Screening and Isolation of Fibrinolytic Protease-Producing Mesophilic Bacteria from Soil Samples Obtained from Slaughterhouses Near Karad

Abstract

This study aimed to screen and isolate fibrinolytic protease-producing mesophilic bacteria from soil samples obtained from slaughterhouses near Karad, Maharashtra, India. Soil samples were strategically collected from various locations within the slaughterhouse premises, including areas near animal pens, waste disposal zones, and surrounding soil patches. Using selective agar plate assays with fibrinogen as the sole carbon and nitrogen source, bacterial colonies exhibiting clear zones of proteolysis were identified, indicating fibrinolytic activity. A total of 50 distinct colonies were initially selected, with 30 showing consistent enzyme activity upon further testing. These isolates were characterized based on colony morphology, Gram staining, and biochemical tests, revealing a predominance of Gram-positive, rod-shaped bacteria. Molecular identification through 16S rRNA gene sequencing classified the isolates into genera Bacillus, Pseudomonas, and Staphylococcus, with Bacillus species being the most prolific fibrinolytic protease producers. Bacillus subtilis isolate BS-12 exhibited the highest fibrinolytic activity, followed by Bacillus cereus isolate BC-22 and Pseudomonas aeruginosa isolate PA-15. Optimization of culture conditions, including temperature, pH, agitation speed, and nutrient supplementation, was performed to enhance protease production. Optimal conditions varied among isolates, with Bacillus species favoring slightly alkaline pH and moderate agitation speeds. Protease activity was quantified using azocasein as a substrate, with the most promising isolates showing enzyme activities ranging from 50 to 150 U/mL. The fibrinolytic proteases demonstrated potential for thrombolytic therapy by efficiently degrading fibrin clots in vitro. Challenges such as enzyme purification, stability, and scalability were noted, highlighting the need for further research to optimize downstream processing and enhance enzyme formulations. Future directions include detailed biochemical and structural characterization of the enzymes, genetic engineering to improve yield and specificity, and testing in diverse industrial and biomedical settings. This study underscores the significance of slaughterhouse environments as reservoirs of biotechnologically valuable microorganisms and contributes to the growing body of knowledge on microbial diversity and enzyme discovery for industrial and medical applications.