Immobilization of Alcohol Dehydrogenase - Development of Support Materials and Immobilization Methods

On the path towards a sustainable society, enzyme catalysis becomes ever more important as an environmentally friendly alternative for the industrial production of chemicals. Enzyme immobilization is a principal technique to facilitate the widespread implementation of enzymes in the industry. Enzyme immobilization enables enzyme reuse and enzyme stabilization for increased viability in continuous operations.

Enzyme immobilization involves the attachment of enzymes to a solid support by physical or chemical interactions. The immobilization method and support are decisive factors in the success of enzyme immobilization systems (i.e., the activity retention, stability, and reusability of the immobilized enzyme). The objective of this project was to investigate enzyme immobilization for applications in inorganic membrane reactors using a holistic approach, where we studied enzyme immobilization techniques and membrane fabrication together to guide the design of immobilization methods and inorganic membranes with properties tailored for enzyme immobilization. Inorganic membranes were supplied by project collaborators, who applied the principles of material science and ceramic engineering for the development and fabrication of the inorganic membranes in a parallel project.

The main motivation for utilizing inorganic support materials for enzyme immobilization is the high stability of the materials, including chemical, thermal, and mechanical stability, which allows a stable operation and enables support regeneration when the process efficiency decreases due to fouling of the support or enzyme deactivation. Besides high stability, inorganic membranes offer several other advantages, such as high permeability and long service life. Furthermore, inorganic membranes have high microbial stability and are well-suited for enzyme immobilization. We started our investigations by studying the interactions between enzymes and different inorganic raw materials that are commonly used for membrane fabrication to identify suitable materials and important design parameters for membrane fabrication. Alcohol dehydrogenase (ADH) was used as a model enzyme and was immobilized on raw powders of aluminum oxide, silicon carbide, titanium oxide, and yttria stabilized zirconia by physical adsorption and covalent bonding. The stability and activity of the immobilized ADH were evaluated based on the properties of the inorganic powders (i.e., surface area, particle size distribution, and surface charge). Enzyme loading on the particles and enzyme activity were greatly affected by the surface area and surface charge of the powders. Aluminum oxide and silicon carbide powders provided suitable conditions for the immobilization of ADH.

Subsequently, aluminosilicate nanofiber membranes were fabricated by project collaborators using electrospinning. The nanofiber membranes offered a large surface area (11.7 m2 g-1), a high porosity, and pore sizes fit for the immobilization of ADH. These properties combined ensured high permeability and high enzyme loading in the membranes —the enzymes could penetrate the membranes but were retained within the nanofiber structure. Similarly, the membranes offered favorable conditions for enzyme immobilization. Up to 96% immobilization efficiency was observed when the enzyme immobilization was conducted in filtration mode, which resulted in enzyme entrapment in the nanofiber membranes. The immobilized enzymes showed high activity, with near-complete substrate conversion in a single pass through the biocatalytic membranes, however, the enzyme stability was limited by leakage from the membranes. Moreover, the nanofiber membranes were extremely fragile, which limited their applications in general. It was proposed to seal the membranes by depositing a layer of polyelectrolytes on the membranes, for example by the layer-by-layer (LbL) assembly method, to prevent enzyme leakage from the membranes and thus maintain high enzyme activity.

We demonstrated how polyelectrolyte LbL assembly could be used to tailor the properties of nanofiltration membranes, including the pore size, membrane thickness, and surface charge, and could thus be used to promote favorable conditions for enzyme immobilization. We studied solute transport in polyelectrolyte multilayer membranes of different thicknesses and pore sizes to evaluate the effects of membrane thickness on solute transport in the membranes, which could have possible implications for enzyme immobilization. We investigated alternative methods for enzyme immobilization on membranes that could simultaneously be used to control the transport properties of the membranes, including immobilization by polyelectrolyte LbL assembly and interfacial polymerization. We described how the polyelectrolyte LbL assembly method could be used as a versatile and reversible method for enzyme immobilization. Similarly, we described how immobilization by interfacial polymerization provided a facile method for the fabrication of stable biocatalytic membranes with controllable properties. In experiments involving the immobilization of ADH on polymeric ultrafiltration membranes by interfacial polymerization, we observed a decrease in water permeability by increasing the polymerization time and thus the degree of cross-linking of the biocatalytic membrane. Furthermore, we observed 100% immobilization efficiency and up to 70% substrate conversion in a single pass through the biocatalytic membranes.

In summary, we investigated the possibility of fabricating inorganic membranes with properties tailored for enzyme immobilization. We applied inorganic nanofiber membranes for the immobilization of ADH and found that the membranes offered excellent conditions for the immobilization of the enzyme, with high enzyme loading and activity. However, the fragility of the membranes limited their applications at this stage. Nevertheless, the promising results should be an encouragement to continue the development of inorganic nanofiber membranes with higher stability and mechanical flexibility. We further demonstrated how membranes surface modification could be used to promote favorable conditions for enzyme immobilization.