New-Generation Antibacterial Agent—Cellulose-Binding Thermostable TP84_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 (phage) TP-84, and it effectively digests host bacteria cell wall. Biofilms, together with antibiotic resistance, are major problems in clinical medicine and industry. The challenge is to keep antibacterial molecules at the site of desired action, as their diffusion leads to a loss of efficacy. The TP84_28 endolysin gene was cloned into an expression-fusion vector, forming a fusion gene <i>cbd_tp84_28_his</i> with a cellulose-binding domain from the cellulase enzyme. The Cellulose-Binding Thermostable TP84_Endolysin (CBD_TP84_28_His) fusion protein was biosynthesized in <i>Escherichia coli</i> and purified. Thermostability and enzymatic activities against various bacterial species were measured by a turbidity reduction assay, a spot assay, and biofilm removal. Cellulose-binding properties were confirmed via interactions with microcellulose and cellulose paper-based immunoblotting. The high affinity of the CBD allows for a 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 a lytic effect against thermophilic bacteria <i>Geobacillus stearothemophilus</i>, <i>Thermus aquaticus</i>, <i>Bacillus stearothermophilus</i>, and <i>Geobacillus</i> ICI and minor effects against mesophilic <i>Bacillus cereus</i> and <i>Bacillus subtilis.</i> 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.