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 counte...

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Published in:Biotechnology and bioengineering 2016-09, Vol.113 (9), p.1862-1872
Main Authors: Bolivar, Juan M., Krämer, Christina E. M., Ungerböck, Birgit, Mayr, Torsten, Nidetzky, Bernd
Format: Article
Language:English
Subjects:
Bioengineering
Bioreactors
Enzymes
Enzymes, Immobilized - chemistry
Enzymes, Immobilized - metabolism
Equipment Design
Escherichia coli
falling-film microreactor
gas/liquid/solid biotransformation
immobilized oxidase
microchannel
Microtechnology - instrumentation
Microtechnology - methods
Oxidoreductases - chemistry
Oxidoreductases - metabolism
Oxygen - analysis
Oxygen - chemistry
Oxygen - metabolism
oxygen mass transfer
Recombinant Fusion Proteins - chemistry
Recombinant Fusion Proteins - metabolism
Zbasic2-binding module
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Summary: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‐Al2O3 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 KLa, comprising a KL of about 7 × 10−5 m · s−1 and a specific surface area of up to 105 m−1. Biotechnol. Bioeng. 2016;113: 1862–1872. © 2016 Wiley Periodicals, Inc. The authors report integrated development and detailed characterization of an advanced falling‐film microreactor (FFMR) for O2‐dependent chemical transformations with immobilized enzymes. Reversible, selective, and oriented protein immobilization was achieved through concurrent designs of the microchannel surface (γ‐Al2O3 wash‐coat layer) and enzyme used (Zbasic2 fusion protein). Advanced non‐invasive optical sensing was applied to measure liquid‐phase oxygen concentrations in both in‐ and out‐flow as well as directly in the microchannels. The immobilized enzyme FFMR offers extremely efficient O2 supply to the biocatalytic reaction in a manner protecting the enzymes agai
ISSN:0006-3592
1097-0290
DOI:10.1002/bit.25969