Enzymatic Synthesis of Sialic Acids in Microfluidics to Overcome Cross-Inhibitions and Substrate Supply Limitations

Obst F, Mertz M, Mehner PJ, Beck A, Castiglione K, Richter A, Voit B, Appelhans D (2021)

Publication Type: Journal article

Publication year: 2021

Journal

ACS Applied Materials and Interfaces American Chemical Society

DOI: 10.1021/acsami.1c12307

Abstract

Multienzymatic cascade reactions are a powerful strategy for straightforward and highly specific synthesis of complex materials, such as active substances in drugs. Cross-inhibitions and incompatible reaction steps, however, often limit enzymatic activity and thus the conversion. Such limitations occur, e.g., in the enzymatic synthesis of the biologically active sialic acid cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). We addressed this challenge by developing a confinement and compartmentalization concept of hydrogel-immobilized enzymes for improving the efficiency of the enzyme cascade reaction. The three enzymes required for the synthesis of CMP-Neu5Ac, namely, N-acyl-d-glucosamine 2-epimerase (AGE), N-acetylneuraminate lyase (NAL), and CMP-sialic acid synthetase (CSS), were immobilized into bulk hydrogels and microstructured hydrogel-enzyme-dot arrays, which were then integrated into microfluidic devices. To overcome the cytidine triphosphate (CTP) cross-inhibition of AGE and NAL, only a low CTP concentration was applied and continuously conveyed through the device. In a second approach, the enzymes were compartmentalized in separate reaction chambers of the microfluidic device to completely avoid cross-inhibitions and enable the use of higher substrate concentrations. Immobilization efficiencies of up to 25% and pronounced long-term activity of the immobilized enzymes for several weeks were realized. Moreover, immobilized enzymes were less sensitive to inhibition and the substrate-channeling effect between immobilized enzymes promoted the overall conversion in the trienzymatic cascade reaction. Based on this, CMP-Neu5Ac was successfully synthesized by immobilized enzymes in noncompartmentalized and compartmentalized microfluidic devices. This study demonstrates the high potential of immobilizing enzymes in (compartmentalized) microfluidic devices to perform multienzymatic cascade reactions despite cross-inhibitions under continuous flow conditions. Due to the ease of enzyme immobilization in hydrogels, this concept is likely applicable for many cascade reactions with or without cross-inhibition characteristics.

Authors with CRIS profile

Michael Mertz Lehrstuhl für Bioverfahrenstechnik Kathrin Castiglione Lehrstuhl für Bioverfahrenstechnik

Involved external institutions

Leibniz-Institut für Polymerforschung Dresden e. V. DE Germany (DE) Technische Universität Dresden DE Germany (DE)

How to cite

APA:

Obst, F., Mertz, M., Mehner, P.J., Beck, A., Castiglione, K., Richter, A.,... Appelhans, D. (2021). Enzymatic Synthesis of Sialic Acids in Microfluidics to Overcome Cross-Inhibitions and Substrate Supply Limitations. ACS Applied Materials and Interfaces. https://doi.org/10.1021/acsami.1c12307

MLA:

Obst, Franziska, et al. "Enzymatic Synthesis of Sialic Acids in Microfluidics to Overcome Cross-Inhibitions and Substrate Supply Limitations." ACS Applied Materials and Interfaces (2021).

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