Present treatments for gastroenteritis include the use of antibiotics, probiotics, or spore solutions, which are derived mainly from microbial communities. To avoid multidrug resistance, probiotic bacteria like Bacillus clausii can be of great importance. In the present study, isolation, purification, and physicochemical characterization and bactericidal effect of an extracellularly secreted, novel protease was performed from B. clausii strain UBBC07. In vitro assays, including well-diffusion assay and gel-overlay assay, were used to confirm the anti-diarrheal potential of isolated protein. Molecular weight and the sequence of purified protease were identified by LC-MS/MS. The purified protein’s molecular mass was 23,460 Da. Peptide mass fingerprinting confirmed the novel protein to be a protease with 36% homology to the existing Din b family protein, with strong metalloprotease-like properties. Physicochemical analysis showed that the protein was stable at a broad range of temperatures and pH and retained up to 50% activity at very high temperatures and extreme pH. The efficacy of protein was tested against two major diarrhea-causing pathogens (Bacillus cereus and Salmonella enterica), and the minimum inhibitory concentration obtained was 9.78 μM. The bactericidal effect of purified protein was assayed using time-kill kinetics, where 105 CFU/ml of B. cereus and S. enterica cells were killed in 4 h. Microscopic analysis of Gram-stained bacterial cells also showed debris formation after protease treatment. Researchers have described the impact of resistant bacteria on the long-term prevalence of diarrhea. This situation inspires researchers to come up with more cost-effective and high-efficacy alternatives like proteases to treat this disease.
Key words: Bacillus clausii, gut disorders, metalloprotease, LC-MS/MS, spores, immunity
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