The study of the activity of lysozyme after its covalent immobilization on detonation-synthesized nanodiamonds

A comparative evaluation of the antibacterial ac-tivity of free lysozyme and lysozyme covalently con-jugated to nanodiamonds was carried out using gram-negative luminescent bacteria Photoba-cterium phosphoreum as model target. The activity of both free and immobilized lysozyme was evalu-ated by changes in optical density of bacterial cell suspension after addition of each of them into the suspension. The surface of nanodiamonds intend-ed for covalent immobilization of lysozyme was ac-tivated with benzoquinone. It was found that the amount of lysozyme immobilized on nanodiamonds depended on the weight ratio of components (en-zyme: nanoparticles) at the stage of covalent bind-ing. Maximum immobilization of lysozyme (up to 1 mg protein per 1 mg nanoparticles) was observed at the weight ratio enzyme: nanodiamonds equal to 6:1. At the increase of protein amount at the stage of covalent immobilization the binding value did not increase. It is suggested that nanoparticle cluster size is likely to be additional factor influencing on covalent binding effectiveness. It was shown that lysozyme covalently conjugated to nanodiamonds exhibited catalytic activity and lysed bacterial cells as confirmed by the decrease in optical density of cell suspension. The addition of chelator of bivalent metal ions - EDTA (at the concentration of 2mM) in the suspension enhanced the action of immobilized enzyme more than twofold. Comparative experi-ments showed that the activity of lysozyme as part of nanodiamonds-enzyme complex was lower than the activity of free lysozyme. At equal concentration (25 mkg/ml) of free and immobilized lysozyme in-jected into cell suspension the same value of the reduction of optical density (by 60-65 %) was regis-tered after 1 and 3 hours, respectively. The causes of observed effect are discussed in the study.