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.

Demystifying the Flow: Biocatalytic Reaction Intensification in Microstructured Enzyme Reactors

“…Differences between A imm calculated and A imm measured are probably attributable to differences in the actual roughness of the glass surfaces used. Changes in surface roughness are ascribable to variable procedures of microchannel fabrication (e.g., chemical etching . By comparing A app to the experimental A imm the effectiveness factor ( η ) of the immobilized enzyme was obtained according to Equation .…”

“…This is important for efficient process development. Although many studies of enzyme immobilization in microreactors have been performed, only few have attempted to determine the upper bounds of A imm and its relationship with STY_max based on the characteristic length …”

Kinetic Analysis of Enzymes Immobilized in Porous Film Arrays

“…14–16 However, studying enzymes immobilized in adherent films eliminates particle damage from aggressive stirring 67 and facilitates integration with confocal microcopy methods. 68–70 The latter is the subject of future work to study the spatial distribution of enzyme, substrate, or pH in the support matrix. Taken together, we offer a quantitative framework to characterize the kinetics of immobilized enzymes and subsequently guide rational design choices (e.g., enzyme loading, geometry) for immobilized-enzyme devices.…”

The Promises and the Challenges of Biotransformations in Microflow

“…High surface‐to‐volume ratio of microchannel reactors offers the possibility to obtain relatively high enzyme loads of biocatalysts attached to the surfaces. It also enables attachment of a single layer of cells (Figure a), oriented single‐layer immobilization of enzymes fused with tags (Figure e), and creation of bacterial biofilms (Figure b), which together with short diffusional paths lead to excellent accessibility of a biocatalyst for the substrate and thereby high biocatalyst productivity.…”

“…[78] Monitoring of enzyme-catalyzed oxidations using dissolved oxygen was performed using either dissolved indicator dye, [82] by oxygen sensing layer deposited on the inner surface of a resealable flow-cell [48] and at the in-and outlet tubing using an external magnet and magnetic nanoparticles covered with the O 2 -sensitive fluorescent dye. [83] Furthermore, surface-attached dyed nanoparticles were used to evaluate the oxygen mass transfer coefficient in the falling film microreactor used for gas-liquid-solid biotransformation. [43] On the other hand, optical pH sensors have been much less implemented in enzymatic microreactors, primarily due to the limited operating range.…”