Discovery and functional characterization of endoglucanases from <i>Coptotermes formosanus</i> with enhanced cellulose hydrolysis via yeast surface display

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 <i>Coptotermes formosanus</i> cDNA library and heterologously expressed in <i>Saccharomyces cerevisiae</i>. 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 <i>in silico</i> and substantially lowered hydrolytic activity both <i>in vitro</i> 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.IMPORTANCEEfficient, low-impact methods to depolymerize cellulose are essential for turning agricultural and forestry residues into renewable chemicals and materials. Here, we report eight novel endoglucanases mined from a termite cDNA library and show that yeast surface display both enhances cellulose hydrolysis and enables enzyme reuse, identifying CTEG6 as a particularly robust candidate for further development. By integrating enzyme characterization, molecular docking, targeted mutagenesis, and fermentation optimization, we pinpoint specific residues (A11, D251, and S258) as key contributors to substrate binding and catalytic performance. These mechanistic insights not only justify prioritizing CTEG6 for further applications but also offer a practical route toward more efficient, sustainable bioconversion technologies for renewable energy, waste valorization, and green manufacturing.