Simultaneously improving the specific activity and thermostability of α-amylase BLA by rational design

• Published in: Bioprocess and biosystems engineering 2022-11, Vol.45 (11), p.1839-1848
• Main Authors: Cui, Xin, Yuan, Xin, Li, Shunyi, Hu, Xinlin, Zhao, Jing, Zhang, Guimin
• Format: Article
• Language: English
• Subjects: Amylases; Biotechnology; Chemistry; Chemistry and Materials Science; Conformation; Conserved sequence; Design; Desizing; Dynamic structural analysis; Engineering; Environmental Engineering/Biotechnology; Enzymes; Food Science; Free energy; Hydrophobicity; Industrial and Production Engineering; Industrial Chemistry/Chemical Engineering; Molecular dynamics; Mutagenesis; Mutants; Mutation; Proteins; Research Paper; Residues; Thermal stability; α-Amylase
• ISSN: 1615-7591; 1615-7605
• DOI: 10.1007/s00449-022-02790-0

Higher activity and alkaline α-amylases are desired for textile desizing and detergent additive. Here, rational design was used to improve the specific activity and thermostability of the α-amylase BLA from Bacillus licheniformis . Seventeen mutants of BLA were designed based on sequence consensus analysis and folding free energy calculation, and characterized by measuring their respective activity and thermostability at pH 8.5. Among them, mutant Q360C exhibited nearly threefold improved activity than that of wild-type and retained a higher residual activity (75% vs 59% for wild-type) after preincubation at 70 ℃ for 30 min. The modeled structures and molecular dynamics simulations analysis demonstrated that the enhanced hydrophobic interaction near residue 360 and reduced disturbance to the conformation of catalytic residues are the possible reasons for the improved thermostability and activity of Q360C. The results suggest that 360th of BLA may act as a hotspot for engineering other enzymes in the GH13 superfamily.