Enhancement of the Isomerization Activity and Thermostability of Cellobiose 2-Epimerase from Caldicellulosiruptor saccharolyticus by Exchange of a Flexible Loop

Cellobiose 2-epimerase (CE) offers a promising enzymatic approach to produce lactulose. However, its application is limited by the unsatisfactory isomerization activity and thermostability. Our study attempted to optimize the catalytic performances of CEs by flexible loop exchange, for which four mutants were constructed using CsCE (CE from Caldicellulosiruptor saccharolyticus) as a template. As a result, all mutants maintained the same catalytic directions as the templates. Mutant RmC displayed a 2.2- and 1.34-fold increase in the isomerization activity and catalytic efficiency, respectively. According to the results of molecular dynamics (MD) simulations, it was revealed that the loop exchange in RmC enlarged the entrance of the active site for substrate binding and benefited proton transfer involved in the isomerization process. Besides, the t1/2 of mutant StC at 70 °C was increased from 29.07 to 38.29 h, owing to the abundance of rigid residues (proline) within the flexible loop of StC. Our work demonstrated that the isomerization activity and thermostability of CEs were closely related to the flexible loop surrounding the active site, which provides a new perspective to engineer CEs for higher lactulose production.