Functionalized porous ceramic microbeads as carriers in enzymatic tandem systems

Porous Al2O3/SiO2 microbeads (MBs) are propped as carriers for enzymes used in flow reactor setups for tandem enzymatic reactions. Glucose oxidase (GOx) and catalase (CAT) perform sequential reactions. GOx catalyzes the conversion of glucose and O2 to gluconic acid and H2O2. CAT decomposes H2O2 to H2O and O2. Spherical MBs were fabricated via ionotropic gelation followed by rapid-sintering (1200 °C/5 min), featuring reasonably high open porosity (∼50%), appropriate specific surface area (30 ± 5 m2/g) and multi-modal pore sizes (d50 = 79 nm) for envisaged enzyme immobilizations. After activation and amino-silanization, GOx and CAT were successfully immobilized onto MBs applying an EDC/sulfo-NHS crosslinking reaction. Under varied pH and temperature conditions, the immobilized enzymes showed advantages towards harsh conditions (pH 2; 70 °C) and demonstrated high activities at neutral pH and 21 ± 2 °C (RT), serving as optimal conditions for flow experiments. By varying flow rates (0.1–6 ml/min) in individual reactor compartments, GOx- and CAT-functionalized MBs achieved satisfiable H2O2 conversion rates at 1.0 ml/min for 24 h. In tandem use, obtained H2O2 is constantly degraded over time, maintaining high enzymatic overall performances for two more operation cycles. The presented strategy is particularly interesting, as it can potentially be transferred to other multi-enzyme reaction systems.