The Role of Cellobiose 2-epimerase in the Production of Oligosaccharides

Cellobiose 2-epimerase is an enzyme responsible for catalyzing the hydroxyl stereoisomerization at the C-2 position of cellobiose's glucose moiety, resulting in the production of 4-O-β-D-glucopyranosyl-D-mannose. Currently, more than 30 epimerases have been identified, with 21 of them being related to sugar epimerases. Most exoglycosidases can react with modified sugars, including acylated and/or phosphorylated carbohydrates. However, only aldose 1-epimerase, maltose 1-epimerase, and cellobiose 2-epimerase utilize unmodified disaccharides as substrates. This technique of generating rare oligosaccharides finds significant applications in various industries, such as pharmaceuticals, food, and agriculture.

Fig.1 Reaction of cellobiose 2-epimerase with mannobiose, cellobiose, and lactose. (Wu, et al., 2019)

Enzymatic Epimerization for Oligosaccharide Manufacture at CD BioGlyco

Enzymatic epimerization is a powerful technology to develop an environmentally friendly process to Manufacture Oligosaccharides, especially epilactose. Our scheme is as follows.

• Isolation and cultivation of bacterial strains

Isolating cellulosic anaerobic bacteria from gastric juice by the anaerobic coiled tube method. Strains that grew well in a modified multisugar medium and have cellobiose 2-epimerase activity are isolated.

• Analysis of the 16S rRNA gene of the strain to confirm the correct source

The genomic DNA of the strains is isolated by the cetyltrimethylammonium bromide procedure. The 16S rRNA gene is amplified by polymerase chain reaction (PCR) using the genomic DNA of the isolate as a template.

• Gene expression and protein purification

After expressing the full gene, the protein is purified by centrifugation, column elution, and ultrafiltration.

• Enzyme activity assay of cellobiose 2-epimerase

Using lactose as the reaction substrate, incubate the reaction mixture with the enzyme at a certain temperature for an appropriate period. Measure the initial rate of epilactose formation from lactose using high-performance liquid chromatography (HPLC).

• Purification and nuclear magnetic resonance spectrum analysis of epilactose

The reaction is carried out in a mixture containing sodium phosphate buffer, lactose, and cellobiose 2-epimerase. After the reaction, the entire reaction mixture is spotted on a silica gel plate, and the color development is repeated in the solvent system. Cut narrow strips and measure carbohydrate mobility. The product is extracted in distilled water and the extract is concentrated in a rotary evaporator. The purified product is analyzed by ¹³C nuclear magnetic resonance (NMR) spectroscopy.

• Resistance to digestive enzymes

Epilactose and sucrose are treated separately with crude intestinal enzyme solution. The resulting solution is quantified by HPLC for undigested carbohydrates.

Fig.2 Enzymatic epimerization process of epilactose. (CD BioGlyco)

Applications

• Enzymatic epimerization is an advanced technology for the production of epilactose.
• Epilactose produced by enzymatic epimerization shows promise as a prebiotic.
• The improvement of enzymatic epimerization helps the progress of oligosaccharide production technology.

Advantages

• The purity of cellobiose 2-exopolymerase is as high as 85% after simple purification.
• Cellobiose 2-exopolymerase efficiently epimerizes 2-epimerize lactose to produce epilactose, a rare oligosaccharide.
• Epilactose produced is resistant to rat intestinal enzymes utilized by human bifidobacteria and stimulates tight junction permeability in Caco-2 cells. This suggests that this rare disaccharide has promise as a prebiotic.

CD BioGlyco provides complete One-Stop Solutions for Carbohydrate Manufacture, and our experts customize experimental schemes according to clients' scientific research projects. Our dedicated team of experts works tirelessly to provide Enzymatic Process for Oligosaccharide Manufacture to meet clients' needs and requirements. We offer enzymatic epimerization service to develop an environmentally friendly process to manufacture oligosaccharides. If you are interested in our services, please contact us for more details without any hesitation.

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