dehydrogenase (YADH) encapsulation. The enzyme loading efficiency, catalytic activities, and the operational and storage stability of immobilized YADH were investigated. Loading efficiency of YADH in ALG gel was 68.8%, and the loading efficiency in ALG-SiO₃ hybrid composites increased significantly, with the highest loading efficiency in calss II ALG-SiO₃ hybrid composite reaching to 93.3%. The catalytic activity and stability of YADH in ALG-SiO₃ hybrid composites was significantly higher than that in ALG gel due to higher enzyme loading efficiency and improved carrier microenvironment. Comparedto other two types of ALG-SiO₃ hybrid composites, class II ALG-SiO₃ hybrid composite showed much better performance due to the more appropriate microenvironment for enzyme encapsulation.As a result, class II ALG-SiO₃ hybrid composite was chosen to coencapsulate three dehydrogenases, including formate dehydrogenase (FateDH), formaldehyde dehydrogenase (FaldDH) and alcohol dehydrogenase (ADH). Carbon dioxide was converted to methanol through a novel three sequential reduction at natural pH and temperature, and methanol yields based on the NADH concentration was as high as 97.2%.