Abstract

Microbial cellulose degradation offers a sustainable route to convert agricultural and forestry residues into value-added chemicals. Here, we report the discovery and functional characterization of eight novel endoglucanases (CTEG1 and CTEG3–CTEG9) from a Coptotermes formosanus cDNA library and heterologously expressed in Saccharomyces cerevisiae. Screening of crude extracts and whole-cell fermentations identified CTEG6 as the top performer (≈30 U/mg at 30°C) and the best whole-cell hydrolytic strain (1.51 mg/mL reducing sugar after 72 h). To investigate the basis of activity variation, we quantified EG gene copy number by quantitative PCR (qPCR) and observed strain-to-strain differences in gene dosage. Structural modeling (AlphaFold2) and flexible induced-fit docking with a carboxymethyl cellulose sodium (CMC-Na) fragment implicated residues A11, D251, and S258 in substrate binding; targeted mutagenesis of these sites (CTEG10: A11S/D251N/S258G) reduced hydrogen-bonding in silico and substantially lowered hydrolytic activity both in vitro and in 72-h fermentations. Under optimized conditions (30°C, pH 6.0), the CTEG6 strain efficiently hydrolyzed CMC-Na, microcrystalline cellulose (MCC), and natural cellulose from corncob powder. Together, these results identify CTEG6 as a promising biocatalyst and provide a theoretical basis for rational enzyme optimization toward scalable biomass conversion.