Characterization of a novel thermostable GH16 β-1,3-1,4-glucanase from Streptomyces lividans with potential industrial applications.
Abstract
A novel GH16 β-1,3-1,4-glucanase, SlGH16, 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. SlGH16 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 SlGH16 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.