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Let the substrate flow, not the enzyme: Practical immobilization of d ‐amino acid oxidase in a glass microreactor for effective biocatalytic conversions
Author(s) -
Bolivar Juan M.,
Tribulato Marco A.,
Petrasek Zdenek,
Nidetzky Bernd
Publication year - 2016
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.26011
Subject(s) - microreactor , immobilized enzyme , chemistry , microchannel , substrate (aquarium) , glucose oxidase , chemical engineering , bioreactor , chromatography , enzyme catalysis , porous glass , catalysis , enzyme , organic chemistry , materials science , nanotechnology , porosity , oceanography , geology , engineering
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 O 2 , 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 O 2 to the solid surface ( Da II ≤ 0.3). Potential of the microreactor for intensifying O 2 ‐dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342–2349. © 2016 Wiley Periodicals, Inc.