Insight into novel biochemical features and function of novel antifungal GH16-β-glucanase from Bacillus velezensis

β-Glucanases play a critical role in degrading fungal cell wall β-glucans, compromising fungal integrity and development. Here we characterize BvlzGluc, a novel β-glucanase from Bacillus velezensis CECT 8237 with potent and specific activity against β-1,3-glucans. The enzyme hydrolyzes yeast-derived β-glucan but is inactive against β-1,6- and β-1,3/1,4-linked polysaccharides, such as laminarin and lichenin, indicating strict substrate specificity. Mass spectrometry of Botrytis cinerea cell wall extracts revealed that BvlzGluc cleaves high-molecular-weight β-glucans into smaller oligosaccharides, supporting its classification as an endo-β-1,3-glucanase. Kinetic analysis yielded a Km of 48 μM and a Vmax of 0.026 μmol/min. Structural modeling showed a GH16 jelly roll fold lacking a classical catalytic groove, and site-directed mutagenesis of two predicted binding residues (Y36 and T216) confirmed their functional relevance. CD spectroscopy revealed that T216A disrupts folding, while Y36A retains structure but shows reduced activity. Despite its compact architecture and divergence from canonical motifs, BvlzGluc demonstrates efficient antifungal function. Its physicochemical simplicity and specificity make it a promising candidate for scalable production and targeted applications in crop protection. This work expands our understanding of bacterial glucanases and underscores the value of structurally minimal enzymes in biocontrol strategies.