A new pathway for lactulose synthesis by N-acetylglucosamine 2-epimerase from Dictyoglomus thermophilum

The disaccharide lactulose is a beneficial compound used in both the pharmaceutical and functional food industries. Its chemical synthesis involves hazardous reaction conditions, and separating byproducts is challenging. Enzymatic catalysis is a novel strategy for lactulose synthesis. Studies have revealed that cellobiose 2-epimerase (CE), which belongs to the N-acetylglucosamine 2-epimerase (AGE) superfamily, can catalyze the isomerization of lactose to produce lactulose. However, no reports on the biosynthesis of lactulose by AGE are available. Here, an AGE from Dictyoglomus thermophilum (DithAGE) was expressed in Escherichia coli using different fusion tags. The recombinant Sumo-DithAGE had the highest isomerization activity. The K182R mutant, which has improved catalytic efficiency and thermal stability, was generated by the substitution of a surface residue. The maximum conversion rates of lactulose catalyzed by K182R were 40.37 % and 35.19 % at 85 °C and 90 °C, respectively, which were 4.49 % and 6.41 % greater than those of the wild-type variant. Owing to their high thermostability, specific activity, and conversion rate, the native DithAGE and the K182R mutant reported here have great potential for lactulose production. We heterologously expressed DithAGE in a microbial host and determined its isomerization activity. The realization of lactulose production by AGE provides a new pathway for enzymatic synthesis.