0b28f49288fd7ee4e725d0a36f380108 pmc_33917878.pdf a3d0901ecc4113e3311a0bc93ed6b9a4cdaa5e35 pmc_33917878.pdf 080462aa1122293baff84028732a9ed1bee2bc0daad389e81dd2b574d23425d5 pmc_33917878.pdf Title: Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties Subject: Plant cystatins (or phytocystatins) comprise a large superfamily of natural bioactive small proteins that typically act as protein inhibitors of papain-like cysteine proteases. In this report, we present the purification and characterization of the first phytocystatin isolated from Moringa oleifera (MoPI). MoPI has a molecular mass of 19 kDa and showed an extraordinary physicochemical stability against acidic pHs and high temperatures. Our findings also revealed that MoPI is one of the most potent cysteine protease inhibitors reported to date, with Ki and IC50 values of 2.1 nM and 5.7 nM, respectively. More interestingly, MoPI presents a strong antimicrobial activity against human pathogens such as Enterococcus faecalis and Staphylococcus aureus. In addition, MoPI also showed important anticoagulant activity, which is an unprecedented property for this family of protease inhibitors. These results highlight the pharmaceutical potential of this plant and its derived bioactive molecules. Keywords: Moringa oleifera; protease; plant protease inhibitor; phytocystatin; papain inhibitor; antibacterial; anticoagulant; bioactive compound Author: Juliana Cotabarren, Santiago Claver, Juan Abreu Payrol, Javier Garcia-Pardo and Walter David Obregón Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Thu Apr 8 12:52:35 2021 CEST ModDate: Fri Apr 9 06:11:37 2021 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 11 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 846947 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- ODMRJP+VnURWPalladioL Type 1 Custom yes yes no 10 0 QHVWFG+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 OOTVTU+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 MPTXAJ+URWPalladioL-BoldItal Type 1 Custom yes yes no 24 0 BAMGYB+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 32 0 SOZKOC+VnURWPalladioL-Bold Type 1 Custom yes yes no 36 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 45 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 73 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 77 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 81 0 YFFKLH+CMSY10 Type 1 Builtin yes yes no 93 0 BOLMEN+SFRM0900 Type 1 Custom yes yes no 106 0 KNJMCL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 118 0 KNJMDN+PalatinoLinotype TrueType WinAnsi yes yes no 121 0 KNJMDP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 124 0 KNJMCL+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 138 0 KNJMDM+PalatinoLinotype CID TrueType Identity-H yes yes yes 141 0 KNJMDN+PalatinoLinotype TrueType WinAnsi yes yes no 147 0 KNJMDP+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 150 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 160 0 CRUZTT+VnURWPalladioL-Italic Type 1 Custom yes yes no 170 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-10-02 03:20:57 CEST RepresentationInformation: pmc_33917878.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2023-03-31 12:09:46 CEST Size: 846947 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 359 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Materials Destination: subsection.2.1 Item: Title: Crude Extract Preparation Destination: subsection.2.2 Item: Title: Identification and Purification of MoPI Destination: subsection.2.3 Children: Item: Title: Partial Purification by Heat Treatment Destination: subsubsection.2.3.1 Item: Title: Affinity Chromatography Destination: subsubsection.2.3.2 Item: Title: Enzymatic Analysis of the Inhibitory Activity against Papain Destination: subsection.2.4 Item: Title: Biological Assays Destination: subsection.2.5 Children: Item: Title: Anticoagulant Activity Destination: subsubsection.2.5.1 Item: Title: Antimicrobial Activity Destination: subsubsection.2.5.2 Item: Title: Statistical Analysis Destination: subsection.2.6 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Isolation and Purification of MoPI Destination: subsection.3.1 Item: Title: Inhibition Kinetics Destination: subsection.3.2 Item: Title: Anticoagulant Activity Destination: subsection.3.3 Item: Title: Antimicrobial Activity Destination: subsection.3.4 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.4 Info: Title: Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties Author: Juliana Cotabarren, Santiago Claver, Juan Abreu Payrol, Javier Garcia-Pardo and Walter David Obregón Subject: Plant cystatins (or phytocystatins) comprise a large superfamily of natural bioactive small proteins that typically act as protein inhibitors of papain-like cysteine proteases. In this report, we present the purification and characterization of the first phytocystatin isolated from Moringa oleifera (MoPI). MoPI has a molecular mass of 19 kDa and showed an extraordinary physicochemical stability against acidic pHs and high temperatures. Our findings also revealed that MoPI is one of the most potent cysteine protease inhibitors reported to date, with Ki and IC50 values of 2.1 nM and 5.7 nM, respectively. More interestingly, MoPI presents a strong antimicrobial activity against human pathogens such as Enterococcus faecalis and Staphylococcus aureus. In addition, MoPI also showed important anticoagulant activity, which is an unprecedented property for this family of protease inhibitors. These results highlight the pharmaceutical potential of this plant and its derived bioactive molecules. 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