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

Abstract

BACKGROUND 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. RESULTS 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 (197.6 s−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. CONCLUSION 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.

Two-Step Purification of Epilactose Produced by Cellobiose 2-Epimerase from Caldicellulosiruptor Saccharolyticus

“…These results are in accordance with the ones reported by Chen and collaborators that performed a 12 h reaction using 25 U/mL of β-galactosidase from Bifidobacterium bifidum and achieved almost a complete elimination of lactose while 94% of the epilactose was recovered from the reaction mixture [7]. Recently, a screening of β-galactosidase enzymes that could be suitable for epilactose purification showed that the most promising enzyme was the one from Bacillus stearothermophilus [17]. The use of this enzyme resulted in the recovery of 98.5% of the epilactose, while 87.6% of the lactose was hydrolyzed.…”

“…To obtain purer epilactose for food applications as potential prebiotic ingredient, the remaining lactose (73.8 ± 0.5% of the mixture) needs to be removed. As the compounds are isomers, its separation is a complex procedure, and the lactose hydrolysis seems to be the most efficient method to eliminate this sugar [7,17]. However, due to the high similarity of the two structures, both lactose and epilactose can be substrates for microbial enzymes, thus limiting the selection of the most suitable catalyst.…”

“…Therefore, under these conditions, the yeast S. cerevisiae was proven to be the most suitable microorganism for monosaccharides removal. In fact, the use of the baker's yeast for monomers consumption and product purification is a common approach [11] and was already used in some epilactose purification strategies [7,17,20].…”