Engineering the substrate-binding pocket of Cellobiose 2-epimerase for enhancing lactulose biosynthesis

Cellobiose 2-epimerase (CE) converts lactose into lactulose and epilactose with high added value and prebiotic benefits. However, the low conversion of lactulose by CsCE limits its industrial application. Inspired by previous work introducing engineered disulfide bonds on the loop of CsCE, we performed saturation mutagenesis and high-throughput screening of all residues within 4 Å of the engineered disulfide bond. The best mutant, CsCE-S173C/F231C/L172S/I178V(M4), showed a 1.3-fold increase in isomerization activity over CsCE-S173C/F231C. Molecular dynamics simulations and binding pocket analysis revealed that mutations reshaped the shape and size of the substrate-binding pocket and increased non-covalent interactions of the protein with the ligand. Binding free energy calculations showed a higher binding affinity of M4 for lactose and lactulose. Therefore, we demonstrated that the catalytic characteristics of CE can be modulated by further manipulating the loop region near the pocket, which is important for improving the catalytic efficiency and promiscuity of the enzyme.