Molecular identification and statistical optimization of endo-α-amylase production by Bacillus licheniformis KeR9 isolated from potato roots for maltooligosaccharide synthesis

A starch-degrading bacterial strain (KeR9) isolated from potato root tissues was identified through whole-genome sequencing using average nucleotide identity (ANI) and DNA-DNA hybridization (DDH) as Bacillus licheniformis . This strain with enzymatic production potential was then deposited in the Genbank project PRJNA1095867. Based on bioinformatic analysis, the KeR9 α-amylase is an enzyme of the GH13_5 glycoside hydrolase subfamily that shares high sequence similarity to reference α-amylases. Its molecular mass was estimated at 58 kDa using ProtParam, which was also experimentally confirmed by SDS-PAGE, revealing a single protein band corresponding to amylolytic activity. KeR9 α-amylase showed to hydrolyze starch into glucose and various maltooligosaccharides, including maltose and maltotriose, via an endo-α-amylase mechanism. The response surface methodology (RSM) approach, in combination with NEMRODW software, was successfully applied to optimize the production parameters. The optimal conditions were determined as follows: starch (8.4718), KH₂PO₄ (1.0837), and MgSO₄·7 H₂O (0.1842), all as g/L; agitation speed (108.91 rpm); and pH (7.04). Under these conditions, the experimental amylase activity of B. licheniformis KeR9 reached 3090.14 ± 15 mU/mL, closely matching the predicted value (3300.25 mU/mL). Compared to the control (331 ± 35 mU/mL), enzyme activity was ten-fold increased, hence confirming the accuracy of the model and the efficiency of the optimization strategy used. Overall, data inferred from the present study show (i) that B. licheniformis KeR9 is a promising candidate for the production of high-performance α-amylase and (ii) that multivariate optimization tools are pertinent to accurately address industrial enzyme requirements.