Marine P. otitidis EGY-NIOF-A1 keratinase-nano molybdenum trioxide- composite as ecofriendly antifouling paint

Marine biofouling remains a persistent operational and environmental challenge, while conventional metal-based antifouling coatings raise ecological concerns due to toxicity and bioaccumulation. In this study, a sustainable enzymatic antifouling strategy was developed by using keratinase overproduced from the marine isolate Pseudomonas otitidis EGY-NIOF-A1 (OQ547301). Cultivation in feather-based medium yielded 0.482 mg/mL protein with 73 units per mL (U/mL) keratinolytic activities. The enzyme demonstrated antioxidant capacity (29% 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging) and antimicrobial activity against Escherichia coli , Staphylococcus aureus , and Enterococcus faecalis , with no hemolytic effects, supporting its biosafety profile. The keratinase gene (1596 bp) was successfully amplified, representing the first reported keratinase sequence from P. otitidis . To enhance catalytic performance, a composite of keratinase with nano‑molybdenum trioxide (MoO 3 NP) was prepared. In vitro assays showed increased proteolytic and keratinolytic activities (3.9 U vs 2.3 U for crude enzyme), consistent with molecular docking simulations revealing stable non-covalent interactions (ΔG = −4.26 kcal/mol). Keratinase-MoO 3 NP-composite was incorporated into marine coatings and evaluated by using standardized panel immersion assays on wood and steel substrates for up to four weeks. Log 10 -transformed colony-forming units (CFU) enumeration combined with factorial general linear modeling demonstrated that keratinase-supplemented coatings significantly reduced microbial colonization relative to blank controls ( p < 0.001), with efficacy modulated by substrate type, immersion duration, enzyme concentration, and paint pH. Notably, sustained antifouling activity was observed over extended exposure, frequently rivaling or exceeding a commercial benchmark. After four weeks of immersion for wood panels, the effect of pH on the keratinase-MoO 3 NP-composite with the Blank for 10% (PKB 2) suppressive action at pH 4 was the highest value of 99.73%. On a steel panel at pH 7, the highest suppressive effect was 97.43% for PKB 2. These findings establish enzyme–nano hybrid coatings as promising environmentally benign alternatives to conventional biocidal antifouling systems and provide mechanistic and statistical validation for their further development. The coordinated overexpression of multiple enzymes enabled by gene cloning approaches represents a promising direction for advancing next-generation antifouling coatings.