Modular microgel flow reactors for automated glycan synthesis with immobilized enzymes

Chemical Engineering Journal, vol. 543, pp. 178229

Abstract

Glycans are biomolecules that play roles in numerous biological processes and are becoming increasingly important for applications in biomedicine, nutrition, pharmacy, and research. Their structural diversity makes glycans attractive target molecules, but also poses a challenge in their synthesis. Compared to chemical synthesis, enzymatic glycan synthesis offers various advantages: high regio- and stereoselectivity, the use of mild reaction conditions, and better ecological and economic sustainability. Automated enzymatic glycan synthesis (AEGS) is an essential development step in making defined glycan structures efficiently accessible on a larger scale. Here, we present a modular Mi crogel continuous flow R eactor for A utomated and continuous G lycan synthesis with immobilized E nzymes (MiRAGE). The enzymes are immobilized in microgels, SpyT-agarose particles, or Ni 2+ -IDA beads using different strategies. The immobilized enzymes are integrated into reactor compartments, which are supplied with substrates via a continuous reaction buffer flow. A semipermeable membrane ensures that the enzymes as well as substrates and products are retained in the module, while by-products can be selectively removed via a crossflow stream. The modular design allows several enzymatic reaction steps to be coupled, automating the synthesis of complex glycan structures. To this end, we demonstrate the concept towards the synthesis of the human milk oligosaccharide LNFPIII, Lewis X , blood group-, and Galili epitopes. In addition, we compare the different immobilization techniques and reactor strategies in terms of consistency and yield. MiRAGE represents a robust, scalable, and versatile approach that not only enables the automated synthesis of complex glycans but can also be transferred to other biocatalytic processes. Key points • Immobilization of SpyCatcher-glycosyltransferases on SpyTag agarose and microgels • Embedment of immobilized glycosyltransferases in a continuous flow bioreactor • Enzymatic glycan synthesis with removal of by-products and retention of biocatalysts

Authors 11

1. Kai Philip Hussnaetter corresponding Aachen Laboratory for Biomaterials

   RWTH Aachen University

   Affiliation as printed

   Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstraße 20, 52074, Aachen, Germany

2. Maria I. Pieper Aachen Functional and Interactive Polymers Institute of Technical and Macromolecular Chemistry

   RWTH Aachen University

   Affiliation as printed

   Functional and Interactive Polymers, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 2, Aachen, 52074, Germany

3. Ulrich Thiele

   Karlsruhe Institute of Technology

   Affiliation as printed

   Karlsruher Institute of Technology (KIT), Institute of Functional Interfaces, Hermann v. Helmholtz, Platz 1, 76344, Eggenstein-Leopoldshafen, Germany

4. Philip Palm Aachen Laboratory for Biomaterials

   RWTH Aachen University

   Affiliation as printed

   Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstraße 20, 52074, Aachen, Germany

5. Robert Burock

   Zentrum für Innovation und Technik in Nordrhein-Westfalen · Max Planck Institute for Dynamics of Complex Technical Systems

   Affiliation as printed

   Max Planck Institute for Dynamics of Complex Technical System, Bioprocess Engineering, Sandtorstrasse 1, 39106, Magdeburg, Germany

   glyXera GmbH, Brenneckestrasse 20 * ZENIT, 39120, Magdeburg, Germany

6. René Hennig

   Zentrum für Innovation und Technik in Nordrhein-Westfalen

   Affiliation as printed

   glyXera GmbH, Brenneckestrasse 20 * ZENIT, 39120, Magdeburg, Germany

7. Katharina Bleher

   Karlsruhe Institute of Technology

   Affiliation as printed

   Karlsruher Institute of Technology (KIT), Institute of Functional Interfaces, Hermann v. Helmholtz, Platz 1, 76344, Eggenstein-Leopoldshafen, Germany

8. Andrij Pich Aachen Functional and Interactive Polymers Institute of Technical and Macromolecular Chemistry

   RWTH Aachen University

   Affiliation as printed

   Functional and Interactive Polymers, Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 2, Aachen, 52074, Germany

9. Matthias Franzreb

   Karlsruhe Institute of Technology

   Affiliation as printed

   Karlsruher Institute of Technology (KIT), Institute of Functional Interfaces, Hermann v. Helmholtz, Platz 1, 76344, Eggenstein-Leopoldshafen, Germany

10. Erdmann Rapp

    Zentrum für Innovation und Technik in Nordrhein-Westfalen · Max Planck Institute for Dynamics of Complex Technical Systems

    Affiliation as printed

    Max Planck Institute for Dynamics of Complex Technical System, Bioprocess Engineering, Sandtorstrasse 1, 39106, Magdeburg, Germany

    glyXera GmbH, Brenneckestrasse 20 * ZENIT, 39120, Magdeburg, Germany

11. Lothar Elling Aachen Laboratory for Biomaterials

    RWTH Aachen University

    Affiliation as printed

    Laboratory for Biomaterials, Institute for Biotechnology and Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstraße 20, 52074, Aachen, Germany