Purification and Biochemical Characterization of a New Protease Inhibitor from Conyza dioscoridis with Antimicrobial, Antifungal and Cytotoxic Effects.
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 ?-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.
Project description:Herbicide resistance is a challenge for modern agriculture further complicated by cases of resistance to multiple herbicides. Conyza bonariensis and Conyza canadensis are invasive weeds of field crops, orchards, and non-cropped areas in many parts of the world. In California, USA, Conyza populations resistant to the herbicides glyphosate and paraquat have recently been described. Although the mechanism conferring resistance to glyphosate and paraquat in these species was not elucidated, reduced translocation of these herbicides was observed under experimental conditions in both species. Glyphosate and paraquat resistance associated with reduced translocation are hypothesized to be a result of sequestration of herbicides into the vacuole, with the possible involvement of over-expression of genes encoding tonoplast transporters of ABC-transporter families in cases of glyphosate resistance or cationic amino acid transporters (CAT) in cases of paraquat resistance. However, gene expression in response to herbicide treatment has not been studied in glyphosate and paraquat resistant populations. In the current study, we evaluated the transcript levels of genes possibly involved in resistance using real-time PCR. First, we evaluated eight candidate reference genes following herbicide treatment and selected three genes that exhibited stable expression profiles; ACTIN, HEAT-SHOCK-PROTEIN-70, and CYCLOPHILIN. The reference genes identified here can be used for further studies related to plant-herbicide interactions. We used these reference genes to assay the transcript levels of EPSPS, ABC transporters, and CAT in response to herbicide treatment in susceptible and resistant Conyza spp. lines. No transcription changes were observed in EPSPS or CAT genes after glyphosate or paraquat treatment, suggesting that these genes are not involved in the resistance mechanism. Transcription of the two ABC transporter genes increased following glyphosate treatment in all Conyza spp. lines. Transcription of ABC transporters also increased after paraquat treatment in all three lines of C. bonariensis. However, in C. canadensis, paraquat treatment increased transcription of only one ABC transporter gene in the susceptible line. The increase in transcription of ABC transporters after herbicide treatment is likely a stress response based on similar response observed across all Conyza lines regardless of resistance or sensitivity to glyphosate or paraquat, thus these genes do not appear to be directly involved in the mechanism of resistance in Conyza spp.
Project description:<h4>Background</h4>Flavonoids are widely used in the market because of their antibacterial, antiviral, and antioxidant activities. But the production speed of flavonoids is limited by the growth of plants. CBL9 (<i>Chaetomium cruentum</i>) is a flavonoid-producing endophytic fungi from <i>Conyza blinii</i> H. Lév, which has potential to produce flavonoids.<h4>Methods</h4>In this study, we isolated total flavonoids from endophytic fungus CBL9 of <i>Conyza blinii</i> H. Lév using macroporous resin D101. The process was optimized by response surface and the best extraction process was obtained. The antioxidant activities of total flavonoids were analyzed in vitro.<h4>Results</h4>It was found that the best parameters were 25 °C pH 2.80, 1.85 h, and the adsorption ratio reached (64.14 ± 0.04)%. A total of 60% ethanol was the best elution solvent. The elution ratio of total flavonoid reached to (81.54 ± 0.03)%, and the purity was 7.13%, which was increased by 14.55 times compared with the original fermentation broth. Moreover its purity could rise to 13.69% after precipitated by ethanol, which is very close to 14.10% prepared by ethyl acetate extraction. In the antioxidant research, the clearance ratio of L9F-M on DPPH, ABTS, •OH, •O<sup>2-</sup>, (96.44 ± 0.04)% and (75.33 ± 0.03)%, (73.79 ± 0.02)%, (31.14 ± 0.01)% at maximum mass concentration, was higher than L9F.<h4>Conclusion</h4>The result indicated using macroporous resin in the extraction of total flavonoid from endophytic fungus is better than organic solvents with higher extraction ratio, safety and lower cost. In vitro testing indicated that the flavonoid extracted by macroporous resin have good antioxidant activity, providing more evidence for the production of flavonoid by biological fermentation method.
Project description:<h4>Background</h4><i>Conyza blinii</i> H. Lév is a medicinal plant that has a variety of pharmacological activities, but its study is at a standstill due to the shortage of resources.<h4>Method</h4>This study utilized the surface sterilization method to isolate endophytic fungi, and they were preliminarily identified by morphology. Flavonoid-producing strains were screened by NaNO<sub>2</sub>-Al(NO)<sub>3</sub> colorimetry and further identified by the ITS sequence. Additionally, we used five antioxidant assays (DPPH, Hydroxyl radical, ABTS, FRAP and T-AOC assays) to systematically evaluate the antioxidant capacity of total flavonoids , and we also determined their antibacterial activity.<h4>Results</h4>In this study, 21 endophytic fungi were isolated from wild <i>Conyza blinii</i> H. Lév for the first time. There were six flavonoid-producing strains, especially CBL11, whose total flavonoid content reached 50.78 ± 2.4 mg/L. CBL12, CBL12-2 and CBL1-1 all exhibited excellent antioxidant activity. The effect of CBL12 was similar to that of ascorbic acid at low concentrations, and its radical scavenging rates for DPPH and ABTS were 94.56 ± 0.29 % and 99.88 ± 0.27%, respectively, while its IC<sub>50</sub> values were only 0.11 ± 0.01 mg/mL and 0.2 ± 0.01 mg/mL. Through LC-MS, we found that CBL12 could produce many high-value flavonoids, such as 3-methoxyflavone, nobiletin, formononetin, scopoletin, and daidzein. Additionally, CBL9 had good antibacterial activity against both gram-positive and gram-negative bacteria. Notably, we obtained the high-yield strains CBL12 and CBL9, which not only had high yields (10.64 ± 1.01 mg/L and 10.17 ± 0.11 mg/L, respectively) but also had excellent biological activity. Hence, the results of this study provide new ideas for endophytic fungi that can be exploited as a source of flavonoids and other medicinal components from <i>Conyza blinii</i> H. Lév. Moreover, this study can serve as a reference for the development of rare medicinal materials.
Project description:Flaxleaf fleabane (<i>Conyza bonariensis</i> [L.] Cronquist) is one of the most difficult weeds to control worldwide. There are more than 150 <i>Conyza</i> species in the world and eight species in Australia. Correct identification of these species can be problematic due to their morphological similarities especially at seedling stage. Developing a robust genetics - based species identification method to distinguish <i>C. bonariensis</i> from other closely related species is important for early control of weeds. We thus examined the chloroplast (cp) genome of <i>C. bonariensis</i>, aiming to identify novel DNA barcodes from the genome sequences, and use the entire cp genome as a super-barcode for molecular identification. The <i>C. bonariensis</i> chloroplast genome is 152,076 bp in size, encodes 133 genes including 88 protein-coding genes, 37 tRNA genes and 8 ribosomal RNA genes. A total of 151 intergenic regions and 19 simple sequence repeats were identified in the cp genome of <i>C. bonariensis</i>, which provides a useful genetic resource to develop robust markers for the genetic diversity studies of <i>Conyza</i> species. The sequence information was used to design a robust DNA barcode <i>rps</i>16 and <i>trnQ-UUG</i> which successfully separated three predominant <i>Conyza</i> species (<i>C. bonariensis</i>, <i>C. canadensis</i>, and <i>C. sumatrensis</i>). Phylogenetic analyses based on the cp genomes of <i>C. bonariensis</i>, <i>C. canadensis</i> and 18 other Asteraceae species revealed the potential of using entire cp genome as a plant super-barcode to distinguish closely-related weed species.
Project description:<i>Conyza bonariensis</i>, flaxleaf fleabane, is a serious weed in Australian agricultural systems, particularly the north-east cropping system. We present the complete chloroplast sequence of <i>C. bonariensis</i> reconstructed from Illumina whole genome shotgun sequencing. This is the first complete chloroplast genome available for genus <i>Conyza</i>. The complete chloroplast sequence is 153,014?bp long, and has the same gene content and structure as other members of the tribe Astereae. A Bayesian phylogeny of the chloroplast coding regions of 18 representatives of Astereae is presented. The <i>C. bonariensis</i> chloroplast genome is deposited at GenBank under accession number MF276802.
Project description:Reducing pesticide application in agricultural land is a major challenge for the twenty-first century. Responses of weed seed's germination and seedling's early development to chemical soil conditions around the seed may be a promising way to aid weed control in a no-till system. Thus, the objective of this work was to test, under controlled conditions, whether different chemical conditions affect the germination and development of horseweed [Conyza canadensis (L.) Cronquist]. We used, as treatment, solutions containing different nutrients (P, K, Ca, and Mg), separately and in combination, and at two pH levels (4.8 and 6.5). Phosphorus alone inhibited horseweed seed germination at ~ 7 times while had ~ 4 times reduction in final germination percentage and germination speed index for both pH tested. Other nutrients tested had a no-effect in germination speed index compared to the control treatment. Potassium alone or associated with other ions (P, Ca, and Mg) at pH 4.8 had a synergistic effect on seedling development (root and shoot length). In the same way, K associated with Mg was synergistic to the root length at pH 6.5. Seeds in the control treatment (distilled water) presented a high germination speed index at pH 6.5, while at low pH this parameter was higher when in association with KMg, PMg and Ca. The findings demonstrate that seed germination traits and seedling development of horseweed depend on nutrient kind exposure and pH conditions in the seed environment. This work suggests that adequate topsoil management (i.e., pH and nutrient availability) may aid to reduce weed germination, because, it consists of an important factor of weed occurrence in agricultural areas.
Project description:Human urinary trypsin inhibitor (UTI) is a proteoglycan composed of one core protein covalently linked to one glycosaminoglycan, which is a low sulfated chondroitin 4-sulfate. It is used as an anti-inflammatory medicine based on the protease inhibitory activity of the core protein. However, functions of the chondroitin sulfate have not been clarified. Recently, we succeeded in remodeling the UTI chondroitin sulfate to hyaluronan to create hyaluronan hybrid UTI, without changing the core protein. Here, we investigated the effect of the remodeled chondroitin sulfate on the activities of serine proteases. Native UTI showed stronger protease inhibitory activity than hyaluronan hybrid UTI or hydrolyzed glycosaminoglycan UTI. Chondroitin 4-sulfate chains with a small peptide derived from the native UTI did not show any protease inhibitory activity. These results suggest that the chondroitin sulfate chain linked covalently to core protein enhances protease inhibitor activity of UTI although the chondroitin sulfate chain itself does not.
Project description:BACKGROUND:Proteases are hydrolytic enzymes that catalyze peptide linkage cleavage reactions at the level of proteins and peptides with different degrees of specificity. This group draws the attention of industry. More than one protease in three is a serine protease. Classically, they are active at neutral to alkaline pH. The serine proteases are researched for industrial uses, especially detergents. They are the most commercially available enzyme group in the world market. Overall, fungi produced extracellular proteases, easily separated from mycelium by filtration. RESULTS:A new basidiomycete fungus CTM10057, a hyperproducer of a novel protease (10,500?U/mL), was identified as Pleurotus sajor-caju (oyster mushroom). The enzyme, called SPPS, was purified to homogeneity by heat-treatment (80?°C for 20?min) followed by ammonium sulfate precipitation (35-55%)-dialysis, then UNO Q-6 FPLC ion-exchange chromatography and finally HPLC-ZORBAX PSM 300 HPSEC gel filtration chromatography, and submitted to biochemical characterization assays. The molecular mass was estimated to be 65?kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Native-PAGE, casein-zymography, and size exclusion by HPLC. A high homology with mushroom proteases was displayed by the first 26 amino-acid residues of the NH2-terminal aminoacid sequence. Phenylmethanesulfonyl fluoride (PMSF) and diiodopropyl fluorophosphates (DFP) strongly inhibit SPPS, revealing that it is a member of the serine-proteases family. The pH and temperature optima were 9.5 and 70?°C, respectively. Interestingly, SPPS possesses the most elevated hydrolysis level and catalytic efficiency in comparison with SPTC, Flavourzyme® 500?L, and Thermolysin type X proteases. More remarkably, a high tolerance towards organic solvent tolerance was exhibited by SPPS, together with considerable detergent stability compared to the commercial proteases Thermolysin type X and Flavourzyme® 500?L, respectively. CONCLUSIONS:This proves the excellent proprieties characterizing SPPS, making it a potential candidate for industrial applications especially detergent formulations.
Project description:Background: Proteases cleave proteins, thereby providing essential amino acids for protein synthesis, and degrade misfolded and damaged proteins to maintain homeostasis. Proteases also serve as signaling molecules, therapeutic agents and find wide applications in biotechnology and pharmaceutical industry. Plant-derived proteases are suitable for many biomedical applications due to their easy availability and activity over a wide range of pH, temperature, and substrates. Moringa oleifera Lam (Moringaceae) is a very common food plant with medicinal property and geographically distributed in tropical countries. Here, we isolate proteases from the leaves of Moringa oleifera and characterize its enzymatic activity. Methods: Proteases were isolated from the aqueous leaf extract of Moringa oleifera by ammonium sulfate precipitation and purified by ion exchange chromatography. Subsequently, the enzyme kinetics was determined using casein as a substrate and calibrated over different pH and temperature range for maximal activity. Results: We obtained purified fraction of the protease having a molecular weight of 51 kDa. We observed that for the maximal caseinolytic activity of the protease, a pH of 8 and temperature of 37ºC was found to be most effective. Conclusion: The plant-derived proteolytic enzymes are finding increasing clinical and industrial applications. We could extract, purify and characterize the enzymatic activity of proteases from the leaves of Moringa oleifera. Further molecular characterization, substrate specificity and activity of the extracted protease are required for determining its suitability as a proteolytic enzyme for various applications.
Project description:Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (M<sup>pro</sup>) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 M<sup>pro</sup> splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.