Functional and structural insights into a cold-active GH64 laminaripentaose-producing β-1,3-glucanase from Candidatus...

β-1,3-Glucans are ubiquitous polysaccharides whose enzymatic hydrolysis yields β-1,3-gluco-oligosaccharides (GOS) with immunomodulatory, antioxidant, and prebiotic properties. These bioactivities highlight their potential as functional food ingredients. However, conventional enzymatic methods often yield structurally heterogeneous GOS, complicating their application. Glycoside hydrolase family 64 (GH64) enzymes are unique in producing laminaripentaose as a single dominant product, enabling the preparation of structurally uniform GOS. Here, a novel GH64 enzyme, CsGH64, from Candidatus saccharibacteria was biochemically and structurally characterized. CsGH64 exhibited strict β-1,3-glucanase activity, producing laminaripentaose at > 80% yield, with optimal activity at pH 6.0 and 20 °C. Kinetic analysis revealed a Km of 27.9 mg mL⁻¹ and a Vmax of 16.3 U mg⁻¹. Comparative structural analysis with previously characterized GH64 enzymes uncovered distinct features underlying its cold-adapted catalytic properties. These results identify CsGH64 as a promising biocatalyst for the food and nutraceutical industries, enabling the efficient and mild production of structurally defined, bioactive oligosaccharides.