Improving the Thermostability of Serine Protease PB92 from Bacillus alcalophilus via Site-Directed Mutagenesis Based on Semi-Rational Design
Open Access
Abstract
Proteases have been widely employed in many industrial processes. In this work, we aimed to improve the thermostability of the serine protease PB92 from Bacillus alcalophilus to meet the high-temperature requirements of biotechnological treatments. Eight mutation sites (N18, S97-S101, E110, and R143) were identified, and 21 mutants were constructed from B-factor comparison and multiple sequence alignment and expressed via Bacillus subtilis. Among them, fifteen mutants exhibited increased half-life (t1/2) values at 65 °C (1.13–31.61 times greater than that of the wild type). Based on the composite score of enzyme activity and thermostability, six complex mutants were implemented. The t1/2 values of these six complex mutants were 2.12–10.05 times greater than that of the wild type at 65 °C. In addition, structural analysis revealed that the increased thermal stability of complex mutants may be related to the formation of additional hydrophobic interactions due to increased hydrophobicity and the decreased flexibility of the structure. In brief, the thermal stability of the complex mutants N18L/R143L/S97A, N18L/R143L/S99L, and N18L/R143L/G100A was increased 4-fold, which reveals application potential in industry.Keywords
Funding Information
- National Natural Science Foundation of China (No. 32260015, No. S202210681008)
- Innovation and Entrepreneurship Training Program for College Students (No. 32260015, No. S202210681008)
This publication has 43 references indexed in Scilit:
- Vibrational entropy differences between mesophile and thermophile proteins and their use in protein engineeringProtein Science, 2014
- Commercial proteases: Present and futureFEBS Letters, 2013
- Contribution of Hydrophobic Interactions to Protein StabilityJournal of Molecular Biology, 2011
- A novel scoring function for discriminating hyperthermophilic and mesophilic proteins with application to predicting relative thermostability of protein mutantsBMC Bioinformatics, 2010
- Enhancing thermostability of a Rhizomucor miehei lipase by engineering a disulfide bond and displaying on the yeast cell surfaceApplied Microbiology and Biotechnology, 2009
- Purification and Characterization of Two Distinct Metalloproteases Secreted by the Entomopathogenic Bacterium Photorhabdus sp. Strain Az29Applied and Environmental Microbiology, 2004
- The double cubic lattice method: Efficient approaches to numerical integration of surface area and volume and to dot surface contouring of molecular assembliesJournal of Computational Chemistry, 1995
- Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocketProtein Engineering, Design and Selection, 1992
- Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilusProtein Engineering, Design and Selection, 1992
- Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinasesProtein Engineering, Design and Selection, 1991