Enzyme immobilization and its applications using nanostructured materials

Abstract

Enzymes are versatile biocatalysts and find increasing applications in various industrial processes including organic synthesis and bioremediation. However, the application of enzymes is often limited by the short catalytic lifetime and by the difficulty in recovery and recycling. Recent breakthroughs in nanotechnology provide more diverse materials and approaches to solve these problems. In this study, different types of nanostructured materials were used as hosts for enzyme immobilization since they provided a large surface area, leading to a higher volumetric enzyme activity. Crowding effect of enzyme molecules in confined spaces was investigated using entrapped enzymes in mesoporous silica. Magnetically separable and highly stable enzyme-palladium nanocomposites were developed for further application into dynamic kinetic resolution. With two different model enzymes, α-chymotrypsin (CT) and lipase (LP), crosslinked enzyme aggregates (CLEAs) were developed in hierarchically-ordered mesocellular mesoporous silica (HMMS) for effective enzyme entrapment and stabilization. The immobilization was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale CLEAs entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). Using this ship-in-a-bottle approach, high loading capacity and greatly improved stability were achieved mainly due to the prevention of enzyme leaching and autolysis. CLEA in one-dimensional bent pores of SBA-15 mesoporous silica did not also leach out. Highly loaded CLEAs in SBA-15 are so stable that the initial enzyme activity was maintained without any loss under rigorous shaking for more than two weeks. Silica nanoparticles prepared by Stöber method were utilized for covalent immobilization of lipase. Glycidyl methacrylate (GMA) and ethylene diamine (EDA) were incorporated onto the surface of the na...