Proteolytic enzymes from Bacillus subtilis AB2 as antibiofilm adjuvants: Bioprocess optimization, mechanistic insights, and synergy with antibiotics.

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 Km 0.31% and Vmax 1.92 U/ml, while keratinase exhibited dual optima (pH 3.0 and ~7.0) with Km 0.27% and Vmax 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.