Sequence-diversity-guided discovery, model-guided mutagenesis, and secretory production of GH5 endoglucanases in Bacillus subtilis

Cellulases are central to lignocellulose deconstruction, but identifying additional glycoside hydrolase family 5 (GH5) enzymes remains slow. Deep-learning predictors have proliferated, yet few studies carry their predictions through experimental validation and testing in a production host. A 23-gene panel was assembled by CatPred-based selection from clustered UniProt sequences (25,148 initial entries) and EglS-seeded homology-search results, supplemented by two comparative routes. Twenty-two candidates were assayed on carboxymethylcellulose (CMC) after expression in Escherichia coli , with EglS as a separate control. AQSP_CL and MACA_CL, for which we found no prior biochemical characterisation, were purified alongside EglS and the thermophilic benchmark Cel5A. Both new enzymes had short-assay optima of 70 °C. AQSP_CL was most active at pH 5.0 and gave an apparent mass-based CMC kinetic-efficiency estimate close to EglS (0.201 versus 0.231 U·mL·mg⁻2); Cel5A was more thermostable. CatPred cellotetraose predictions showed no detected correlation with lysate CMC activity in eight assayed candidates with reconciled identities ( ρ = 0.10, p = 0.82). AQSP_CL had the highest fraction of poses meeting the operational distance criterion in ensemble docking (52.0% versus 28.7–39.0%), although the differences were not resolved at the conformer level and Vina scores did not distinguish the enzymes. In first-round screening, M65Q had the highest measured lysate activity among single substitutions (1.61-fold wild type). A few-shot model trained on first-round data nominated the second-round library, but additive predictions did not generalise to combinations. Signal peptide SP3 increased extracellular activity of parental AQSP_CL in Bacillus subtilis approximately eightfold over the signal-peptide-free construct. The study links discovery of two GH5 endoglucanases to experimental assessment of computational nominations, lead-enzyme development and extracellular production. AQSP_CL provides a new scaffold for further biomass-deconstruction research. The CMC and flask-scale measurements establish enzyme properties and extracellular recovery; biomass-conversion performance remains to be evaluated.