The growing demand for biocatalysts in biomass processing highlights the necessity of enhancing the thermostability of glycoside hydrolases. However, improving both thermostability and activity is often hindered by trade-offs between backbone rigidity and the flexibility of substrate-binding regions. In this study, Bacillus subtilis cellulase and β-glucanase were engineered using a two-step process incorporating the computational tools Pythia and ESM-2, which were found complementary in improving stability and activity. The engineered cellulase and β-glucanase exhibited increases in their apparent melting temperatures (5.8 °C and 8.4 °C), accompanied by up to a 1.5-fold increase in initial activities. At 50 °C, while the wild-type cellulase lost 60% of its activity after 24 h and wild-type β-glucanase lost activity completely in 2 h, the engineered cellulase-M5 retained its initial activity, and β-glucanase-M7 displayed a 2.2-fold increase in its half-life. Structural analysis indicated that Pythia-identified mutations likely enhanced backbone robustness through refined polar and hydrophobic interactions, while beneficial mutations from ESM-2 appeared to affect polysaccharide-binding regions. This two-step computational redesign offers a promising approach for optimizing both thermostability and activity in glycoside hydrolases and other enzyme families with extensive sequence diversity.
Keywords: Activity; Cellulase; Computational redesign; Thermostability; β-Glucanase.
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