Characterization of BpLPMO10 from Bacillus paralicheniformis: A bi-functional AA10 LPMO with chitin-preferential activity and strong exo-enzyme synergy.

LPMOs are copper metalloenzymes that facilitate the conversion of challenging polysaccharides like cellulose and chitin. This study focuses on the AA10 family LPMO gene (BpLPMO10) cloned from the chitin-degrading bacterium Bacillus paralicheniformis and expressed in Escherichia coli. Using the 2,6-DMP assay, the optimal reaction conditions for BpLPMO10 were determined to be pH 8.0 and 30 °C, with ascorbic acid as the optimal electron donor. Substrate binding assays revealed affinity for β-chitin, colloidal chitin, phosphoric acid-swollen cellulose (PASC), and microcrystalline cellulose (MCC), but not for α-chitin. FTIR, SEM, and XRD analyses demonstrated oxidative cleavage of crystalline regions in β-chitin and MCC, reducing crystallinity and altering surface morphology. MALDI-TOF MS further verified the simultaneous oxidation of both substrates, consistent with C1-type cleavage. Notably, BpLPMO10 exhibited unprecedented synergism with exo-chitinases SmChiA and SmChiB on β-chitin, boosting reducing sugar yields by 82-84 %, the highest synergy reported for bifunctional AA10s. Additionally, the incorporation of cellulase (Celluclast® 1.5 L) resulted in a 13 % increase in reducing sugar yields from PASC and a 36 % increase from MCC. Molecular dynamics simulations revealed stronger binding to chitohexaose than cellohexaose, attributed to enhanced interactions with 11 key residues. These findings establish BpLPMO10 as a high-efficiency, dual-activity LPMO with exceptional β-chitin synergism, advancing enzymatic biomass conversion strategies.