Identification of a thermostable cellobiose 2-epimerase from Caldicellulosiruptor sp. Rt8.B8 and production of epilactose using Bacillus subtilis.

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Epilactose, a potential prebiotics, was derived from lactose through enzymatic catalysis. However, production and purification of epilactose are currently difficult due to powerless enzymes and inefficient downstream processing steps. The encoding gene of cellobiose 2-epimerase (CE) from Caldicellulosiruptor sp. Rt8.B8 was cloned and expressed in Escherichia coli BL21(DE3). The enzyme was purified and it was suitable for industrial production of epilactose from lactose without by-products, because of high kcat (197.6s-1 ) and preferable thermostability. The Rt8-CE gene was further expressed in the Bacillus subtilis strain. We successfully produced epilactose from 700 g L-1 lactose in 30.4% yield by using the recombinant Bacillus subtilis whole cells. By screening of a β-galactosidase from Bacillus stearothermophilus (BsGal), a process for separating epilactose and lactose was established, which showed a purity of over 95% in a total yield of 69.2%. In addition, a mixed rare sugar syrup composed of epilactose and d-tagatose was successfully produced from lactose through the co-expression of l-arabinose isomerase and β-galactosidase. Our study shed light on the efficient production of epilactose using a food-grade host expressing a novel CE enzyme. Moreover, an efficient and low-cost process was attempted to obtain high purity epilactose. In order to improve the utilization of raw materials, the production process of mixed syrup containing epilactose and d-tagatose with prebiotic properties produced from lactose was also established for the first time. © 2021 Society of Chemical Industry.