Toward Automated Enzymatic Glycan Synthesis in a Compartmented Flow Microreactor System

Abstract

Immobilized microfluidic enzyme reactors (IMER) are of particular interest for automation of enzyme cascade reactions. Within an IMER, substrates are converted by paralleled immobilized enzyme modules and intermediate products are transported for further conversion by subsequent enzyme modules. By optimizing substrate conversion in the spatially separated enzyme modules purification of intermediate products is not necessary, thus shortening process time and increasing space-time yields. The IMER enables the development of efficient enzyme cascades by combining compatible enzymatic reactions in different arrangements under optimal conditions and the possibility of a cost-benefit analysis prior to scale-up. These features are of special interest for automation of enzymatic glycan synthesis. We here demonstrate a compartmented flow microreactor system using six magnetic enzyme beads (MEBs) for the synthesis of the non-sulfated human natural killer cell-1 (HNK-1) glycan epitope. MEBs are assembled to build compartmented enzyme modules, consisting of enzyme cascades for the synthesis of uridine 5'-diphospho-α-d-galactose (UDP-Gal) and uridine 5'-diphospho-α-d-glucuronic acid (UDP-GlcA), the donor substrates for the Leloir glycosyltransferases β4-galactosyltransferase and β3-glucuronosyltransferase, respectively. Glycan synthesis was realized in an automated microreactor system by a cascade of individual enzyme module compartments each performing under optimal conditions. The products were analyzed inline by an MS-system connected to the microreactor. The high synthesis yield of 96% for the non-sulfated HNK-1 glycan epitope indicates the excellent performance of the automated enzyme module cascade. Furthermore, combinations of other MEBs for nucleotide sugars synthesis with MEBs of glycosyltransferases have the potential for a fully automated and programmed glycan synthesis in a compartmented flow microreactor system.

Multi-Enzyme Systems in Flow Chemistry

“…Heinzler et al developed a cascade of immobilized microfluidic enzyme reactors where intermediate products are transported for further conversion in subsequent reactor modules under optimal conditions for the production of glycan [117]. Six different enzymes, namely galactokinase, UDP-sugar pyrophosphorylase, UDP-glucosedehydrogenase, NADH oxidase, β1,4-galactosyltransferase and glucuronosyltransferase, were immobilized on magnetic particles and loaded to each microreactor compartment at the best loading yield (g immobilized enzyme per L of settled beads).…”

Advances in Magnetic Nanoparticles Engineering for Biomedical Applications—A Review

“…Coating (for example by poly-l-lysine) to obtain stable nonpyrogenic MNP suspension [115] product homogeneity quality reduction by concentration gradients and hot spots in the reaction flask [25,51] enhanced quality due to homogeneous morphology, narrow size distribution [25,67,116] high within one bacteria strain but strain variation possible [52][53][54]95] reproducibility, production rate and scale-up capability significant batch to batch variations in size, morphology, and magnetic properties [25,111,[117][118][119], poor scaling up capability. A reported study from Lin et al showed a production rate of 4.73 g/h for microfluidic synthesis comparing to 1.4 g/h for conventional synthesis with the same conditions [89] continuous production, no batch-to-batch variation, high scale-up capability high at the defined environmental conditions [92], mg/(L • day) production rate [52], high scale-up capability, though challenging due to long term bacteriostatic growth conditions [38,40,46,78] clogging not applicable microchannel-wall blocking during nucleation or by agglomeration [77,104,[120][121][122] not applicable automation poor feasible/integratable [66,123,124] capability of online characterization not applicable for batch, though magnetic characterization of whole batches by magnetic particle spectroscopy is feasible parameter control and synthesis adjustment feasible during synthesis, control of magnetic parameters by magnetic particle spectroscopy [25,125] and NMR [126] cost low, common lab equipment expensive microreactor fabrication expensive specialized equipment…”

Continuous Flow Organophosphorus Chemistry

“…A recent contribution by Elling, Franzreb et al on the development of a continuous flow system for biocatalyzed cascade reactions towards non-sulfated human natural killer cell-1 (HNK-1) glycan epitope (29, Figure 10) included the preparation of uridine diphosphate galactose (28) as an intermediate, however with a slightly different approach [53] than Wang et al [52] Here, the preparation of 28 from 27 relied on the direct use of UTP and UDP-sugar pyrophosphorylase (USP). The entire process sequence relied on six enzymes immobilized on magnetic parti- Figure 8.…”

“…Chemical structure of the non-sulfated human natural killer cell-1 (HNK-1) glycan epitope. [53] Other specific applications involving flow dephosphorylation reactions were also reported. For instance, Wang et al de-scribed the use of a three-enzyme cascade featuring a capillary monolithic reactor for the degradation of DNA strands into single nucleosides.…”