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  • Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects.

Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects.

Molecules (Basel, Switzerland) (2020-11-26)
Aida Karray, Mona Alonazi, Slim Smaoui, Philippe Michaud, Dina Soliman, Abir Ben Bacha
ABSTRACT

The main objective of the current study was the extraction, purification, and biochemical characterization of a protein protease inhibitor from Conyzadioscoridis. Antimicrobial potential and cytotoxic effects were also examined. The protease inhibitor was extracted in 0.1 M phosphate buffer (pH 6-7). Then, the protease inhibitor, named PDInhibitor, was purified using ammonium sulfate precipitation followed by filtration through a Sephadex G-50 column and had an apparent molecular weight of 25 kDa. The N-terminal sequence of PDInhibitor showed a high level of identity with those of the Kunitz family. PDInhibitor was found to be active at pH values ranging from 5.0 to 11.0, with maximal activity at pH 9.0. It was also fully active at 50 °C and maintained 90% of its stability at over 55 °C. The thermostability of the PDInhibitor was clearly enhanced by CaCl2 and sorbitol, whereas the presence of Ca2+ and Zn2+ ions, Sodium taurodeoxycholate (NaTDC), Sodium dodecyl sulfate (SDS), Dithiothreitol (DTT), and β-ME dramatically improved the inhibitory activity. A remarkable affinity of the protease inhibitor with available important therapeutic proteases (elastase and trypsin) was observed. PDInhibitor also acted as a potent inhibitor of commercial proteases from Aspergillus oryzae and of Proteinase K. The inhibitor displayed potent antimicrobial activity against gram+ and gram- bacteria and against fungal strains. Interestingly, PDInhibitor affected several human cancer cell lines, namely HCT-116, MDA-MB-231, and Lovo. Thus, it can be considered a potentially powerful therapeutic agent.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Thrombin from bovine plasma, lyophilized powder, ≥2,000 NIH units/mg protein (E1%/280 = 19.5)
Sigma-Aldrich
Cathepsin B from bovine spleen, lyophilized powder, ≥10 units/mg protein
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Collagenase from Clostridium histolyticum, lyophilized powder, ≥125 CDU/mg solid (CDU = collagen digestion units), 0.5-5.0 FALGPA units/mg solid
Sigma-Aldrich
Protease from Bacillus sp., liquid, ≥16 U/g
Sigma-Aldrich
Protease from Bacillus sp., liquid, ≥8 U/g
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Elastase from human leukocytes, lyophilized powder, ≥50 units/mg protein (Bradford)
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Proteinase K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology
Sigma-Aldrich
Protease from Bacillus licheniformis, Type VIII, lyophilized powder, 7-15 units/mg solid
Sigma-Aldrich
Protease from Bacillus licheniformis, ≥2.4 U/g
Sigma-Aldrich
Protease from Aspergillus oryzae, ≥500 U/g
Sigma-Aldrich
α-Chymotrypsin from bovine pancreas, (TLCK treated to inactivate residual tryspin activity), Type VII, essentially salt-free, lyophilized powder, ≥40 units/mg protein