Collagenase and keratinase are two important proteolytic enzymes with recognized applications in biotechnology and medicine, particularly in the enzymatic removal of necrotic tissue and the control of infection. In the present work, a soil isolate of Bacillus subtilis strain AB2 (PX453297.1) was optimized for enzyme production under different nutritional and physicochemical conditions. The enzymes were recovered by ammonium sulphate precipitation and dialysis, examined by SDS-PAGE and zymography, and further assessed for pH and temperature optima, stability, the influence of metal ions, and kinetic parameters. Maximum collagenase activity (4.41 ± 0.22 U/ml) was observed at 37°C and pH 7.5 in a glucose–peptone medium, whereas keratinase production was enhanced between 37 and 40°C at pH 7.5 in lactose–peptone medium. Protein bands of approximately 55 and 33 kDa were detected, representing 6.2- and 5.5-fold purification. Collagenase showed an alkaline optimum (pH 10.0, 37–45°C) with K_m 0.31% and V_max 1.92 U/ml, while keratinase exhibited dual optima (pH 3.0 and ~7.0) with K_m 0.27% and V_max 0.84 U/ml. Biofilm assays revealed that collagenase reduced pre-formed biomass by 62–68% and viable counts by 1.1–1.7 log₁₀, clearly outperforming keratinase (41–57%, 0.7–1.2 log₁₀). When combined with conventional antibiotics, both enzymes potentiated activity, with notable synergy between collagenase and oxacillin against Staphylococcus aureus (FICI 0.31–0.37), ciprofloxacin against Pseudomonas aeruginosa (FICI 0.37–0.50), and meropenem against Klebsiella pneumoniae (FICI 0.28–0.44). These results indicate that B. subtilis AB2 produces collagenase and keratinase with distinct biochemical characteristics and strong antibiofilm properties, underscoring their promise as adjuncts in chronic wound care as well as in industrial applications.

Keratinase kerZJ is a multifunctional protease with potential as a feed additive and functional ingredient. Here we performed an integrated multi‑omics evaluation of its biosafety and impact on gut homeostasis in mice. Our findings confirm that kerZJ is well-tolerated, with no evidence of systemic toxicity or intestinal epithelial damage. Integrated transcriptomic and proteomic analyses revealed that kerZJ reinforces intestinal barrier integrity by upregulating extracellular matrix components, including collagen IV, and modulates mucosal immunity by enhancing B-cell activation and antimicrobial peptide defenses without inducing inflammation. Furthermore, kerZJ administration led to a significant upregulation of digestive enzymes and a dose-dependent increase in short-chain fatty acids production. Microbiome analysis showed that while high-dose kerZJ altered community composition, it enriched for beneficial taxa like Lactobacillaceae and did not induce dysbiosis. These results demonstrate that kerZJ safely enhances gut barrier function, promotes a favorable immune and metabolic environment, and fosters a resilient gut ecosystem, supporting its development as a safe feed additive and nutraceutical component.