Enzyme Selection in Chemical Enzymatic Method

Chemoenzymatic methods have emerged as the preferred and cost-effective approach for oligosaccharide synthesis, effectively addressing the challenges associated with chemical methods. Key catalysts play a crucial role in the reversible conversion of cellobiose and cellodextrin into α-D-glucose 1-phosphate, glucose, and cello oligosaccharides, respectively. The chemoenzymatic synthesis involving these phosphorylases follows an inverse phosphorylase mechanism, which bears similarities to inverse glycoside hydrolases. This mechanism involves direct displacement reactions catalyzed by general acid/base, with phosphate nucleophilically attacking in phosphorylases. And water-derived hydroxides are deprotonated by general base catalysts in hydrolases. Moreover, general acid catalysts contribute protons to glycoside oxygens, leading to product release with a reverse anomeric configuration.

Fig 1 Strategies for chemoenzymatic synthesis of carbohydrates. (Li, et al., 2019)

Chemoenzymatic Process for Oligosaccharide Manufacture at CD BioGlyco

Leveraging our extensive expertise in the field, CD BioGlyco offers reliable and advanced Technologies for Oligosaccharide Manufacture. We provide a chemoenzymatic approach to synthesize human milk oligosaccharide backbone structures. This process involves utilizing engineered β-N-acetylhexosaminase as a catalyst and active oxazoline as a donor molecule in a transglycosylation reaction. Through this method, the human milk oligosaccharide precursor lacto-N-triose II and three regioisomers are synthesized using an in situ chemoenzymatic cascade-generated oxazoline.

Additionally, CD BioGlyco provides high-quality production processes for cellobiose and cellulodextrin. The following outlines the production process of cellobiose and cellodextrins.

• Cloning, expression, and purification of cellobiose phosphorylase and cellodextrin phosphorylase
• Enzyme activity assay
• Synthesis of oligosaccharides by reverse phosphorolysis

Donor specificity in reverse phosphorolysis is determined using α-glycosyl 1-phosphate under optimal conditions with either glucose (with cellobiose phosphorylase) or cellobiose (with cellodextrin phosphorylase).

• Synthesis of oligosaccharides via sugar synthase-type reactions
• Separation of oligosaccharides by high-performance liquid chromatography (HPLC)

After heat inactivation and desalting of the product used for purification, the product is purified by HPLC, and the purity is verified by thin-layer chromatography (TLC).

• Structure determination of oligosaccharides

The structures of the oligosaccharide products are analyzed using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy.

Fig.2 Production process of cellobiose and cellulodextrin. (CD Bioglyco, 2023)

Applications

• Vaccine development: Chemoenzymatic processes are integral to the production of glycoconjugate vaccines. By combining the specificity of enzymes with chemical synthesis, researchers can create well-defined oligosaccharide antigens that mimic the surface structures of pathogens.
• Glycan array fabrication: Chemoenzymatic approaches are pivotal in constructing glycan arrays for high-throughput screening.
• Drug discovery and development: Chemoenzymatic processes contribute to drug discovery by enabling the synthesis of glycan-based small molecule inhibitors. Enzymes are utilized to selectively modify specific positions on oligosaccharide structures, generating compounds that disrupt glycan-protein interactions involved in disease pathways. These compounds can serve as lead compounds for the development of novel therapeutics.

Advantages

• Chemoenzymatic methods have played a significant role in the synthesis of precisely structured oligosaccharides, enriching our knowledge of their biological functions and potential therapeutic applications.
• By effectively blending the advantages of chemical and enzymatic synthesis, the chemoenzymatic method offers a potent and versatile approach to oligosaccharide production.
• Due to its effectiveness and cost-efficiency, the chemoenzymatic method stands out as a preferred strategy for synthesizing oligosaccharides.

CD BioGlyco has been focusing on providing the One-Stop Solution for Carbohydrate Manufacture. Our Oligosaccharide Manufacture Service has been widely recognized by researchers, and we will continue to explore the needs of different clients. If you require more information, please do not hesitate to contact us.

• Physical-/Chemical Process for Oligosaccharide Manufacture
• Enzymatic Process for Oligosaccharide Manufacture
• Separation Process for High-Purity Oligosaccharide Manufacture