Nanobiocatalysts Based on Protease Conjugates with Carboxylated Multi-Walled Carbon Nanotubes for Combating Bacterial Biofilms

The use of hydrolytic enzymes is one of the most promising methods for combating bacterial biofilms. However, the use of native enzymes is limited by the rapid loss of activity under unfavorable conditions. Immobilization of enzymes on carbon nanoparticles enhances their stability, allows for biocatalyst reuse, and creates a synergistic effect due to the intrinsic antimicrobial properties of the nanomaterials. The aim of this investigation was to create and comparatively analyze conjugates of acid and alkaline proteases with carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) and to assess their effect on the formation and destruction of E. coli VKM B-3858D biofilms. The immobilization efficiency and kinetics of enzyme adsorption on the support were quantified by determining the protein concentration using the Bradford assay. The morphology and dispersion of the resulting conjugates were analyzed using atomic force microscopy (AFM). Protease activity was determined by a modified Anson method using the Folin–Ciocalteu reagent. Biofilm biomass was determined using crystal violet staining. The binding efficiency of the acid protease to MWCNTs-COOH was shown to reach 93%, which is significantly higher than that of the alkaline protease. The highest degree of immobilization was observed at a protein concentration of 117–338 μg/mL (10–20 mg/mL of the enzyme preparations). The interaction of the acid protease with the carbon nanoparticles increased dispersion, reducing the size of aggregates from ~1 μm to ~68 nm. As a result, acid protease conjugates with MWCNTs-COOH significantly reduced the biofilm biomass compared to both the enzyme-free control and the native enzyme. Alkaline protease, unlike the acid protease, destroys mature biofilms, and immobilization on MWCNTs-COOH enhances this ability. Native alkaline protease and acid protease conjugates with MWCNTs-COOH are effective in combating the biofilm formation of Gram-negative bacteria, while alkaline protease conjugates are suitable for disrupting mature biofilms.