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

Abstract

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: 600 μm; depth: 200 μm). 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 Zbasic2 and the channel surface contains a negatively charged γ-Al2 O3 wash-coat layer. Non-covalent wall attachment of the chimeric Zbasic2 _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(-4) m. Direct O2 concentration measurements at the channel surface demonstrated that the liquid side mass transfer coefficient (KL ) for O2 governed the overall gas/liquid/solid mass transfer and that the O2 transfer rate (≥0.75 mM · 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 KL a, comprising a KL of about 7 × 10(-5) m · s(-1) and a specific surface area of up to 10(5) m(-1) . Biotechnol. Bioeng. 2016;113: 1862-1872. © 2016 Wiley Periodicals, Inc.

Process intensification for O2‐dependent enzymatic transformations in continuous single‐phase pressurized flow

“…Using a falling‐film microreactor for the GOX reaction, Illner, Hofmann, Löb, and Kragl () obtained STY of ~80 mM/min at 20–30% of conversion at low TON (2 × 10 3 ). Using the same type of reactor, we reported a STY of up to 45 mM/min, however, at low TON of soluble GOX and low conversion (Bolivar, Krämer, et al, ). In both cases (Bolivar, Krämer, et al, ; Illner et al, ), the equivalent TOF (TON/residence time) was below the TOF of the catalyst at air‐saturated conditions (~1 × 10 4 min −1 ).…”

“…Using the same type of reactor, we reported a STY of up to 45 mM/min, however, at low TON of soluble GOX and low conversion (Bolivar, Krämer, et al, ). In both cases (Bolivar, Krämer, et al, ; Illner et al, ), the equivalent TOF (TON/residence time) was below the TOF of the catalyst at air‐saturated conditions (~1 × 10 4 min −1 ). Boundaries in terms of STY and catalyst productivity were theoretically discussed in a seminal study by Dencic and coworkers (Dencic, Meuldijk, et al, ).…”

“…k L a values of up to 30 min −1 were reported for segmented gas–liquid flow in microchannels (Kashid et al, ). A falling‐film microreactor operated in continuous countercurrent gas–liquid flow showed a k L a of 450 min −1 (Bolivar, Krämer, Ungerböck, Mayr, & Nidetzky, ). These developments notwithstanding, important engineering problems remain.…”

Let the substrate flow, not the enzyme: Practical immobilization of d‐amino acid oxidase in a glass microreactor for effective biocatalytic conversions

“…The low solubility of O 2 at atmospheric pressure obviously limits the immediate output from reactions in single‐phase flow. This could be overcome by using either gas‐liquid multiphase flow systems (Bolivar et al, ; Karande et al, ), or a liquid phase strongly enriched in molecular oxygen (Gemoets et al, ). In particular, the microchannel flow reactor represents an apparatus appropriate and safe for high‐pressure process operation to enhance the O 2 concentration in solution under single‐phase flow conditions (Keybl and Jensen, ; Vanoye et al, ; Verboom, ).…”

Conscious coupling: The challenges and opportunities of cascading enzymatic microreactors

“…Active and passive mixing of miscible and immiscible phases can be very efficiently achieved in microsystems [98,99]. Multiphase microreactors offer therefore an interesting approach for taking advantage of phase boundaries such as gas-liquid, liquid-liquid and liquid-solid interfaces for intensifying biocatalytic reactions and transport in continuous flow processes [28,[100][101][102][103].…”