Improved biosynthesis of lactulose by a novel thermostable cellobiose-2-epimerase generated through semi-rational...

Cellobiose-2-epimerase is considered an ideal biocatalyst for the strictly thermodynamic synthesis of lactulose. However, its poor thermolability limits its industrial application. Herein, we successfully developed a novel thermostable cellobiose-2-epimerase for improving lactulose biosynthesis from lactose. The A242L mutant, derived from Dictyoglomus thermophilum (DiCE), was generated using semi-rational mutagenesis after computer-aided design via the FireProt web server. After systematic enzymatic characterization and optimization, A242L mutant exhibited a 123.5% T_1/2 in thermostability at 80 °C relative to the wild-type enzyme, and the E. coli BL21/pET28a(+)-A242L mutant harvested a maximal lactulose yield of 64.52%, representing a 51.38% improvement at a substrate concentration of 200 g/L for the whole-cell biotransformation reaction conducted at pH 7.0 and 80 °C for 4 h. Molecular docking and molecular dynamics (MD) simulation results indicated that the enhanced thermostability of the enzyme was closely related to the structure and rigidity of its flexible loops. The enhanced thermostability allows A242L to tolerate higher temperatures for extended periods, driving the reaction towards lactulose production, which is suitable for industrial lactulose production under harsh conditions.