title: Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions.
abstract: Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica-binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d-amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10(-5)  nmol/mm(2) ). Surface attachment of the enzyme was completely stable under flow. The operational half-life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d-Met into α-keto-γ-(methylthio)butyric acid was characterized in single-pass and recycle reactor configurations, employing in-line measurement of dissolved O2 , and off-line determination of the keto-acid product. Reaction-diffusion time-scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII  ≤ 0.3). Potential of the microreactor for intensifying O2 -dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342-2349. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, Graz, A-8010, Austria.
authors.forename: Juan M
authors.initials: JM
authors.lastname: Bolivar
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, Graz, A-8010, Austria.
authors.forename: Marco A
authors.initials: MA
authors.lastname: Tribulato
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, Graz, A-8010, Austria.
authors.forename: Zdenek
authors.initials: Z
authors.lastname: Petrasek
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, Graz, A-8010, Austria. bernd.nidetzky@tugraz.at.|Austrian Centre of Industrial Biotechnology (ACIB), Graz, Austria. bernd.nidetzky@tugraz.at.
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D000327:Adsorption; D019149:Bioreactors; D002384:Catalysis; D003605:D-Amino-Acid Oxidase / Q000737:chemistry*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005898:Glass / Q000737:chemistry*; D044085:Microfluidics / Q000295:instrumentation*; D010100:Oxygen / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry; D013379:Substrate Specificity; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D012822:Silicon Dioxide; D003605:D-Amino-Acid Oxidase; D010100:Oxygen
keywords: enzymatic oxidation; flow microreactor; glass; immobilization; microfluidics; silica-binding module