Exploration of Egyptian Soil Microbiomes Reveals <em>Citrobacter freundii </em>ASN as a Novel Source for a Direct-Acting Fibrinolytic Serine Protease

Background: Thrombotic disorders remain a leading cause of global mortality. The limitations of current thrombolytic drugs necessitate the search for novel fibrinolytic enzymes from unexplored microbial sources. This study aimed to isolate and characterize potent fibrinolytic enzyme-producing bacteria from unique soil environments in Egypt. Methods: Bacterial isolates from four Egyptian biotopes were primarily screened on fibrin agar plates. The most potent isolate was identified via 16S rRNA gene sequencing. Enzyme production was optimized using a one-factor-at-a-time approach followed by Response Surface Methodology. The enzyme was purified using ammonium sulfate precipitation and column chromatography. Its biochemical properties were characterized, and its mode of action was determined using plasminogen-free fibrin plates and fibrinogenolytic assays. Results: A potent fibrinolytic strain, ASN, was isolated from Nile Delta farmland and identified as Citrobacter freundii. It demonstrated the highest fibrinolytic activity (clear zone of 24 mm; specific activity of 480 U/mg) among 75 isolates. Enzyme production was optimized to 2,320 U/mg, with soluble starch, beef extract, and CaCl₂ as key factors. The enzyme, purified to homogeneity (5.1-fold, 65% yield), was a 31 kDa monomeric serine protease. It exhibited optimal activity at pH 8.0 and 45 °C, with remarkable stability over a broad pH (6.0-10.0) and temperature (up to 50 °C) range. Crucially, the enzyme, designated Frecinase, demonstrated direct fibrinolytic action in plasminogen-free assays and effectively degraded the Aα and Bβ chains of fibrinogen first, indicating its specific fibrinogenolytic nature. Conclusions: This is the first report of a direct-acting fibrinolytic serine protease from Citrobacter freundii. The robust biochemical properties and specific fibrinogenolytic pattern of Frecinase highlight its significant potential as a promising candidate for thrombolytic drug development. Our findings underscore the value of exploring under-investigated ecological niches for novel biocatalysts.