From mold to mill: StachCel5, a novel thermoalkaliphilic endoglucanase from Stachybotrys chartarum for pulp fiber biorefining

StachCel5, a novel endo-β-1,4-glucanase from Stachybotrys chartarum IBT 7711, was identified through genome mining and exhibited 65 % sequence identity with known glycoside hydrolase family 5 (GH5) cellulases. The gene was codon-optimized, excluding its native signal peptide, and heterologously expressed in Komagataella phaffii under the control of constitutive glyceraldehye-3-phosphate dehydrogenase (GAP) promoter. The purified enzyme displayed a high specific activity of 287.6 U/mg on carboxymethyl cellulose (Na-CMC), retained over 87 % activity across pH 4-7, and maintained 77 % residual activity after 60 min at 50 °C. Structural modeling predicted a canonical (β/α)₈ TIM-barrel fold, with Glu228 and Glu335 as conserved catalytic residues. Application trials on industrial hardwood and softwood pulps demonstrated that enzymatic pre-treatment with StachCel5 enhanced fiber fibrillation, reduced refining energy, and improved mechanical properties. In softwood pulp, the enzyme enabled equivalent tensile strength (67.8 Nm/g) as untreated controls with 25 % fewer refining revolutions (4500 vs 6000), reflecting significant energy savings. Improved tensile and burst indices, and preserved fiber integrity (zero-span tensile index) confirm the biorefining potential of StachCel5. These results establish StachCel5 as a thermostable and pH-tolerant biocatalyst suitable for enzymatic fiber modification in pulp and paper processing under industrially relevant conditions.