Semirational Design and Immobilization Synergistically Enhance Barnase Activity and Stability

Barnase, derived from Bacillus amyloliquefaciens, is a key enzyme in biocatalysis with widespread applications in pharmaceutical synthesis. However, its stability under extreme conditions, such as high temperatures and extreme pH, limits its industrial applications. Therefore, enhancing both its catalytic efficiency and stability through genetic engineering has become a critical focus of research. In this study, AlphaFold was employed to predict the structure of Barnase, followed by molecular docking and molecular dynamics simulations using GROMACS to design and construct 24 mutants. The results demonstrated that the enzymatic activity of the S28H and D101 K mutants increased by 75.28% and 71.86%, respectively, while the stability of D101 K declined under high temperatures. To address this, D101 K was immobilized onto a ZIF-8 carrier. Under optimized immobilization conditions (1.5 M 2-methylimidazole, 1.5 mL enzyme solution, 20 °C), ZIF-8@D101 K exhibited significantly enhanced thermal stability and pH adaptability. Recycling experiments showed that 96.21% of its activity was retained after three cycles, and 72.47% after eight cycles, demonstrating superior reusability and stability, making it more suitable for industrial applications.