Peptide-mediated stabilization of <i>Trichoderma reesei</i> Cel7A for enhanced thermal stability in bioethanol production

Improving the thermal stability of <i>Trichoderma reesei</i> Cel7A, a key cellulase for bioethanol production, is crucial for reducing costs and enhancing efficiency under industrial conditions. While Cel7A functions optimally at moderate temperatures (40–50 °C), its activity diminishes at higher ranges (50–80 °C). This study integrates Molecular Dynamics (MD) simulations, Machine Learning (ML) predictions, and Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) free energy calculations to develop a peptide-mediated stabilization strategy. Analysis of root mean square fluctuations (RMSF) at 350K identified thermally sensitive, flexible regions. Surface-accessible area calculations pinpointed a stretch of residues (TYPTNETSTPG) for targeted mutations, generating 69,984 peptides. Screening with ML-based DeepPurpose, clustering, and similarity analyses yielded 15 candidates. Docking and ΔSASA calculations highlighted three promising peptides (Peptide-7, Peptide-10, and Peptide-15) with binding free energies of −27.33, −11.07, and −7.36 kcal/mol, respectively. Density Functional Theory (DFT), MD simulations, and QM/MM analyses revealed Peptide-15 as the most stable candidate, displaying strong polar interactions, high dipole moment (447.49 Debye), and consistent energetic stabilization. Its integration with Cel7A achieved thermal stability, suggesting that peptide-based stabilization is a viable method to enhance enzyme performance in bioethanol systems under heat stress.