Structure-Guided Tunnel Engineering to Improve the Catalytic Efficiency of a Cellobiose 2-Epimerase for Efficient Lactulose Synthesis

Cellobiose 2-epimerase (CE) can convert lactose to lactulose, which is important for the enzymatic production of lactulose. However, its poor isomerization activity limits industrial application. In this study, a tunnel engineering strategy was developed based on analyzing the difference in bottleneck residue between Casa-WT (CE from <i>Caldicellulosiruptor saccharolyticus</i>) and Casa-M5 (Casa-WT with five-point mutation). The finally obtained Casa-M7 (Casa-M5/182<i>S</i>/230A) displayed 2.06- and 1.42-fold increases in the isomerization activity and catalytic efficiency, respectively. Further molecular dynamics simulation revealed that Casa-M7 had an expected narrower tunnel shape and adequate conformation distribution, in line with catalytic efficiency. Our study showed that the tunnel shape is crucial for the isomerization activity of CEs, which provides a new perspective for modifying CEs with higher catalytic efficiency for lactulose production.