Large-scale purification of epilactose using a semi-preparative HPLC system

Abstract

Epilactose (4-O-β-d-galactopyranosyl-d-mannopyranose) is a rare disaccharide derived from lactose (4-O-β-d-galactopyranosyl-d-glucopyranose) and possesses prebiotic properties. Epilactose is needed for biological studies in high amounts and purified form. The purification of epilactose is challenging because it behaves similarly to lactose, the starting substance of the epilactose production process. Epilactose can be produced both chemically and enzymatically, using cellobiose 2-epimerases for the latter. In this study, a process for semi-preparative epilactose purification is presented. Firstly, epilactose was produced in a buffered system using recombinant cellobiose 2-epimerase from Flavobacterium johnsoniae. After the enzymatic conversion, some of the residual lactose was removed by crystallisation at 6 °C for 72 h. Subsequently, the lactose still remaining in the conversion mixture was separated from epilactose by ligand-exchange chromatography. The conditions for the most effective chromatographic separation of lactose and epilactose were determined using a design of experiments approach with surface response methodology. The best conditions led to an epilactose purity of 99 % with a total yield of 51 %.