Extraction Of Water-soluble Proteins Research Articles

A Study of Complex Cell Disruption Methods of Intracellular Water-Soluble Protein Extraction from Chlorella sorokiniana

Microalgae biomass is a promising source of plant protein that has potential applications in the food, cosmetics and pharmaceutical industries. However, the presence of a strong cell wall in microalgae cells creates difficulties in obtaining these proteins. This study aims to establish an efficient protocol for disruption of Chlorella sorokiniana microalgae cells to improve the extraction of intracellular water-soluble proteins. C. sorokiniana microalgae cells were disintegrated using ultrasound, microwave radiation, and enzymatic lysis with lysozyme. Ultrasound was the most effective of the cell disruption methods applied separately, increasing the yield of water-soluble proteins by up to 11.4% compared to the control sample. When ultrasound and enzyme were used consecutively, a synergetic effect was observed, as the yield of intracellular water-soluble proteins significantly exceeded (up to 35%) the result achieved by using a single disruption method. The combined effect of physical and biochemical cell disruption methods on the extraction of intracellular water-soluble proteins was also investigated. It was established that several methods applied sequentially to disrupt cells produced a combined, synergetic effect. A mechanism of sequential action of two cell disruption methods, explaining the synergy and increased yield of water-soluble intracellular proteins, is proposed.

Влияние сортовой вариабельности на эффективность водной экстракции белка из нативных семян льна

Currently, flaxseeds as a source of essential micronutrients and biologically active substances are considered as raw materials for the enrichment of food systems. The high content of protein in flaxseeds (18–25%) is the basis for the development of a variety of protein products. The aim of the work is to study the efficacy of water extraction of flaxseeds of different varieties to create healthy food products. The objects of the study were different varieties of flaxseeds, flaxseed cake, and flaxseeds without mucilage. The removal of mucilage from flaxseeds was carried out by water extraction with distilled water at T 50℃ for 60 minutes. For the extraction, flaxseeds were washed and flaxseed cake was moistened up to a moisture content of 80%. The moistened raw materials were crushed and mixed with an extractant (0.6% NaCl solvent) in a ratio 1:10. The process of water extraction was carried out at T 40℃ for 60 minutes. The determination of the characteristics for the protein extracts isolated from different varieties of flaxseeds and flaxseed cake showed that the highest protein yield was obtained from the varieties of fiber flaxseed: 54–56%. The high protein yield can be explained by the higher protein content in the studied flaxseeds. Lower extraction efficiency from flax oilseed flax (35–44%) may point to the influence of high fat content due to the formation of protein-lipid complexes. The protein yield from flaxseed cake was 26.42%, which is explained by the low content of the water-soluble fraction of proteins in this raw material. The pre-removal of mucilage polysaccharides from flaxseeds is followed by a competitive extraction of water-soluble proteins, which reduced the yield of proteins in the following extraction (15.72 and 20.34%). Therefore, the water extraction of flaxseed raw materials is an economically affordable method for obtaining products containing protein fractions in the native state. The obtained protein extracts are a prospective basis for developing a variety of plant-based drinks with high biological value, plant based dairy products, and dry protein products.

Sequential ultrasound-microwave assisted extraction of water soluble proteins from Eurycoma longifolia roots

Even though Eurycoma longifolia was extensively studied, few researches investigated the protein content in its water extracts with the consideration of establishing the most suitable extraction method to increase their yields with high efficiency and less time consumption. The study applied a sequential extraction method to increase the yields of water soluble proteins (WSPs) in E. longifolia root extracts by the application of two nonconventional extraction methods, Microwave assisted extraction (MAE) and ultrasound assisted extraction (UAE). The study was established by circumscribed central composite designs (CCCDs) to indicate the optimum extraction conditions and the corresponding maximum WSPs yields for both the methods by investigating the MAE factors which were temperature (T); microwave power (W) and irradiation time (i) and UAE factors temperature (T); ultrasonic intensity (UI); and sonication time (s). The optimum conditions of MAE (T: 54°C, W: 301W and i: 15 min) led to the WSPs yield 23.101±1.647%.The optimum conditions of UAE (T: 46°C; UI: 1.84 W.cm”2 and s: 24 min) led to 24.181±0.321%. These predicted optimum conditions were then employed for the two proposed sequential extraction method, sequential ultrasound-microwave assisted extraction (SUMAE) and sequential microwave-ultrasound assisted extraction (SMUAE). Results revealed that the highest WSPs yield (27.172±1.086%) was obtained by initiating the sequential extraction with UAE for 10 minutes followed by MAE treatment for 5 minutes was the best extraction process. Therefore, SUMAE illustrated more efficiency than SMUAE. This concluded that the SUMAE is a more efficient extraction process than the one-step nonconventional extraction methods and was nominated for the upscaling of extracting proteins from E. longifolia roots in pilot and industrial scales.

Comparative analysis of protein profiles of aqueous extracts from marine sponges and assessment of cytotoxicity on different mammalian cell types

Marine natural products extracted from sponges represent a new source for drug discovery. Here we describe a simple method for preparing aqueous extracts from 7 Mediterranean demosponges, which allowed the extraction of water-soluble compounds, such as proteins by homogenization of sponge tissue in phosphate buffered saline (PBS).The comparative analysis by SDS-PAGE showed differences in number of bands, bandwidth and intensity among the sponges analyzed. The PAS/silver staining revealed a substantial and different glycoprotein assortment among the demosponges studied.To further study the biological activities present in the sponge extracts, we determined the non-cytotoxic doses on four different mammalian cell types demonstrating that the optimal non-cytotoxic doses were cell type- and extract-dependent.In conclusion, the extraction method described in this paper represents a fast and efficient procedure for the extraction of water-soluble proteins from marine sponges. Furthermore, the cell viability data suggest the feasibility of this method for the direct in vitro cell-based assays.

Mild disintegration of the green microalgae Chlorella vulgaris using bead milling

In this work, the mild disintegration of the microalgae Chlorella vulgaris for the release of intracellular products has been studied. By means of bead milling the microalgae suspensions were successfully disintegrated at different biomass concentrations (25–145gDWkg−1) over a range of agitator speeds (6–12ms−1). In all cases over 97% of cell disintegration was achieved resulting in a release of water soluble proteins. A clear optimum rate of disintegration and protein release was observed at an agitator speed of 9–10ms−1 regardless of the biomass concentration. Selective extraction of water soluble proteins was observed as proteins released sooner than cell disintegration took place. Proteins could be released at 85% lower energy input than for cell disintegration resulting in specific energy consumptions well below 2.5kWhkgDW−1.

Effect of pH and postmortem aging on protein extraction from broiler breast muscle

This study determined the effects of extraction buffer pH and postmortem aging on the extraction of salt-soluble and water-soluble proteins from broiler pectoralis muscle. Deboned broiler breast fillets were collected at 4 h postmortem, packaged, and then stored at 4°C until 1, 5, or 8 d postmortem. After the designated aging period, salt-soluble and water-soluble protein extractions were performed using buffers at 7 different pH levels (pH 5.4, 6.4, 6.9, 7.2, 7.5, 8.0, 9.0). Protein concentrations of the extracts were measured and SDS-PAGE analysis was performed. Salt-soluble protein concentration increased (P < 0.0001) as buffer pH increased from pH 5.4 to 6.9 and then remained unchanged from pH 6.9 to 9.0. Water-soluble protein concentration increased (P < 0.0001) as buffer pH increased from pH 5.4 to 7.2 and then remained unchanged from pH 7.2 to 9.0. There was not a significant extraction buffer pH by aging treatment interaction for the total protein concentration of either the salt-soluble or water-soluble protein extracts. The protein concentrations of salt-soluble extracts were similar at both 1 and 8 d postmortem but lower (P < 0.0001) at 5 d postmortem. The protein concentrations of water-soluble extracts were similar at both 1 and 5 d postmortem, but higher (P < 0.0001) at 8 d. Both extraction buffer pH and postmortem aging influenced the SDS-PAGE protein profiles of salt-soluble and water-soluble protein extracts from breast muscles. Data demonstrate that postmortem aging and extraction buffer pH influence both the total amount and the composition of the myofibrillar and sarcoplasmic proteins that can be extracted from broiler breast fillets.

Relative quantitation of ricin in Ricinus communis seeds by image processing

Quantitative and qualitative assays for specific seed proteins could be used as a screening tool in breeding for improved seed characteristics. These assays could be especially useful for selection of castor seeds for altered expression of the toxin ricin. The technique described here includes extraction of water soluble proteins from partial seed samples so that the remaining seed is viable and can be used for genetic evaluation. Individual proteins are resolved by SDS-PAGE and identified by MALDI-TOF/TOF mass spectrometry. Once toxin proteins are correlated with bands in stained SDS-PAGE gels, image processing is used to estimate the relative quantities of these proteins in extracts from individual seeds. The results indicate that this method can serve as a relatively inexpensive and reliable assay for ricin content in castor endosperm tissues and this approach could facilitate the selection of low ricin castor varieties.

Temperature-responsive ionic liquid/water interfaces: relation between hydrophilicity of ions and dynamic phase change

Phase separation between ionic liquids (ILs) and molecular liquids is of interest physico-chemically, and also has industrial relevance. IL/water mixtures are of great interest in many fields. Unlike static phase separation between IL and water, dynamic shifts of IL/water mixtures between a homogeneous mixture and separate phases have a wide variety of applications. The miscibility of ILs with water generally increases upon heating, and a few ILs undergo a lower critical solution temperature (LCST)-type phase transition with water in which the separated biphases become miscible upon cooling. As the phase transition is controlled by changing the temperature by a few degrees, the LCST-type phase response of IL/water mixtures makes it possible to use ILs as solvents in various energy-saving processes. Since many hydrophilic ILs do not undergo phase separation with water, we aim to determine the necessary conditions under which hydrophobic ILs undergo the phase transition. Based on physico-chemical analysis of many hydrophobic ILs that undergo a phase separation after mixing with water, we find there is a particular range of "hydrophilicity" of these hydrophobic ILs within which the LCST-type phase transition is possible. Accordingly, a hydrophilicity index (HI) of ILs is proposed, in terms of the number of water molecules in the separated IL phase. The HI value proves to be a good indicator of the phase behaviour of IL/water mixtures, as well as their phase transition temperature. Potential application of the LCST-type phase change to the selective extraction of water-soluble proteins is also summarised.

Protein extraction and 2‐DE of water‐ and lipid‐soluble proteins from bovine pericardium, a low‐cellularity tissue

Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl beta-D-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.

Extraction and Separation of Water-Soluble Proteins from Different Wheat Species by Acidic Capillary Electrophoresis

Optimization of protein extraction and a capillary zone electrophoresis method for water-soluble protein analysis in wheat is described. The optimal separation was obtained with a 50 microm i.d. x 27 cm (20 cm to detector) uncoated capillary filled with 0.1 M phosphoric acid/beta-alanine, pH 2.5, buffer containing urea (1 M), 0.05% (w/v) hydroxypropylmethylcellulose, and 20% (v/v) acetonitrile. Separation was carried out at 15 kV and 35 degrees C for 9 min. Extract stability was also investigated within 2 h from the extraction. Good visual peak parameters and a higher sensitivity can be obtained when 30% ethanol is used as an extraction medium. The method was successfully used to analyze extracts obtained from whole and refined meals of six Triticum spp. Moreover, the described methodology could be applied to the discrimination of species with different ploidy levels and to the detection of durum wheat adulteration, as well as to screen wheat collections for enzymes involved with the quality of wheat derivatives.

Effect of high-pressure treatment on processing quality of tilapia meat fillets

Tilapia fillets were treated under 50–300 MPa at 25 °C to examine the change of their processing quality. The gel strength from the tilapia meat treated at 50 MPa for 1 h was 1.2 times as that of fresh meat, but there was a 25% decrease for that treated at 250 MPa for 1 h. Extraction of water-soluble proteins was barely affected by high-pressure treatment; however, that of salt-soluble proteins decreased 60% after treating at 250 MPa for 1 h. The actomyosin was extracted below 10% for the meat treated beyond 200 MPa. The liability to denaturation indicates that pressure treatment was applicable to tilapia processing not for storage.

Proteomic Mapping of Brain Plasma Membrane Proteins

Proteomics is potentially a powerful technology for elucidating brain function and neurodegenerative diseases. So far, the brain proteome has generally been analyzed by two-dimensional gel electrophoresis, which usually leads to the complete absence of membrane proteins. We describe a proteomic approach for profiling of plasma membrane proteins from mouse brain. The procedure consists of a novel method for extraction and fractionation of membranes, on-membrane digestion, diagonal separation of peptides, and high-sensitivity analysis by advanced MS. Breaking with the classical plasma membrane fractionation approach, membranes are isolated without cell compartment isolation, by stepwise depletion of nonmembrane molecules from entire tissue homogenate by high-salt, carbonate, and urea washes followed by treatment of the membranes with sublytic concentrations of digitonin. Plasma membrane is further enriched by of density gradient fractionation and protein digested on-membrane by endoproteinase Lys-C. Released peptides are separated, fractions digested by trypsin, and analyzed by LC-MS/MS. In single experiments, the developed technology enabled identification of 862 proteins from 150 mg of mouse brain cortex. Further development and miniaturization allowed analysis of 15 mg of hippocampus, revealing 1,685 proteins. More that 60% of the identified proteins are membrane proteins, including several classes of ion channels and neurotransmitter receptors. Our work now allows in-depth study of brain membrane proteomes, such as of mouse models of neurological disease.

Purification and characterization of a 22-kDa microsomal protein from rat parotid gland which is phosphorylated following stimulation by agonists involving cAMP as second messenger.

Stimulation of secretion in exocrine glands by agonists involving cAMP as second messenger leads to the phosphorylation of the ribosomal protein S6 (protein I) and two other particulate proteins with apparent molecular masses of 24 kDa (protein II) and 22 kDa (protein III) [Jahn, R., Unger, C. & Söling, H. D. (1980) Eur. J. Biochem. 112, 345-352]. This report describes the purification and characterization of protein III. Solubilization studies indicate that protein III is an intrinsic membrane protein. It could be extracted from the endoplasmic reticulum membrane only with Triton X-100, SDS or concentrated formic or acetic acid. The purification of this protein involved extraction of the microsomes with Triton X-100, removal of the detergent by acetone precipitation, extraction of water-soluble proteins, lipids and lipoproteins, and preparative SDS polyacrylamide gel electrophoresis. The protein has a basic pI (greater than 8.7). For determination of the amino acid composition of protein III and for sequencing of its amino-terminal portion, the protein was electroeluted out off the gel, the detergent removed and the protein finally purified by reversed-phase HPLC. Protein III could be phosphorylated in vitro by the catalytic subunit of the cAMP-dependent protein kinase to a degree of approximately 0.14 mol phosphate/mol protein. The only phosphopeptide obtained after in vitro phosphorylation and subsequent tryptic or chymotryptic digestion was identical with the phosphopeptide obtained after stimulation of intact rat parotid gland lobules with isoproterenol. The sequence of this peptide was Lys-Leu-Ser(P)-Glu-Ala-Asp-Asn-Arg. It was confirmed by an analysis of the synthetic peptide following in vitro phosphorylation with cAMP-dependent protein kinase. The first 41 N-terminal residues of protein III were sequenced. So far no sequence homology with other known peptides or proteins could be found.

Separation and quantitation of ribulose-1,5-bisphosphate carboxylase-oxygenase in spinach leaves by high-performance hydrophobic-interaction chromatography

A rapid and highly efficient method is described for the separation and quantitation of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBPCase; E.C. 4.1.1.39) in spinach leaves by hydrophobic-interaction chromatography. A TSK Phenyl 5 PW column was used for the separation of RuBPCase with magnesium sulphate solutions as the mobile phase. Different initial concentrations of the salt, pH values, buffer substances, and column temperatures were studied. After extraction of water-soluble proteins from powdered spinach leaves with 50 m M Bicine (pH 7.8) at a leaf-to-buffer ratio of 0.25 (g/ml), and after centrifugation of the homogenate, the supernatant was directly injected into the chromatographic column for the quantitative determination of RuBPCase. The chromatographic peak for RuBPCase was identified by its enzymatic activity and further characterized by stop-flow spectroscopy and by gradient polyacrylamide gel electrophoresis, with and without sodium dodecyl sulphate. The calibration curve for RuBPCase was linear for concentrations up to 300 μg of loaded enzyme. The recovery of the enzyme was greater than 90% in terms of activity.

Characteristics of hiproly barley II. Quantification of two proteins contributing to its high lysine content

Quantitative extraction of salt and water soluble proteins from Hiproly, CI 4362 and Bomi barley was performed and their content of the lysine rich SP II albumin determined by a single radial immunodiffusion assay. The elevated content of SP II albumin in Hiproly accounted for 37% of the difference in crude protein lysine between Hiproly and Bomi and for 19% between Hiproly and its low lysine sisterline CI 4362. Apart from the lysine rich SP II albumin, other proteins contribute to the high lysine content of Hiproly.