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

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.

What’s new in flow biocatalysis? A snapshot of 2020–2022

“…For the trienzymatic synthesis of sialic acid, N-acyl-D-glucosamine 2-epimerase, N-acetylneuraminate lyase, and cytidine monophosphate (CMP)-sialic acid synthetase, were immobilized into bulk hydrogels and microstructured hydrogel-enzyme-dot arrays, which were then integrated into microfluidic devices. This study demonstrates how immobilizing enzymes in (compartmentalized) microfluidic devices can circumvent cross-inhibitions occurring under continuous conditions (Obst et al, 2021). Advances in the development of new controllable modular immobilization strategies were also reported.…”

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

“…122 The immobilization of three enzymes required for the synthesis of CMP-Neu5Ac within hydrogels as a method to avoid CTP cross-inhibition of N-acyl-D-glucosamine 2epimerase (AGE) and N-acetylneuraminate lyase (NAL) has been described. 123 Utilizing a confinement and compartmentalization strategy enabled higher substrate concentrations to be used. Immobilization has also been used to improve the overall efficiency of sialyl galactoside synthesis using a two-step route from Neu5Ac.…”

“…These reactions were carried out on a preparative (10 mL) scale at 2 mM substrate concentration, and the immobilized enzyme could be reused up to five times 122. The immobilization of three enzymes required for the synthesis of CMP-Neu5Ac within hydrogels as a method to avoid CTP cross-inhibition of N-acyl-D-glucosamine 2epimerase (AGE) and N-acetylneuraminate lyase (NAL) has been described 123. Utilizing a confinement and compartmentalization strategy enabled higher substrate concentrations to be used.…”

Co‐Immobilization of GOD & HRP on Y‐Shaped DNA Scaffold and the Regulation of Inter‐Enzyme Distance

“…[13] Traditional multienzymes coimmobilization methods, including adsorption, covalent binding, and cross-linking, are all random coimmobilization, which is difficult to accurately regulate the spatial position of enzyme and the inter-enzyme distance and thus results in low cascade efficiency. [10,14] To solve this problem and make multiple enzymes to generate substrate channeling effect, [15] various new strategies and artificial scaffolds are developed to immobilize multiple enzymes, [13,16,17,18] such as layer-by-layer coimmobilization strategy to coimmobilize glucose oxidase (GOD) and horseradish peroxidase (HRP), [19] surface displayed fusion technology to establish multienzymes complex, [14] MOF co-encapsulation strategy to co-immobilize GOD and HRP, [20] etc. Recently, Mafferis et al also utilized the nano chamber "cluster" to control the distance between GOD and lactoperoxidase, effectively improving the cascade efficiency.…”