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Browsing by Author "Ahmed, Asmaa A."

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    A putative cytotoxic serine protease from Salmonella typhimurium UcB5 recovered from undercooked burger
    (2023) Kotb, Essam; El-Nogoumy, Baher A.; Alqahtani, Haifa A.; Ahmed, Asmaa A.; Haifa A. Alqahtani; Algarudi, Sakina M.; Almahasheer, Hanan
    A putative virulence exoprotease designated as UcB5 was successfully purified from the bacterium Salmonella typhimurium to the electrophoretic homogeneity with 13.2-fold and 17.1% recovery by hydrophobic, ion-exchange, and gel permeation chromatography using Phenyl-Sepharose 6FF, DEAE-Sepharose CL-6B, and Sephadex G-75, respectively. By applying SDS-PAGE, the molecular weight was confirmed at 35 kDa. The optimal temperature, pH, and isoelectric point were 35 °C, 8.0, 5.6 ± 0.2, respectively. UcB5 was found to have a broad substrate specificity against almost all the tested chromogenic substrates with maximal affinity against N-Succ-Ala-Ala-Pro-Phe-pNA achieving Km of 0.16 mM, Kcat/Km of 3.01 × 105 S−1 M−1, and amidolytic activity of 28.9 µmol min−1 L−1. It was drastically inhibited by TLCK, PMSF, SBTI, and aprotinin while, DTT, β-mercaptoethanol, 2,2′-bipyridine, o-phenanthroline, EDTA, and EGTA had no effect, which suggested a serine protease-type. Also, it has shown a broad substrate specificity against a broad range of natural proteins including serum proteins. A cytotoxicity and electron microscopy study revealed that UcB5 could cause subcellular proteolysis that finally led to liver necrosis. For this, future research should focus on using a combination of external antiproteases and antimicrobial agents for the treatment of microbial diseases instead of using drugs alone.
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    Screening for chitin degrading bacteria in the environment of Saudi Arabia and characterization of the most potent chitinase from Streptomyces variabilis Am1
    (2023) Batool M. AlGarudi; Alabdalall, Amira H.; Safa K. AlZuwaid; Amira Alabdalall; Aldakeel, Sumayh A.; Essam Kotb; Ibtisam Mohammed Ababutain; Algarudi, Sakina M.; Ahmed, Asmaa A.; Albarrag, Ahmed M.
    Forty-six promising chitinolytic isolates were recovered during a screening for chitinolytic bacteria in the environment of Saudi Arabia. The top three isolates belonged to the genus Streptomyces. Streptomyces variabilis Am1 was able to excrete the highest amount of chitinases, reaching the maximum at 84 h with 0.5% yeast extract and nitrogen source and 2% galactose as a carbon source. Purification of chitinase by DEAE-Cellulose and Sephadex G75 improved the specific activity to 18.6-fold and the recovery to 23.8% and showed a mass at 56 kDa. The optimal catalysis of the purified chitinase was at 40 °C and pH 8 with high thermostability and pH stability as reflected by a midpoint temperature value of 66.6 °C and stability at pH 4–9. The protein reagents SDS, EDTA, and EGTA significantly inhibited the enzyme and the EDTA-chelated chitinase restored its activity after the addition of Fe2+ ions suggesting a metallo-chitinase type with ferric ions as cofactors. Chitinase exerted high antifungal activity against some phytopathogenic fungi. Interestingly, the tested Streptomyces were able to produce chitosan nanocubes along with chitosan from chitin degradation which may be an additional power in their antifungal activity in nature. This work also reveals the importance of unexplored environments as a pool of promising microorganisms with biotechnological applications.