In silico engineering of Trichoderma harzianum acetylesterase for enhanced thermal stability and efficiency

Acetylesterase, produced by Trichoderma harzianum, plays a crucial role in deacetylating hemicellulose during pulp production. Thermostable variants of this enzyme, although rare, can significantly enhance industrial efficiency by retaining activity at high temperatures. This research aims to design a thermostable variant of acetylesterase from T. harzianum IOC-3844 (EC 3.1.1.6) using computational methods. A 3D model of the enzyme was created, and stabilizing mutations were identified. Among over 250 screened mutants, a double mutant (D75C, E296L) was recognized as the most promising variant. During 200 ns of molecular dynamics simulations at 37 °C and 70 °C, the model outperformed the native enzyme. At 70 °C, there was a 30.7% decrease in the mean RMSD (0.43 Å), a 9.5% reduction in the mean RMSF (0.06 Å), a 1.5% decrease in the mean Rg (0.29 Å) and a 3.4% reduction in the mean SASA (4.6 nm²). The mutant also displayed 36.5% more mean intramolecular hydrogen bonds and a 42.3% improvement in mean interaction energy (21.96 Kcal/mol). The PCA results showed that the double mutant maintains the native enzyme's structure at high temperatures. This variant exhibits enhanced thermal stability and catalytic activity, making it a promising eco-friendly alternative to harmful chemicals, such as chlorine, in paper production.