Recombinant depolymerase DP21 from Klebsiella phage GADU21 exerts Dual-Antibiofilm and biofilm-disrupting activity against multiple bacterial species

Phage-derived depolymerases are promising antibiofilm enzymes. In this study, intact and disintegrated GADU21 phage were first tested for their ability to disrupt mature biofilm using the crystal violet assay. Disintegrated phage was more active on biofilms than intact phage. The dp21 gene encoding the depolymerase was then amplified from GADU21, cloned into pET30a, and expressed in E. coli BL21. Purified rDP21 (∼105 kDa) was characterized for antibiofilm activity at different pH values, residual activity following thermal pre-incubation, and Mg2+ dependence, with biofilm degradation and prevention capacity also shown by SEM and CLSM. rDP21 exhibited antibiofilm activity at pH 4–10 and retained partial activity after pre-incubation at temperatures up to 70 °C. Importantly, rDP21 did not affect planktonic growth of the tested bacteria, supporting a matrix-targeting rather than bactericidal mode of action, while significantly reducing preformed biofilm biomass and inhibiting biofilm formation by P. aeruginosa, K. pneumoniae, E. coli, P. mirabilis, S. epidermidis, S. aureus and E. faecalis by 58-59%, 49-50%, 46-47%, 30-34%, 33-31%, 30-24%, and 26-33%, respectively. SEM and CLSM images confirmed pronounced disruption of biofilm. These properties support its potential use as an effective antibiofilm agent.