(654i) Self-Immobilized Cross-Linked Enzyme Aggregates for Biocatalysis

Enzymes have many advantages over inorganic catalysts but they are very sensitive to temperature and pH. Recently, we showed that enzymes can be immobilized as cross-linked enzyme aggregate (CLEAs) without using any solid carriers to improve their stability. Firstly, millifluidic reactors with two laminar flows of an enzyme solution and an organic solvent were employed to prepare uniform CLEAs of cellulase and laccase. CLEAs of cellulase had a size between 200 and 400 nm and were highly uniform. They can be collected on silica gels or entrapped in a membrane as practical biocatalysts to hydrolyze cellulose with an activity up to 86% of the free cellulase. Because of their large size, CLEAs can also be encapsulated in calcium alginate beads without any leaching problems. This features allowed CLEAs of cellulase to be reused and recycled up to 10 times for hydrolysis of cellulose. Similarly, CLEAs of laccase were prepared and entrapped in a membrane for oxidization of phenolic compounds. Trypan blue, a dye molecule, can be degraded by using membrane-bound CLEAs continuously for 96 h. Finally, glucose oxidase (GOx) and horseradish peroxidase (HRP) were co-immobilized as combi-CLEAs by using a coaxial-flow millifluidic reactor. The combi-CLEAs were used to catalyze cascade chemical reactions and minimize the decomposition of hydrogen peroxide in the presence of catalase. As a result, the overall reaction rate increased by 14 times compared to free enzyme mixtures in the presence of 6.3 mM of catalase. The combi-CLEAs were further exploited to develop colorimetric glucose assays with a limit of detection of 0.5 mM and a linear range up to 27.8 mM. In the future, millifluidic reactors can be used to prepare CLEAs with tunable sizes and properties for various industrial applications.