Antibiotic resistance and the colonization of resistant bacteria such as Staphylococcus aureus on surfaces, often in the form of biofilms, prolong hospitalization periods and increase mortality, thus is a significant concern for healthcare providers. To prevent biofilm formation, the inadequate concentration of using nanoparticles as antibacterial coating agents is one of the major obstacles. This study aimed to design a hypervalency TiO 2 nanocomposite as a reserved base to carry a high amount of active antibacterial agents such as lysostaphin via a biotin-streptavidin-biotin bridge. The utilization of the streptavidin-biotin system could increase the abundance of lysostaphin. Lysostaphin was expressed in Escherichia coli and purified. Both recombinant lysostaphin and titanium oxide nanocomposite were conjugated with biotin and linked to a streptavidin bridge. The kinetics and activity of the enzyme were examined after each step utilizing N-acetylhexaglycine as a substrate. Physical characteristics of nanoparticles containing lysostaphin were determined using AFM, SEM, FTIR, and zeta potential. The results showed changes in size, charge, and morphology of the nanoparticles following the lysostaphin attachment. Also, the stability and kinetics of the active biological enzymes on nanoparticles were reexamined following 8 months of storage. Exploiting this approach, various biotinylated antibacterial agents could be prepared and rapidly immobilized on a nanoparticle as an active net against related infectious agents.

Introduction: Staphylococcus aureus is a source of nosocomial infections and one of the significant concerns in patients with indwelling devices. Lysostaphin is a bacterially produced endopeptidase with a unique activity on S. aureus. Plasma, the fourth state of the material, consists of charged ions, free electrons, and activated neutral species. Biomedical applications of cold plasma are rapidly growing due to its capacity to treat heat-sensitive objects such as polymeric materials and biological samples. It activates surfaces by etching them to stabilize proteins. The direct effect of cold atmospheric plasma on the eradication of microorganisms have been investigated. However, there is no report on immobilizing antibiotic agents. Methods: In this study, the lysostaphin protein was expressed and purified using Ni-NTA column, then the purified enzyme was immobilized on glass surfaces pretreated with cold atmospheric plasma for 150 s, 200 s, and 300 s. The antimicrobial activity of immobilized lysostaphin on S. aureus was approved by in vitro analysis. Results: The 300 s plasma treatment confirmed to be the best time arrangement for more lysostaphin immobilization, shown by Atomic Force Microscopy. Conclusion: Our results showed that passive adsorption to the treated surface does not affect the structure and subsequent antimicrobial function of the recombinant protein compared to the standard proteins.