New-Generation Antibacterial Agent – Cellulose-Binding Thermostable TP-84 Fusion Endolysin

The increasing antibiotic resistance among bacteria challenges the biotech industry to search for new antibacterial molecules. Endolysin TP84_28 is a thermostable, lytic enzyme, encoded by the bacteriophage TP-84, and it effectively digests host bacteria cell wall. Biofilms, together with antibiotic resistance, are major problems in clinical medicine and industry. The primary challenge is to keep antibacterial molecules at the site of desired action, as their diffusion leads to loss of efficacy. The gene encoding TP84_28 endolysin was cloned into a custom expression-fusion vector, forming a fusion gene cbd_tp84_28_his with a cellulose binding domain from the cellulase enzyme. The CBD_TP84_28_His protein was biosynthesized in Escherichia coli and purified. Thermostability and enzymatic activity against various bacterial species were measured by turbidity reduction assay, spot assay, and biofilms removal. Cellulose binding properties were confirmed via interaction with microcellulose and cellulose paper-based immunoblotting. The high affinity of CBD allowed for high concentration of the fusion enzyme at desired target sites such as cellulose-based wound dressings, artificial heart valves and food packaging. CBD_TP84_28_His exhibits lytic effect against thermophilic bacteria Geobacillus stearothemophilus, Thermus aquaticus, Bacillus stearothermophilus and minor effects against mesophilic Bacillus cereus, Bacillus subtilis. CBD_TP84_28_His retains full activity after preincubation in the temperatures of 30-65°C and exhibits significant activity up to its melting point at 73°C. CBD_TP84_28_His effectively reduces biofilms. These findings suggest that integrating CBDs into thermostable endolysins could enable the development of targeted antibacterial recombinant proteins with diverse clinical and industrial applications.