Characterization of BpLPMO10 from Bacillus paralicheniformis: A bi-functional AA10 LPMO with chitin-preferential...

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.