Screening, identification, engineering, and characterization of Bacillus-derived α-amylase for effective tobacco starch degradation.

In this study, two high-performing α-amylase-producing strains, CK3-5 and A8-1 were successfully isolated and characterized, which were taxonomically confirmed as Bacillus velezensis through whole-genome sequencing and bioinformatics. Bioinformatic sequence analysis and molecular docking revealed the catalytic triad (Asp173-Glu208-Asp274) essential for α-amylase function. Through metabolic engineering, the recombinant strain BAX-5/PT17amy(A8-1)SP was developed, which exhibited the highest α-amylase activity of 1440 U/mL upon fermentation optimization, marking a 9.2-fold enhancement over the wild-type strain A8-1, and it successfully degraded 6 % of the starch in the tobacco leaves within 48 h, while the content of 13 harmful substances, including acetamide, pyridine, and acetonitrile, was reduced by 8.6 % to 25.2 %. This study reveals a novel α-amylase gene from B. velezensis and establishes an efficient expression system in B. amyloliquefaciens, offering valuable insights for industrial α-amylase production.