Antimicrobial enzymes, either specific cell lytic enzymes or wide-spectrum bacterial destroying enzymes such as proteases and oxidative enzymes, can be immobilized onto or incorporated into a variety of materials for the development of self-decontaminating surfaces (Figure SD-1). These surfaces have potential applications in antifouling, environmental decontamination, and food packaging.
Figure SD-1. Antimicrobial activity of enzyme-based nanoconjugates and coatings. Antimicrobial enzymes are immobilized onto nanomaterials to form enzyme nanoconjugates, which can be further incorporated into paints and films to generate antimicrobial coatings. Bacterial cells can be killed efficiently upon contact with the conjugates or coatings.
Lysostaphin (Lst) is a bacteriolysin that is highly active against Staphylococcus aureus and related species, including the methicillin-resistant S. aures (MRSA) strains. We conjugated Lst to multiwalled carbon nanotubes (MWNTs) through a PEG12 linker, and observed >99% cell killing in 10 min. The incorporation of the Lst conjugate into a latex paint film formed a highly stable, nonleachable, surface-active antistaphylococcal coating that eradicated normal S. aureus cells and was highly efficient in killing different MRSA strains (Figure SD-2)1.
Figure SD-2. Antistaphylococcal activity lysostaphin-CNT-paint films against four different strains of methicillin-resistant Staphylococcus aureus (MRSA). The plot shows % viability in case of lysostaphin-containing films normalized to that obtained for control paint samples.
Ply500 is an endolysin against Listeria monocytogenes, one of the major pathogens contaminating the food supply chain and causing severe infections and death. We covalently attached Ply500 to the FDA-approved SNPs, and achieved ~5-log killing of Listeria innocua (a reduced pathogenic surrogate) in 24 h (Figure SD-3). The conjugate, while stabilizing the enzyme, completely decontaminated lettuce leaves inoculated with L. innocua. In addition, affinity binding of Ply500 to edible crosslinked starch nanoparticles killed L. innocua by 3 log units in 24 h2.
Figure SD-2. (Left) Schematic representation of oriented affinity immobilization of MBP-linker10Ply500 fusion protein on crosslinked starch particles via MBP. (Right) Activity of Ply500 against isolated L. innocua using a viable plate counting assay after 3 h (grey), 6 h (white) and 24 h (black). L. innocua was grown in BHI media for 7 h.
Bactericidal and Sporicidal Paints and Coatings
We have developed self-cleaning surfaces based on CNTs to prevent biofouling (Figure SD-3). The bioactive nanomaterials are prepared by attaching enzymes, such as proteases3, haloperoxidases4, and perhydrolase (AcT)5 onto SWNTs and MWNTs, and dispersing them into polymeric/paint films to yield antimicrobial coatings (Figure SD-4). In the case of proteases (e.g., trypsin and subtilisin Carlsberg), simple physical adsorption of proteases onto SWNTs enabled incorporation of ~30 times higher catalytic activity than on graphite powder controls and preserved more than 90% of initial activity over 30 days in an aqueous buffer. Protease-SWNT conjugates can inhibit the adsorption of proteins on surfaces and has potential to be applied to medical implants, ship hulls, and hospital walls and equipment.
Figure SD-3. Schematic of the bacterial biofilm formation process. Bacteria attach to a surface, proliferate and secrete ECM components, resulting in a complex biofilm matrix including planktonic bacteria and persister cells. The major approaches to combat biofilms are described in the three boxes.
Figure SD-4. Mechanisms of reagent-dependent antimicrobial enzymes. (a) Antimicrobial system combining glucose oxidase (GOx) with (halo)peroxidase or perhydrolase for producing potent antimicrobial agents (red colored compounds). (b) Laccase-catalyzed reaction for generating I2.
CPO-MWNT conjugates, mixed with a commercial ecofriendly paint to fabricate, generated ~1.4 mM HOCl within 30 min, resulting in killing >99.99% of S. aureus and E. coli (Figure SD-5) In this system, the production of HOCl by haloperoxidases makes them useful candidates for disinfection, sanitization, and decontaminating formulations. Similarly, perhydrolase (AcT) was covalently immobilized onto oxidized MWNTs in the presence of an amphiphilic PEG spacer. The AcT–PEG–MWNT conjugates were then incorporated into a latex-based paint. The paint showed a 6-log reduction in the viability of spores of B. cereus and B. anthracis Sterne within 1 h (Figure SD-6)6. AcT-containing paint composites also exhibited antiviral activity; >4-log reduction in the titer of influenza virus (X-31) (initially challenged with 107 PFU/mL) was observed within 10 min (Figure SD-7)6. The crosslinked AcT-based conjugates were highly stable and maintained activity up to 180 days without observed loss of its activity. Laccase was also immobilized onto MWNTs and subsequently mixed with a commercial latex paint to generate an antimicrobial biocatalytic coating4. The generation of I2 from laccase-based paint coating led to 6-log reduction within 30 min for S. aureus and 60 min for E. coli. Furthermore, an optimized laccase-based paint formulation was able to kill >90% of B. cereus and B. anthracis spores within 1 h. Thus, the immobilization of laccase on nanomaterials offers environmentally benign approaches to generate surface decontaminating coatings.
Figure SD-5. Rate of generation of HOCl by CPO; and Bactericidal activity of CPO catalyzed generation of hypochlorous acid (HOCl) against (Left) S. aureus and (Middle) E. coli. (♦ PBS control, ■ H2O2, ▲ CPO + H2O2 + NaCl). Experimental Conditions: Bacterial cells, 106 CFU/mL; CPO, 5 µg/mL; H2O2, 2 mM; NaCl, 100 mM. (Right) Bactericidal activity of CPO-MWNT conjugates containing paint composites against S. aureus (□) and E. coli (■). Experimental Conditions: Bacterial cells, 106 CFU/mL; CPO, 0.14% (w/w); H2O2, 2 mM; NaCl, 100 mM (* more than 99.99% killing).
Figure SD-6. Sporicidal activity of AcT-PEG-MWNT containing paint composites (0.30%, w/w, AcT) with urea hydrogen peroxide (UHP) and propylene glicol diacetate (PGD) against B. cereus spores (♦ control, ■ 200 mM UHP, ▲ 400 mM UHP, □ 0.30% (w/w) AcT with 200 mM UHP and Δ 0.30 % (w/w) ACT with 400 mM UHP). Experimental conditions: Spores, 106 CFU/mL; PGD, 100 mM; UHP, 200-400 mM, catalase, 100 U/mL and sodium thiosulfate, 0.05% (w/w).
Figure SD-7. Kinetics of antiviral activity of AcT-PEG-MWNT containing paint composites against Influenza A virus strain X-31 (inset displays antiviral activity as a function of H2O2 concentration; ♦ control and ■ 0.30%, w/w, AcT). Experimental conditions: virus, 4x107 PFU/mL; PGD, 10 mM; H2O2, 5 mM; catalase, 100 U/mL and sodium thiosulfate, 0.05% (w/w).
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