Mining, Identification, and Engineering of <i>Db</i> Cel5A, a Thermostable Processive Endoglucanase with Potential for One-Step Cellulose Hydrolysis

Cellulose is the most abundant lignocellulosic resource and requires efficient cellulases for bioconversion. In this study, <i>Db</i>Cel5A, a highly stable processive endoglucanase from <i>Dictyoglomaceae bacterium</i>, was identified via database mining and expressed in <i>Escherichia coli</i>. <i>Db</i>Cel5A exhibited remarkable thermostability, with a <i>T</i>_m of 79.9 °C and a half-life of approximately 30 h at 75 °C. High salt tolerance is a notable characteristic. A product profile dominated by glucose and cellobiose further highlights <i>Db</i>Cel5A's strong potential for one-step saccharification. We employed AI-assisted protein engineering and obtained the optimal mutant K27G, which exhibited a 2.5-fold increase in catalytic efficiency, higher substrate affinity, and enhanced thermostability (<i>T</i>_m = 81.9 °C). Molecular dynamics simulations unveiled the underlying mechanism, and its practical applicability was confirmed by the effective degradation of sugar cane bagasse. Overall, <i>Db</i>Cel5A represents a promising biocatalyst and provides an effective strategy for the discovery and engineering of high-performance cellulases.