Lytic polysaccharide monooxygenases (LPMOs) play a crucial role in the enzymatic depolymerization of cellulose through oxidative cleavage of the glycosidic bond in the highly recalcitrant crystalline cellulose region. Improving the activity of LPMOs is of considerable importance for second-generation biorefinery. In this study, we identified a beneficial amino acid substitution (N526S) located in the cellulose binding module (CBM) of HcLPMO10 (LPMO of Hahella chejuensis) using directed evolution. The improved variant HcLPMO10 M1 (N526S) exhibits 2.1-fold higher activity for the H2O2 production, 2.7-fold higher oxidation activity, and 1.9-fold higher binding capacity toward cellulose compared with those of the wild type (WT). Furthermore, M1 shows 2.1-fold higher activity for degradation of crystalline cellulose in synergy with cellulase, compared to the WT. Structural analysis through molecular modeling and molecular dynamics (MD) simulation revealed that the substitution N526S located in the CBM likely stabilizes the cellulose binding surface and enhances the binding capacity of HcLPMO10 to cellulose, thereby enhancing enzyme activity. These findings demonstrate the important role of the CBM in the catalytic function of LPMO.
Keywords: cellulose binding module (CBM); cellulose degradation; directed evolution; lytic polysaccharide monooxygenase (LPMO); oxidative cleavage; protein engineering.