Department of Biology, College of Science, Imam Abdulrahman Bin Faisal University (IAU), P.O. Box 1982, Dammam, 31441, Saudi Arabia.
Basic & Applied Scientific Research Center, Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam, 31441, Saudi Arabia.
Sci Rep. 2023 Mar 9;13(1):3926. doi: 10.1038/s41598-023-29847-8.
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 K of 0.16 mM, K/K of 3.01 × 10 S M, and amidolytic activity of 28.9 µmol min L. 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.
假定的毒力外切蛋白酶 UcB5 被成功地从沙门氏菌属中纯化出来，通过疏水性、离子交换和凝胶过滤层析，分别使用 Phenyl-Sepharose 6FF、DEAE-Sepharose CL-6B 和 Sephadex G-75，电泳纯度达到 13.2 倍，回收率为 17.1%。通过 SDS-PAGE，分子量确认为 35 kDa。最佳温度、pH 值和等电点分别为 35°C、8.0、5.6±0.2。UcB5 对几乎所有测试的显色底物都具有广泛的底物特异性，对 N-Succ-Ala-Ala-Pro-Phe-pNA 的最大亲和力达到 K 为 0.16 mM，K/K 为 3.01×10 S M，酰胺酶活性为 28.9 µmol min L。它被 TLCK、PMSF、SBTI 和抑肽酶严重抑制，而 DTT、β-巯基乙醇、2,2'-联吡啶、邻菲啰啉、EDTA 和 EGTA 没有影响，这表明它是一种丝氨酸蛋白酶。此外，它对包括血清蛋白在内的广泛天然蛋白质具有广泛的底物特异性。细胞毒性和电子显微镜研究表明，UcB5 可导致亚细胞蛋白酶解，最终导致肝坏死。因此，未来的研究应集中于使用外源性抗蛋白酶和抗菌剂的组合来治疗微生物疾病，而不是单独使用药物。
