Development of a fully integrated falling film microreactor for gas-liquid-solid biotransformation with surface immobilized O₂-dependent enzyme

Microstructured flow reactors are powerful tools for the development of multiphase biocatalytic transformations. To expand their current application also to O₂-dependent enzymatic conversions, we have implemented a fully integrated falling film microreactor that provides controllable countercurrent gas-liquid phase contacting in a multi-channel microstructured reaction plate. Advanced non-invasive optical sensing is applied to measure liquid-phase oxygen concentrations in both in- and out-flow as well as directly in the microchannels (width: 600μm; depth: 200μm). Protein-surface interactions are designed for direct immobilization of catalyst on microchannel walls. Target enzyme (here: d-amino acid oxidase) is fused to the positively charged mini-protein Z_basic2 and the channel surface contains a negatively charged γ-Al₂O₃ wash-coat layer. Non-covalent wall attachment of the chimeric Z_basic2_oxidase resulted in fully reversible enzyme immobilization with fairly uniform surface coverage and near complete retention of biological activity. The falling film at different gas and liquid flow rates as well as reactor inclination angles was shown to be mostly wavy laminar. The calculated film thickness was in the range 0.5-1.3×10^-4m. Direct O₂ concentration measurements at the channel surface demonstrated that the liquid side mass transfer coefficient (K_L) for O₂ governed the overall gas/liquid/solid mass transfer and that the O₂ transfer rate (≥0.75mM·s^-1) vastly exceeded the maximum enzymatic reaction rate in a wide range of conditions. A value of 7.5 (±0.5)s^-1 was determined for the overall mass transfer coefficient K_La, comprising a K_L of about 7×10^-5m·s^-1 and a specific surface area of up to 10⁵m^-1.