Improving the activity and stability of Bacillus clausii alkaline protease using directed evolution and molecular dynamics simulation

• Published in: Enzyme and microbial technology Vol. 147; p. 109787
• Main Authors: Li, Jialin, Jiang, Luying, Cao, Xue, Wu, Yifan, Lu, Fuping, Liu, Fufeng, Li, Yu, Liu, Yihan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.06.2021
• Subjects: Alkaline protease; Alkaline stability; Molecular dynamics simulation; Specific activity; Thermostability; Specific activity; Thermostability; Molecular dynamics simulation; Alkaline stability; Alkaline protease
• ISSN: 0141-0229; 1879-0909
• DOI: 10.1016/j.enzmictec.2021.109787

•B. clausii alkaline protease (PRO) mutant with better properties was obtained.•The PRO mutant demonstrated a 9 times specific activity of wild type at 60 °C.•The PRO mutant possessed improved thermostability and alkaline stability.•Molecular dynamics clarified the mechanism of PRO mutant with improved properties.•It would be beneficial for rational design to engineer PRO mutants for application. Detergent enzymes have been developed extensively as eco-friendly substitutes for the harmful chemicals in detergent. Among them, alkaline protease accounts for a large share of detergent enzyme sales. Thus, improving the specific activity of alkaline protease could play an important role in reducing the cost of detergent enzymes. In our study, alkaline protease from Bacillus clausii (PRO) was used to construct a mutant library through directed evolution using error-prone PCR, and a variant (G95P) with 9-fold enhancement in specific activity was obtained. After incubation at a pH of 11.0 for 70 h, G95P maintained 67 % of its maximal activity, which was 46 % more than wild-type PRO (WT), indicating that G95P has better alkaline stability than WT. The thermostability of G95P was also enhanced, as G95P achieved 17 % initial activity after incubation for 50 h at 60 °C, while WT lost its activity. The MD simulation results verified that variant G95P possessed improved stability of its Gly95-Gly100 loop region and Arg19-Asp265 salt bridge, leading to enhanced stability and catalytic efficiency. This work enhances the understanding of the structure-function relationship of PRO and provides an academic foundation for improving the enzymatic properties of PRO to satisfy industrial requirements using protein engineering.