Structural and Functional Characterization of a GH64 β-1,3-Glucanase from <i>Massilia violaceinigra</i> Reveals an Exolike Endocleavage Mode for Laminaripentaose Production

β-1,3-Glucans are widespread in plants, algae, fungi, and certain bacteria, and their enzymatic hydrolysis yields β-1,3-gluco-oligosaccharides (GOS) with diverse biological activities. However, the structural heterogeneity of GOS mixtures complicates investigations of their structure-activity relationships. Members of glycoside hydrolase family 64 (GH64) are unique in that, despite being classified as endo-β-1,3-glucanases, they predominantly produce a single product─laminaripentaose. Here, we biochemically characterized a GH64 enzyme, <i>Mv</i>gh64, from <i>Massilia violaceinigra</i>. <i>Mv</i>gh64 exhibited high specificity toward β-1,3-glucans and produced laminaripentaose as the major product (>85%). Structural analysis via X-ray crystallography, coupled with GPC and MS analysis of the products, revealed features indicative of an exolike endo cleavage mechanism, including a tunnel-like active site formed by region 1 and loop 1. These findings provide mechanistic insight into GH64 substrate specificity and establish a framework for engineering glycosidases to produce uniform, high-degree-of-polymerization oligosaccharides.