Strategies for Automated Enzymatic Glycan Synthesis (AEGS)

Hussnaetter, Kai Philip; Palm, Philip; Pich, Andrij; Franzreb, Matthias; Rapp, Erdmann; Elling, Lothar

doi:10.1016/j.biotechadv.2023.108208

Cited by 5 publications

(5 citation statements)

References 152 publications

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“…The superordinate goal is not only the reuse of enzymes but also the establishment of a fully automated and scalable synthesis of customized glycans. 28,29 There are various methods for the immobilization of enzymes. According to Sheldon, 30 enzymes can be crosslinked to each other, or bound to a support material by entrapment or postattachment.…”

Section: Introductionmentioning

confidence: 99%

“…Only a few GTs have been immobilized for their use in glycan synthesis, − making it all the more important to take this step to enable more economical synthesis of glycans. The superordinate goal is not only the reuse of enzymes but also the establishment of a fully automated and scalable synthesis of customized glycans. , …”

Section: Introductionmentioning

confidence: 99%

“…However, covalent binding can result in chemical or conformational changes in the enzyme, reducing its activity. , Therefore, using binding techniques, where the reactive group interacts selectively with a very specific part of the enzyme, can be favorable. , For example, Zou et al have developed a method using sortase-mediated covalent ligation for enzyme immobilization . Protein fusions such as Halo-Tag or SNAP-Tag can be used for reaction with chlorine-functionalized ligands or benzyl guanine . The SpyTag (SpyT) SpyCatcher (SpyC) technology links the two components through the formation of an isopeptide bond …”

Section: Introductionmentioning

confidence: 99%

“…35 Protein fusions such as Halo-Tag 37 or SNAP-Tag 38 can be used for reaction with chlorine-functionalized ligands or benzyl guanine. 29 The SpyTag (SpyT) SpyCatcher (SpyC) technology links the two components through the formation of an isopeptide bond. 39 The carrier for the immobilization of enzymes is chosen dependent on the required properties.…”

Section: Introductionmentioning

confidence: 99%

“…42 In this work, we aim at the synthesis of microgels with immobilized Leloir glycosyltransferases (GTs) and work toward a fully automated enzymatic glycan synthesis (AEGS) in an enzyme reactor. We apply the knowledge about the immobilization of GTs previously comprehensively summarized by Hussnaetter et al 29 as the basis for our work and combine it with experimental data.…”

Section: Introductionmentioning

confidence: 99%

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Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis

Sommerfeld,

Palm,

Hussnaetter

et al. 2024

Biomacromolecules

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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 β4GalT and α3GalT in an enzymatic reaction with high yields. Moreover, a cascade of β4GalT and β3GlcNAcT was successfully implemented. These results pave the way toward a modular membrane bioreactor for automated glycan synthesis containing the presented biocatalytic microgels.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Microgels with Immobilized Glycosyltransferases for Enzymatic Glycan Synthesis

Sommerfeld,

Palm,

Hussnaetter

et al. 2024

Biomacromolecules

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Multienzymatic Cascades and Nanomaterial Scaffolding—A Potential Way Forward for the Efficient Biosynthesis of Novel Chemical Products

Hooe,

Smith,

Dean

et al. 2023

Advanced Materials

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Synthetic biology is touted as the next industrial revolution as it promises access to greener biocatalytic syntheses to replace many industrial organic chemistries. Here, it is shown to what synthetic biology can offer in the form of multienzyme cascades for the synthesis of the most basic of new materials—chemicals, including especially designer chemical products and their analogs. Since achieving this is predicated on dramatically expanding the chemical space that enzymes access, such chemistry will probably be undertaken in cell‐free or minimalist formats to overcome the inherent toxicity of non‐natural substrates to living cells. Laying out relevant aspects that need to be considered in the design of multi‐enzymatic cascades for these purposes is begun. Representative multienzymatic cascades are critically reviewed, which have been specifically developed for the synthesis of compounds that have either been made only by traditional organic synthesis along with those cascades utilized for novel compound syntheses. Lastly, an overview of strategies that look toward exploiting bio/nanomaterials for accessing channeling and other nanoscale materials phenomena in vitro to direct novel enzymatic biosynthesis and improve catalytic efficiency is provided. Finally, a perspective on what is needed for this field to develop in the short and long term is presented.

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