The impact of extraction processes on the physicochemical, functional properties and structures of bamboo shoot protein.

This study aimed to extract bamboo shoot protein (BSP) using different extraction approaches and compare their functional and physicochemical properties with commercial protein ingredients, including whey protein and soy protein isolates. The extraction methods including alkali extraction (AE), salt extraction (SE), and phosphate-aided ethanol precipitation (PE) were used. An enhanced solvent extraction method was utilized in combination, resulting in a significant improvement in the protein purity, which reached 81.59 %, 87.36 %, and 67.08 % respectively. The extraction methods had significant effects on the amino acid composition, molecular weight distribution, and functional properties of the proteins. SE exhibited the best solubility and emulsification properties. Its solubility reached up to 93.38 % under alkaline conditions, and the emulsion stabilized by SE with enhanced solvent extraction retained 60.95 % stability after 120 min, which could be attributed to its higher protein content, higher surface hydrophobicity, and relative more stable and organized protein structure. All three BSP samples demonstrated better oil holding capacity, while the SE sample showed comparable functional properties to soy protein such as foaming and emulsifying properties. These findings indicate the potential of BSP as an alternative plant protein ingredient in the food industry.

Two-Dimensional Gel Electrophoresis for Protein Separation of Plant Samples.

This chapter provides a description of the procedure for two-dimensional electrophoresis that can be performed for any given gel size and isoelectric focusing range. This will enable the operator to recognize critical steps and gain sufficient information to generate 2D images suitable for computer-assisted analysis of 2D-gel, as well as mass spectrometry analysis for protein identification and characterization.

One-Dimensional Acrylamide Gel Electrophoresis for Analysis of Plant Samples.

Polyacrylamide gel electrophoresis (PAGE) is a widely used technique for separating proteins from complex plant samples. Prior to the analysis, proteins must be extracted from plant tissues, which are rather complex than other types of biological material. Different protocols have been applied depending on the protein source, such as seeds, pollen, leaves, roots, and flowers. Total protein amounts must also be determined before conducting gel electrophoresis. The most common methodologies include PAGE under native or denaturing conditions. Both procedures are used consequently for protein identification and characterization via mass spectrometry. Additionally, various staining procedures are available to visualize protein bands in the gel, facilitating the software-based digital evaluation of the gel through image acquisition.

Sample Preparation and Phosphopeptide Enrichment for Plant Phosphoproteomics via Label-Free Mass Spectrometry.

Phosphopeptide enrichment is the main bottleneck of every phosphorylation study. Therefore, in this chapter, a general workflow tries to overbridge the hurdles of plant sample handling from sample collection to protein extraction, protein solubilization, enzymatic digestion, and enrichment step prior to mass spectrometry. The workflow provides information to perform global proteomics as well as phosphoproteomics enabling the researcher to use the protocol in both fields.

Modification of the physicochemical, functional, biochemical and structural properties of a soursop seed (Annona muricata L.) protein isolate treated with high-intensity ultrasound.

The obtained seeds from fruit processing are considered by-products containing proteins that could be utilized as ingredients in food manufacturing. However, in the specific case of soursop seeds, their usage for the preparation of protein isolates is limited. In this investigation a protein isolate from soursop seeds (SSPI) was obtained by alkaline extraction and isoelectric precipitation methods. The SSPI was sonicated at 200, 400 and 600 W during 15 and 30 min and its effect on the physicochemical, functional, biochemical, and structural properties was evaluated. Ultrasound increased (p < 0.05) up to 5 % protein content, 261 % protein solubility, 60.7 % foaming capacity, 30.2 % foaming stability, 86 % emulsifying activity index, 4.1 % emulsifying stability index, 85.4 % in vitro protein digestibility, 423.4 % albumin content, 83 % total sulfhydryl content, 316 % free sulfhydryl content, 236 % α-helix, 46 % ß-sheet, and 43 % ß-turn of SSPI, in comparison with the control treatment without ultrasound. Furthermore, ultrasound decreased (p < 0.05) up to 50 % particle size, 37 % molecular flexibility, 68 % surface hydrophobicity, 41 % intrinsic florescence spectrum, and 60 % random coil content. Scanning electron microscopy analysis revealed smooth structures of the SSPI with molecular weights ranging from 12 kDa to 65 kDa. The increase of albumins content in the SSPI by ultrasound was highly correlated (r = 0.962; p < 0.01) with the protein solubility. Improving the physicochemical, functional, biochemical and structural properties of SSPI by ultrasound could contribute to its utilization as ingredient in food industry.

Efficacy of maturase K and rpL20 protein extracted from C. procera leaves on Anophelesstephensi.

The present work is carried out to protein isolation, purification, and characterization from leaves, stem, and seed of C. procera and to evaluate the larvicidal potential on Anopheles stephensi. The whole protein was isolated using protein extraction buffer and precipitated by ammonium sulphate and larvicidal active protein was purified by the column chromatography. The homogeneity of larvicidal protein was confirmed by the SDS-PAGE. The identification of protein was done by the HPLC and LC-MS/ESI-MS. The crude protein from leaves showed 100% mortality of 3rd instar larvae of An. stephensi at the concentration of 5.5 mg/ml after 24 h of exposure. The crude protein from stem showed 25% mortality and no mortality observed was observed in seed protein. The leaves crude protein was further purified by ion exchange chromatography and eluted fractions were tested for larvicidal potential. The purified single protein fractions L2 and L3 from C. procera leaves showed 100% mortality at concentration of 0.06 mg/ml. The homogeneity of purified protein was confirmed by SDS-PAGE and two bands of 26 kDa and 15 kDa protein were observed. The peptide sequence "R.SQMLENSFLIENVMKR.L" was identified in the trypsin digested homogenous protein fraction L2 and "R.DRGSQKR.N" peptide sequence in L3 fraction by LC-MS/ESI-MS. The CprL2 peptide showed the sequence similarity with the protein maturase K and CprL3 peptide showed the sequence similarity with ribosomal protein L20 of C. procera. The conserved functional domain was also identified in both the CprL2 and CprL3 peptide. The identified proteins showed strong larvicidal efficacy at very low concentration. The identified proteins are novel and natural larvicidal agents against An. stephensi and hence can be used to control the malaria.

Expression and Purification of Recombinant Bowman-Birk Trypsin Inhibitor from Foxtail Millet Bran and Its Anticolorectal Cancer Effect In Vitro and In Vivo.

Trypsin inhibitors derived from plants have various pharmacological activities and promising clinical applications. In our previous study, a Bowman-Birk-type major trypsin inhibitor from foxtail millet bran (FMB-BBTI) was extracted with antiatherosclerotic activity. Currently, we found that FMB-BBTI possesses a prominent anticolorectal cancer (anti-CRC) activity. Further, a recombinant FMB-BBTI (rFMB-BBTI) was successfully expressed in a soluble manner in host strain Escherichia coli. BL21 (DE3) was induced by isopropyl-ß-d-thiogalactoside (0.1 mM) at 37 °C for 3.5 h by the pET28a vector system. Fortunately, a purity greater than 93% of rFMB-BBTI with anti-CRC activity was purified by nickel-nitrilotriacetic acid affinity chromatography. Subsequently, we found that rFMB-BBTI displays a strikingly anti-CRC effect, characterized by the inhibition of cell proliferation and clone formation ability, cell cycle arrest at the G2/M phase, and induction of cell apoptosis. It is interesting that the rFMB-BBTI treatment had no obvious effect on normal colorectal cells in the same concentration range. Importantly, the anti-CRC activity of rFMB-BBTI was further confirmed in the xenografted nude mice model. Taken together, our study highlights the anti-CRC activity of rFMB-BBTI in vitro and in vivo, uncovering the clinical potential of rFMB-BBTI as a targeted agent for CRC in the future.

Rice proteins: A review of their extraction, modification techniques and applications.

Rice protein is highly nutritious and easy to digest and absorb. Its hydrolyzed peptides have significant effects on lowering blood pressure and cholesterol. First, a detailed and comprehensive explanation of rice protein extraction methods was given, and it was found that the combination of enzymatic and physical methods could improve the extraction rate of rice protein, but it was only suitable for laboratory studies. Second, the methods for improving the functional properties of rice protein were introduced, including physical modification, chemical modification, and enzymatic modification. Enzymatic modification of the solubility of rice protein to improve its functional properties has certain limitations due to the low degree of hydrolysis, the long time required, the low utilization of the enzyme, and the possible undesirable taste of the product. Finally, the development and utilization of rice protein was summarized and the future research direction was suggested. This paper lists the advantages and disadvantages of various extraction techniques, points out the shortcomings of existing extraction techniques, aims to fill the gap in the field of rice protein extraction, and then provides a possible improvement method for the extraction and development of rice protein in the future.

Extractability and chromatographic separation of proteins from potato (Solanum tuberosum L.) trimmings.

By-products from the potato processing industry, like potato trimmings, are sustainable sources of proteins. Here, a size-exclusion high performance liquid chromatography (SE-HPLC) method was applied to simultaneously determine the extractability and aggregation state of proteins from three batches of potato trimmings of different cultivars. Obtained SE-HPLC profiles allowed distinguishing between the patatin and protease inhibitor fractions of potato proteins. Moreover, only 75% of the crude proteins could be extracted in phosphate buffer containing sodium dodecyl sulfate and a reducing agent, indicating the presence of physical extraction barriers. Ball milling for 5 min significantly increased protein extractability, but prolonged treatment resulted in aggregation of native patatin and a reduced protein extractability. Microwave-dried trimmings had a lower protein extractability than freeze-dried trimmings. In future research, the SE-HPLC method can be used to examine changes in potato protein (fractions) as a result of processing.

Extraction of Soluble Proteins from Leaves.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

Fibrillization of lentil proteins is impacted by the protein extraction conditions and co-extracted phenolics.

Legume proteins can be induced to form amyloid-like fibrils upon heating at low pH, with the exact conditions greatly impacting the fibril characteristics. The protein extraction method may also impact the resulting fibrils, although this effect has not been carefully examined. Here, the fibrillization of lentil protein prepared using various extraction methods and the corresponding fibril morphology were characterized. It was found that an acidic, rather than alkaline, protein extraction method was better suited for producing homogeneous, long, and straight fibrils from lentil proteins. During alkaline extraction, co-extracted phenolic compounds bound proteins through covalent and non-covalent interactions, contributing to the formation of heterogeneous, curly, and tangled fibrils. Recombination of isolated phenolics and proteins (from acidic extracts) at alkaline pH resulted in a distinct morphology, implicating a role for polyphenol oxidase also in modifying proteins during alkaline extraction. These results help disentangle the complex factors affecting legume protein fibrillization.

Affinity gel synthesis from the p-aminobenzoic acid derivative 4-amino-2-methylbenzoic acid and purification of polyphenol oxidase from various plant sources.

The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (Ki: 0.07 ± 0.03 µM), mushroom (Ki: 0.7 ± 0.3 µM), and eggplant (Ki: 4.8 ± 1.2 µM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (Ki: 1112 ± 324 µM), mushroom (Ki: 567 ± 81 µM), and eggplant (Ki: 2016.7 ± 805.6 µM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO.

A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Fern proteins show promise against crop pests.

These and other ancient plants may provide alternatives to chemical insecticides.

Isolation of Protein Complexes from Tobacco Leaves by a Two-Step Tandem Affinity Purification.

Protein purification is an essential method for understanding protein function, as many biochemical and structural techniques require a high concentration of isolated protein for analysis. Yet, many studies of protein complexes are hampered by our inability to express them recombinantly in model systems, generally due to poor expression or aggregation. When studying a protein complex that requires its host cellular environment for proper expression and folding, endogenous purification is typically required. Depending on the protein of interest, however, endogenous purification can be challenging because of low expression levels in the host and lack of knowledge working with a non-model expression system, resulting in yields that are too low for subsequent analysis. Here, we describe a protocol for the purification of protein complexes endogenous to Nicotiana benthamiana directly from leaf tissue, with yields that enable structural and biochemical characterization. The protein complex is overexpressed in Nicotiana benthamiana leaves via agroinfiltration, and the protein-packed leaves are then mechanically ground to release the complex from the cells. The protein complex is finally purified by a simple two-step tandem affinity purification using distinct affinity tags for each complex member, to ensure purification of the assembled complex. Our method yields enough protein for various biochemical or structural studies. We have previously used this protocol to purify the complex formed by an innate immune receptor native to tobacco, ROQ1, and the Xanthomonas effector XopQ, and to solve its structure by single-particle cryo-electron microscopy-we use this example to illustrate the approach. This protocol may serve as a template for the purification of proteins from N. benthamiana that require the plant's cellular environment and are expressed at low levels. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression of the protein complex in leaf tissue Basic Protocol 2: Tandem affinity purification of the ROQ1-XopQ complex.

Cytotoxic Cyclotides from Anchietea pyrifolia, a South American Plant Species.

Cyclotides are mini-proteins with potent bioactivities and outstanding potential for agricultural and pharmaceutical applications. More than 450 different plant cyclotides have been isolated from six angiosperm families. In Brazil, studies involving this class of natural products are still scarce, despite its rich floristic diversity. Herein were investigated the cyclotides from Anchietea pyrifolia roots, a South American medicinal plant from the family Violaceae. Fourteen putative cyclotides were annotated by LC-MS. Among these, three new bracelet cyclotides, anpy A-C, and the known cycloviolacins O4 (cyO4) and O17 (cyO17) were sequenced through a combination of chemical and enzymatic reactions followed by MALDI-MS/MS analysis. Their cytotoxic activity was evaluated by a cytotoxicity assay against three human cancer cell lines (colorectal carcinoma cells: HCT 116 and HCT 116 TP53-/- and breast adenocarcinoma, MCF 7). For all assays, the IC50 values of isolated compounds ranged between 0.8 and 7.3 µM. CyO17 was the most potent cyclotide for the colorectal cancer cell lines (IC50, 0.8 and 1.2 µM). Furthermore, the hemolytic activity of anpy A and B, cyO4, and cyO17 was assessed, and the cycloviolacins were the least hemolytic (HD50 > 156 µM). This work sheds light on the cytotoxic effects of the anpy cyclotides against cancer cells. Moreover, this study expands the number of cyclotides obtained to date from Brazilian plant biodiversity and adds one more genus containing these molecules to the list of the Violaceae family.

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila.

Cyclotides and acyclic versions of cyclotides (acyclotides) are peptides involved in plant defense. These peptides contain a cystine knot motif formed by three interlocked disulfide bonds, with the main difference between the two classes being the presence or absence of a cyclic backbone, respectively. The insecticidal activity of cyclotides is well documented, but no study to date explores the insecticidal activity of acyclotides. Here, we present the first in vivo evaluation of the insecticidal activity of acyclotides from Rinorea bengalensis on the vinegar fly Drosophila melanogaster. Of a group of structurally comparable acyclotides, ribe 31 showed the most potent toxicity when fed to D. melanogaster. We screened a range of acyclotides and cyclotides and found their toxicity toward human red blood cells was substantially lower than toward insect cells, highlighting their selectivity and potential for use as bioinsecticides. Our confocal microscopy experiments indicated their cytotoxicity is likely mediated via membrane disruption. Furthermore, our surface plasmon resonance studies suggested ribe 31 preferentially binds to membranes containing phospholipids with phosphatidyl-ethanolamine headgroups. Despite having an acyclic backbone, we determined the three-dimensional NMR solution structure of ribe 31 is similar to that of cyclotides. In summary, our results suggest that, with further optimization, ribe 31 could have applications as an insecticide due to its potent in vivo activity against D. melanogaster. More broadly, this work advances the field by demonstrating that acyclotides are more common than previously thought, have potent insecticidal activity, and have the advantage of potentially being more easily manufactured than cyclotides.

Leucine-Rich, Potent Anti-Bacterial Protein against Vibrio cholerae, Staphylococcus aureus from Solanum trilobatum Leaves.

A 24 kDa leucine-rich protein from ion exchange fractions of Solanum trilobatum, which has anti-bacterial activity against both the Gram-negative Vibrio cholerae and Gram-positive Staphylococcus aureus bacteria has been purified. In this study, mass spectrometry analysis identified the leucine richness and found a luminal binding protein (LBP). Circular dichroism suggests that the protein was predominantly composed of α- helical contents of its secondary structure. Scanning electron microscopy visualized the characteristics and morphological and structural changes in LBP-treated bacterium. Further in vitro studies confirmed that mannose-, trehalose- and raffinose-treated LBP completely inhibited the hemagglutination ability towards rat red blood cells. Altogether, these studies suggest that LBP could bind to sugar moieties which are abundantly distributed on bacterial surface which are essential for maintaining the structural integrity of bacteria. Considering that Solanum triolbatum is a well-known medicinal and edible plant, in order to shed light on its ancient usage in this work, an efficient anti-microbial protein was isolated, characterized and its in vitro functional study against human pathogenic bacteria was evaluated.

Extraction and deproteinization process of polysaccharide from purple sweet potato.

Extraction and deproteinization process of polysaccharide from purple sweet potato (PPSP) were optimized via the response surface methodology (RSM). The results indicated that the optimal conditions of extraction in hot water of PPSP were as follows: The extraction temperature was 120℃, the extraction time was 2.5 hr, and the solid-liquid ratio was 1∶10 (g/ml). The optimal conditions of Sevage deproteinization were as under the oscillation time was 20 min, the deproteinization times was twice, and polysaccharide solution-Sevage reagent ratio was 1:1 (ml/ml). The extraction yield of PPSP was 3.32%, and the protein removal rate was 93.14% in such a condition.