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

## TL;DR

This study isolates a protease from gut bacteria that can destroy Blastocystis parasites, offering a natural treatment option.

## Contribution

First study to test a bacterial serine protease's anti-Blastocystis activity from the gut.

## Key findings

- The protease from P. aeruginosa FZM498 showed optimal activity at 35°C and pH 8.0.
- The enzyme caused structural collapse of Blastocystis sp. cysts in vitro.
- It exhibited plasmin-like activity with the lowest Km value against D-Val-Leu-Lys-pNA.

## Abstract

The intestine is considered a habitat for both bacteria and parasites. In this study, many fecal bacterial isolates and the protozoan Blastocystis 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 16SrDNA gene sequencing as P. aeruginosa 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 (T1/2 = 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 Vmax and Km dynamic constants against azocasein were 36.232 U/mL and 0.0072 mM, respectively. It exhibited the lowest Km value against the synthetic substrate D-Val-Leu-Lys-pNA among all substrates, indicating a plasmin-like activity. Interestingly, when tested against Blastocystis 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-Blastocystis activity of an extracted bacterial serine protease from the gut. This could be a promising, eco-friendly, natural alternative as an anti-Blastocystis 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-Blastocystis efficacy as a potential natural and ecologically friendly antiparasitic therapy.