Isolation and Characterization of a Biocontrol Serine Protease from Pseudomonas aeruginosa FZM498 Involved in Antagonistic Activity Against Blastocystis sp. Parasite

The intestine is considered a habitat for both bacteria and parasites. In this study, many fecal bacterial isolates and the protozoan <i>Blastocystis</i> sp. were recovered from stool samples of individuals with gastrointestinal conditions. Isolated bacteria were tested for extracellular protease production, and the most potent producer was identified by <i>16SrDNA</i> gene sequencing as <i>P. aeruginosa</i> FZM498. The enzyme was extracted and purified to electrophoretic homogeneity by the DEAE-Sepharose ion-exchanger and SDS-PAGE revealed a major band at 42.15 KDa. It exhibited maximal activity at 35 °C with thermostability at 60 °C (<i>T</i>_1/2 = 200.04 min). It was most active at pH 8.0 and stable at 5.0-9.5. Enzymatic activity was greatly stimulated in the presence of Fe²⁺ ions, but was repressed by Zn²⁺ and Hg²⁺ ions. Inhibition by PMSF, TLCK, aprotinin, benzamidine, and SBTI protease reagents suggests a serine protease family. The <i>V</i>_max and <i>K</i>_m dynamic constants against azocasein were 36.232 U/mL and 0.0072 mM, respectively. It exhibited the lowest <i>K</i>_m value against the synthetic substrate D-Val-Leu-Lys-pNA among all substrates, indicating a plasmin-like activity. Interestingly, when tested against <i>Blastocystis</i> sp., cysts appeared progressively shrunken, ruptured, and mycelial-like, indicating complete structural collapse with leakage of intracellular contents. The importance of this research is that it is the first study to test the anti-<i>Blastocystis</i> activity of an extracted bacterial serine protease from the gut. This could be a promising, eco-friendly, natural alternative as an anti-<i>Blastocystis</i> agent. The objective of this study was to isolate, purify, and biochemically characterize an extracellular serine protease produced by gut-associated bacteria, as well as to assess its in vitro anti-<i>Blastocystis</i> efficacy as a potential natural and ecologically friendly antiparasitic therapy.