Characterization of a novel thermostable GH16 β-1,3-1,4-glucanase from Streptomyces lividans with potential industrial applications

A novel GH16 β-1,3-1,4-glucanase, Sl GH16, was identified from Streptomyces lividans and exhibits considerable thermostability and a broad pH tolerance, retaining appreciable activity under industrially relevant high-temperature and extreme pH conditions. Sl GH16 preferentially hydrolyzes barley β-glucan to produce low-molecular-weight oligosaccharides (DP3–DP5), offering some potential for functional feed and oligosaccharide production. Integrative structural analyses, including AlphaFold3 modeling, molecular docking, and molecular dynamics simulations, revealed that two disulfide bonds (Cys161-Cys169, Cys181-Cys186) and the central aromatic residue Phe248 act as important stabilizing elements, preserving substrate-binding integrity and overall protein rigidity. Site-directed mutagenesis confirmed the important roles of these residues in catalytic efficiency and thermal resilience. The combination of moderate stability, precise substrate recognition, and efficient oligosaccharide generation positions Sl GH16 as a promising candidate biocatalyst for high-temperature industrial applications. This study not only elucidates the structural determinants of GH16 enzyme thermostability but also provides a mechanistic reference for rational protein engineering aimed at enhancing enzyme performance under harsh processing conditions.