Investigations on transglycosylation reactions of glycosidases and glycosynthases for the syntheses of (poly-)N-acetyllactosamine glycans

  • Untersuchungen zu Transglykosylierungsreaktionen von Glykosidasen und Glykosynthasen für die Synthesen von (poly-)N-Acetyllaktosamin Glykanen

Henze, Manja; Elling, Lothar (Thesis advisor); Schwaneberg, Ulrich (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


Growing ecological concern drives researchers’ interest to enlarge the "synthetic playing fields" in bio-driven catalysis of carbohydrates. The challenge is to fill in the gaps between naturally occurring properties of a biocatalyst and requirements of an industrial process. In the sense of this demand a next generation of robust biocatalysts featuring minimized product hydrolysis, uncommon reaction temperature or media composition is pursued with the overall goal to reach a high product yield. Hence, glycoside hydrolases are excellent catalysts to enable a green synthesis of high-value sugar structures due to their usability in large-scale production and cost-effective, purchasable commodities. This thesis highlights the exploitation of glycoside hydrolases from Bacillus circulans and Pyrococcus woesei for the syntheses of (poly-)N-acetyllactosamine (LacNAc) derivatives. LacNAc is a fundamental motif in mammalian glycoproteins that has high potential for pharmaceutical applications. However, the synthesis of LacNAc structures in transgalactosylation of β-galactosidases is often accompanied by hydrolysis of the donor substrate and product. To overcome this drawback two fundamental strategic directions, protein- and reaction-engineering, exist. In particular, the development of nucleophile mutants of glycosidases (glycosynthases) has gained attention following the protein engineering route. Based on the protein structure of the mesophilic Bacillus circulans β-galactosidase (BgaC) from glycoside hydrolase family 35, the catalytic glutamic acid at position 233 was exchanged by non-nucleophilic amino acids to generate a glycosynthase. Here, eliminated hydrolytic activity with perpetuated transglycosylation performance was accomplished with His6BgaC/Glu233Gly. A systematic synthesis of LacNAc type 1 (3Galβ1-3GlcNAcβ1-, lacto-N-biose) structure was executed by optimizing key parameters like enzyme concentration, donor/acceptor ratio and acceptor concentration. In fact, the resulting active glycosynthase His6BgaC/Glu233Gly was shown to be suitable for the stereo- and regioselective synthesis of β1-3-linked galactosides with different N-acetylglucosamine acceptor substrates in moderate-to-high yields. Moreover, the functionalized lacto-N-biose compounds represent the starting point for coupling reactions onto biomaterial surfaces. In combination with two recombinant glycosyltransferases, β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori (β3GlcNAcT) and human β1,4-galactosyltransferase-1 (β4GalT-1), the glycosynthase His6BgaC/Glu233Gly enabled the individual modular design of type 1 (3Galβ1-3GlcNAcβ1-) and type 2 (3Galβ1-4GlcNAcβ1-) LacNAc oligomers by sequential or one-pot reaction processes. For the first time, a poly-LacNAc type 1 oligomer and mixed type 2/type 1 poly-LacNAc oligomers were obtained. The second part of this thesis addressed the hyperthermophilic β-glycoside hydrolase from Pyrococcus woesei. Initially, microwave-assisted synthesis - as a key technology in green chemistry - was successfully applied in transgalactosylation reactions with lactose as donor substrate. Reactions under microwave irradiation, performed at a temperature far below the optimal temperature of this thermozyme, resulted in higher product yields accompanied by decreased side products. This sophisticated experimental set-up shows a general synthesis strategy with a hyperthermophilic biocatalyst using heat-sensitive substrates. Finally, the combination of microwave heating technology and molecular engineering of this thermophilic glycosidase was a logical consequence to round off the investigations on different enzymatic strategies with glycosidases. Therefore, three active glycosynthases (Glu414Gly, Glu414Ser, Glu414Ala) of the hyperthermophilic glycosidase were created. Remarkably, all variants showed transglycosylation capability with three different α-glycosyl fluorides (αGalF, αGlcF, αManF). Subsequently, a detailed examination of optimal reaction conditions with αGalF was performed to improve the product yield of LacNAc type 2. In addition, transgalactosylation reactions with the hyperthermophilic glycosynthase Glu414Ala and the heat-labile glycosyl fluoride led to increased product yields under microwave irradiation compared to reactions under thermal heating at comparable temperatures. In summary, the presented results demonstrate the power of rational protein design and reaction engineering for the production of tailor-made N-acetyllactosamine glycan structures using glycosidases and glycosynthases.


  • Laboratory for Biomaterials [162820]
  • Department of Biology [160000]
  • Chair of Biotechnology [162610]