Novel Cellobiose 2-Epimerase from Thermal Aquatic Metagenome for the Production of Epilactose

Epilactose is a prebiotic molecule that exerts a bifidogenic effect and increases calcium and iron absorption in the small intestine. This study identifies a novel cellobiose 2-epimerase gene (ceM) by investigating metagenomic data generated from a thermal aquatic habitat. The computation of secondary and tertiary structure analysis, molecular docking, and MD simulation analysis indicated the protein CE_M to be a novel cellobiose 2-epimerase. The gene was expressed in Escherichia coli, followed by biochemical characterization of the purified protein. CE_M is capable of transforming lactose into the high-value rare sugar, epilactose, in a wide range of temperatures (4-70 °C) and pH (6.0-10.0). The enzyme was exposed to 50 °C, and hardly a 10% loss in activity was recorded after 32 h of heat treatment, suggesting that CE_M is a thermostable protein. CE_M is a kinetically highly efficient enzyme, with a turnover number of 9832 ± 490 s^-1 for lactose to epilactose epimerization. The maximum conversion yield of 26% epilactose was obtained in 15 min of catalytic reaction. Further, the enzyme was successfully tested to transform lactose in milk and whey samples.