〔Abstract〕 To construct the K64 capsule depolymerase recombinant protein , Dep44 , and investigate its potential application against carbapenem-resistant Klebsiella pneumoniae ( CRKP) infections . Methods The de- polymerase-encoding phage vB _Kpn_HF1013 ( GenBank : PP803128) was isolated and genomically analyzed to screen for candidate depolymerases . The recombinant protein Dep44 was constructed and functionally verified for depolymerase activity. Dep44 sensitive range was validated and Dep44 antimicrobial activity was assessed by bio- film disruption and serum sterilization assays . Results The tail spike protein of phage vB_Kpn_HF1013 exhibited depolymerase activity and recombinant protein Dep44 specifically degraded K64 CRKP capsule . Biofilm eradication assays demonstrated that recombinant Dep44 at both 2 μg/mL and 10 μg/mL significantly disrupted bacterial bio- films relative to the control . Serum bactericidal assays showed that Dep44 exhibited synergistic activity with serum , dependent on the complement system , as Dep44 alone lacked bactericidal properties . Conclusion Dep44 effec- tively targets and degrades K64 CRKP capsule , disrupts biofilms , and enhances serum bactericidal activity , high- lighting its potential for managing K64 CRKP infections and clearing biofilms from medical devices .