The capsular exopolysaccharides (EPS) surrounding the
bacterial cells are essential for protection against recognition by host
defense mechanisms and are important virulence factors for pathogens. The EPS
barrier can be destroyed by phage-associated proteins with depolymerization
activities. It makes bacterial cells more accessible to the effect of natural
defense mechanisms or antibiotics. Utilizing depolymerases of lytic phages is a
promising yet challenging antimicrobial therapy.
The aim of this study was to prepare recombinant phage
protein and to evaluate its enzymatic activity.
The predicted extracellular depolymerase gene of Klebsiella pneumoniae phage was
amplified and cloned in the pEXP-5-CT/TOPO® expression vector (Invitrogen).
Correct construct, verified by sequencing was expressed in E. coli BL21(DE3)
pLysS cells. The recombinant depolymerase was purified by chromatography using
Ni-NTA His•Bind® Resins gravity columns (Piercenet). According to the amino
acid sequence, the molecular weight of this enzyme was calculated to be at 94
kDa. It was showed that enzyme, encoded by Klebsiella
pneumoniae phage degraded purified capsule and removed the capsular
polysaccharide from the surface of bacterial cells. These results indicate the
possibility of utilizing such enzyme as a promising tool for degradation of the
bacterial exopolysaccharides.
