Surfactant-tolerance evolution of Bacillus clausii protease for enhancing activity and stability by reshaping the substrate access tunnel

• Published in: Colloids and surfaces, B, Biointerfaces Vol. 245; p. 114289
• Main Authors: Ma, Xiangyang, Wang, Liya, Chen, Jingyu, Guo, Enping, Zheng, Hongchen, Zhao, Lei, Lu, Fuping, Liu, Yihan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2025
• Subjects: Alkaline protease; Catalytic efficiency; Stability; Substrate access tunnel; Surfactant; Substrate access tunnel; Catalytic efficiency; Stability; Surfactant; Alkaline protease
• ISSN: 0927-7765; 1873-4367; 1873-4367
• DOI: 10.1016/j.colsurfb.2024.114289

Alkali proteases are crucial in numerous industries, especially in the laundry industry, but their inactivation by surfactants limits their effectiveness. This study employed substrate access tunnel engineering to improve the performance of WT bcPRO in surfactants. By modifying the key residues in the substrate pocket, the best variant N212S showed higher stability and activity in both AES and LAS. Molecular dynamics (MD) simulations provided insights into the enhanced stability and activity. The Asn212Ser mutation weakened the anti-correlation motion, increased the number of hydrogen bonds between amino acid residues, and made the protein structure more compact, contributing to its stability. Additionally, the mutation extended the substrate access tunnel and enabled additional interactions with the substrate, enhancing its catalytic activity in surfactants. This study demonstrates a strategy for reshaping the substrate access tunnel to improve protease stability and activity in surfactant environments, offering a promising protease candidate for the laundry industry. •Tunnel engineering was used to modify the protease from Bacillus clausii (WT bcPRO).•The best mutant N212S showed enhanced stability in both AES and LAS than WT bcPRO.•· N212S showed 45 % and 60 % higher activity than WT bcPRO in AES and LAS, respectively.•More interactions formed between amino acid residues make the structure more stable.•The improvement of activity is owing to higher binding energy and more interactions with substrates.