Supplementary material for proteases from Bacillus

Our study was based on the hypothesis that whey, a dairy side-stream with high nutritional content and low cost, can serve as an efficient substrate for the screening and fermentation of protease-producing Bacillus strains, and that the produced proteases can hydrolyze whey proteins into low-molecular-weight peptides and amino acids with functional and industrial potential. To test this hypothesis, soil samples were collected and screened on whey-based media. From 25 isolates, two strains showed the strongest proteolytic activity and were identified by 16S rRNA sequencing as Bacillus amyloliquefaciens LK1 and Bacillus subtilis LK2. Both strains secreted extracellular serine proteases with molecular weights around 30 kDa and isoelectric points near 8.5. Enzyme production was optimized by varying incubation times and aeration rates. Maximum protease activity was observed after 48 h of cultivation, reaching 748 U/L for LK1 and 650 U/L for LK2 when sweet whey was used as the growth medium, demonstrating whey’s value as a fermentation substrate.

Biochemical characterization showed that the enzymes were active across a wide pH range (6–10) with optima at pH 8–9, and temperature optima of 60–65 °C. Both retained stability for several hours at 55 °C but were completely inhibited by PMSF and partially by EDTA/EGTA, confirming they are subtilisin-like serine proteases. LK1 displayed greater tolerance to salts and detergents compared to LK2, a trait advantageous for industrial applications. Application studies demonstrated that LK1 efficiently hydrolyzed both sweet and acid whey proteins, producing small peptides and free amino acids, and in the case of acid whey, achieving complete hydrolysis of high-molecular-weight proteins. Comparative assays with commercial proteases showed that LK1 performed as effectively as some industrial enzymes and surpassed others, highlighting its unique potential. The resulting whey protein hydrolysates are enriched in peptides with known antioxidant, antimicrobial, antihypertensive, and immunomodulatory properties, underscoring their relevance in functional foods and nutraceutical applications.

The data were gathered through a combination of microbiological screening, phylogenetic analysis, fermentation optimization, enzyme purification, SDS-PAGE and isoelectric focusing, biochemical stability assays, and functional hydrolysis experiments. Together, these methods provided a robust dataset linking microbial strain selection to enzyme properties and functional outcomes. In summary, our findings confirm that whey can be successfully valorized as a cultivation and application medium for protease-producing Bacillus strains. The enzymes produced, particularly from B. amyloliquefaciens LK1 demonstrate strong potential for industrial applications in whey hydrolysis and bioactive peptide generation. This work supports both enzyme technology development and circular bioeconomy strategies for dairy waste valorization.