Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis.

Glycans, composed of linked monosaccharides, play crucial roles in biology and find diverse applications. Enhancing their enzymatic synthesis can be achieved by immobilizing enzymes on materials such as microgels. Here, we present microgels with immobilized glycosyltransferases, synthesized through droplet microfluidics, immobilizing enzymes either via encapsulation or postattachment. SpyTag-SpyCatcher interaction was used for enzyme binding, among others. Fluorescamine and permeability assays confirmed enzyme immobilization and microgel porosity, while enzymatic activities were determined using HPLC. The potential application of microgels in cascade reactions involving multiple enzymes was demonstrated by combining ß4GalT and α3GalT in an enzymatic reaction with high yields. Moreover, a cascade of ß4GalT and ß3GlcNAcT was successfully implemented. These results pave the way toward a modular membrane bioreactor for automated glycan synthesis containing the presented biocatalytic microgels.

Characterization of Galectin Fusion Proteins with Glycoprotein Affinity Columns and Binding Assays.

Galectins are ß-galactosyl-binding proteins that fulfill essential physiological functions. In the biotechnological field, galectins are versatile tools, such as in the development of biomaterial coatings or the early-stage diagnosis of cancer diseases. Recently, we introduced galectin-1 (Gal-1) and galectin-3 (Gal-3) as fusion proteins of a His6-tag, a SNAP-tag, and a fluorescent protein. We characterized their binding in ELISA-type assays and their application in cell-surface binding. In the present study, we have constructed further fusion proteins of galectins with fluorescent protein color code. The fusion proteins of Gal-1, Gal-3, and Gal-8 were purified by affinity chromatography. For this, we have prepared glycoprotein affinity resins based on asialofetuin (ASF) and fetuin and combined this in a two-step purification with Immobilized Metal Affinity chromatography (IMAC) to get pure and active galectins. Purified galectin fractions were analyzed by size-exclusion chromatography. The binding characteristics to ASF of solely His6-tagged galectins and galectin fusion proteins were compared. As an example, we demonstrate a 1.6-3-fold increase in binding efficiency for HSYGal-3 (His6-SNAP-yellow fluorescent protein-Gal-3) compared to the HGal-3 (His6-Gal-3). Our results reveal an apparent higher binding efficiency for galectin SNAP-tag fusion proteins compared to His6-tagged galectins, which are independent of the purification mode. This is also demonstrated by the binding of galectin fusion proteins to extracellular glycoconjugates laminin, fibronectin, and collagen IV. Our results indicate the probable involvement of the SNAP-tag in apparently higher binding signals, which we discuss in this study.

Strategies for Automated Enzymatic Glycan Synthesis (AEGS).

Glycans are the most abundant biopolymers on earth and are constituents of glycoproteins, glycolipids, and proteoglycans with multiple biological functions. The availability of different complex glycan structures is of major interest in biotechnology and basic research of biological systems. High complexity, establishment of general and ubiquitous synthesis techniques, as well as sophisticated analytics, are major challenges in the development of glycan synthesis strategies. Enzymatic glycan synthesis with Leloir-glycosyltransferases is an attractive alternative to chemical synthesis as it can achieve quantitative regio- and stereoselective glycosylation in a single step. Various strategies for synthesis of a wide variety of different glycan structures has already be established and will exemplarily be discussed in the scope of this review. However, the application of enzymatic glycan synthesis in an automated system has high demands on the equipment, techniques, and methods. Different automation approaches have already been shown. However, while these techniques have been applied for several glycans, only a few strategies are able to conserve the full potential of enzymatic glycan synthesis during the process - economical and enzyme technological recycling of enzymes is still rare. In this review, we show the major challenges towards Automated Enzymatic Glycan Synthesis (AEGS). First, we discuss examples for immobilization of glycans or glycosyltransferases as an important prerequisite for the embedment and implementation in an enzyme reactor. Next, improvement of bioreactors towards automation will be described. Finally, analysis and monitoring of the synthesis process are discussed. Furthermore, automation processes and cycle design are highlighted. Accordingly, the transition of recent approaches towards a universal automated glycan synthesis platform will be projected. To this end, this review aims to describe essential key features for AEGS, evaluate the current state-of-the-art and give thought- encouraging impulses towards future full automated enzymatic glycan synthesis.