Two-step computational redesign of Bacillus subtilis cellulase and β-glucanase for enhanced thermostability and activity

• Published in: International journal of biological macromolecules 2024-12, Vol.285, p.138274, Article 138274
• Main Authors: Zhang, Huan, Zhu, Tong, Zhai, Qinglin, Chen, Qiansi, Zhang, Xuanshuo, Chen, Yiqiang, He, Wei, Li, Jingjing, Fan, Jianqiang, Tao, Jiemeng, Hu, Xingchuan, Qi, Lingfeng, Wang, Chaochao, Liao, Kuanqi, Chen, Yanchun, Cui, Yinglu, Chen, Shanyi, Wu, Bian
• Format: Article
• Language: English
• Subjects: Activity; Cellulase; Computational redesign; Thermostability; β-Glucanase
• ISSN: 0141-8130; 1879-0003; 1879-0003
• DOI: 10.1016/j.ijbiomac.2024.138274

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.