Synergetic Antibacterial Tannic Acid/Bi-Enzyme-Based Coatings: Layer-by-Layer and One-Pot Films

Bacterial biofilms present a significant challenge, from hospital-acquired infections to biofouling in industrial systems, causing severe impacts on healthcare and placing heavy economic burdens worldwide. In this work, we developed sustainable antibacterial coatings based on tannic acid (TA) and a mixture of two antibacterial enzymes, amyloglucosidase (AMG) and glucose oxidase (GOX)solutions. After the deposition of the polyethylene imine precursor layer on the substrate, TA and AMG-GOX-based films were built at acidic pH by the (i) layer-by-layer (LbL) and (ii) one-pot deposition method. Both films combine the effect of TA, released at physiological pH, and hydrogen peroxide (H₂O₂), produced from starch through a cascade of enzymatic reactions. AMG converts starch into glucose, and then GOX oxidizes the glucose, generating H₂O₂. Complete eradication of planktonic and adherent Staphylococcus aureus (S. aureus) was achieved on TA/AMG-GOX LbL films with eight bilayers and TA-AMG-GOX one-pot films. In addition, compared with an uncoated surface, 98% inhibition of S. aureus biofilm formation was observed in a starch-containing environment and 41% in the starch-free medium. We showed that a synergistic action of the released TA, combined with the production of H₂O₂, catalyzed by a dual enzymatic reaction, occurs in the bacterial environment. This study outlines an antibacterial mechanism that can serve as a reference for developing future strategies aimed at targeting the biofilm matrix, instead of starch, as a substrate to trigger the enzymatic cascade reactions, by using cellulase instead of AMG.