Effect of sorbed water on the thermal stability of soybean protein

A soybean protein isolate (SPI), and its beta-conglycinin and glycinin componets were obtained from defatted soybean flour by applying dissolution and precipitation based on the difference in their solubility depending on each isoelectric point. The purity evaluated by SDS-PAGE of the beta-conglycinin and glycinin preparations was about 84% and 80%, respectively, resulting in a clear difference in the pH dependence on solubility. A BET plot derived from the water sorption isotherm at 25 degrees C showed that the amount of the monolayer adsorption of these preparations was about 6-9%, the value for the beta-conglycinin preparation being about 1.5 times higher than that for the glycinin preparation. The beta-conglycinin and glycinin preparations were respectively denatured at around 75 degrees C and 86 degrees C in the presence of excess water, whereas the denaturation temperature of both preparations was markedly increased by decreasing sorbed water content below 40%, corresponding well with the unfrozen water content.     

Novel method using phytase for separating soybean beta-conglycinin and glycinin

A novel method for separating soybean beta-conglycinin and glycinin from defatted soymilk by a phytase treatment was developed. Phytase was added to defatted soymilk (1000FYT/100 g of protein) at pH 6.0, and the mixture incubated for 1 h at 40 degrees C. This procedure separated beta-conglycinin and glycinin without needing a reducing agent or cooling into the soluble and insoluble fractions, respectively. Simultaneously, most of the phytate in both proteins was removed.     

Purification and characterization of mRNA from soybean seeds. Identification of glycinin and beta-conglycinin precursors

Poly(A)-rich RNA was isolated from developing soybean seeds (Glycine max (L.) Merr.) and fractionated on linear log sucrose gradients. Two major fractions sedimenting at 18 S and 20 S were separated and then purified by further sucrose gradient fractionation. Both fractions were active as messengers when added to a rabbit reticulocyte lysate protein synthesis system. The 18 S fraction caused proteins migrating primarily to the 60,000-dalton region of a sodium dodecyl sulfate gel to be produced, while translation of the 20 S fraction preferentially directed the synthesis of polypeptides similar in size to the alpha and alpha' subunits of beta-conglycinin. Evidence that many of the 60,000-dalton polypeptides were related to glycinin and the high molecular weight 20 S translation products were related to beta-conglycinin was obtained by immunoprecipitation using monospecific antibodies against glycinin and beta-conglycinin, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immunoprecipitated products revealed that the glycinin precursor region contained at least three different size components and that the family of glycinin precursors had larger apparent molecular weight (58,000-63,000) than the disulfide-linked complexes between acidic and basic glycinin subunits (57,000). Unlike the disulfide-linked glycinin complexes which were cleaved by disulfide reduction, glycinin precursors were insensitive to reducing agents. The alpha and alpha' subunits synthesized in vitro also had slightly larger apparent molecular weights than purified alpha and alpha' standards.     

Proteome rebalancing in soybean seeds can be exploited to enhance foreign protein accumulation

Seeds possess a high intrinsic capacity for protein production that makes them a desirable bioreactor platform for the manufacture of transgenic products. One strategy to enhance foreign protein production involves exchanging the capacity to produce intrinsic proteins for the capacity to produce a high level of foreign proteins. Suppression of the alpha/alpha' subunit of beta-conglycinin storage protein synthesis in soybean has been shown previously to result in an increase in the accumulation of the glycinin storage protein, some of which is sequestered as proglycinin into de novo endoplasmic reticulum (ER)-derived protein bodies. The exchange of glycinin for conglycinin is quantitative, with the remodelled soybeans possessing a normal protein content with an altered proteome. The green fluorescent protein (GFP)-kdel reporter was transferred in a construct using the glycinin promoter and terminator to mimic glycinin gene expression. When expressed in soybean seeds, GFP-kdel accreted to form ER-derived protein bodies. The introgression of GFP-kdel into the alpha/alpha' subunit of the beta-conglycinin suppression background resulted in a fourfold enhancement of GFP-kdel accumulation to > 7% (w/w) of the total protein in soybean seeds. The resulting seeds accumulated a single population of ER membrane-bound protein bodies that contained both GFP-kdel and glycinin. Thus, the collateral proteome rebalancing that occurs with the suppression of intrinsic proteins in soybean can be exploited to produce an enhanced level of foreign proteins.     

Accumulation of high levels of free amino acids in soybean seeds through integration of mutations conferring seed protein deficiency

Soybean ( Glycine max [L.] Merr.) seeds are rich in protein, most of which is contributed by the major storage proteins glycinin (11S globulin) and beta-conglycinin (7S globulin). Null mutations for each of the subunits of these storage proteins were integrated by crossbreeding to yield a soybean line that lacks both glycinin and beta-conglycinin components. In spite of the absence of these two major storage proteins, the mutant line grew and reproduced normally, and the nitrogen content of its dry seed was similar to that for wild-type cultivars. However, protein bodies appeared underdeveloped in the cotyledons of the integrated mutant line. Furthermore, whereas free amino acids contribute only 0.3-0.8% of the seed nitrogen content of wild-type varieties, they constituted 4.5-8.2% of the seed nitrogen content in the integrated mutant line, with arginine (Arg) being especially enriched in the mutant seeds. Seeds of the integrated mutant line thus appeared to compensate for the reduced nitrogen content in the form of glycinin and beta-conglycinin by accumulating free amino acids as well as by increasing the expression of certain other seed proteins. These results indicate that soybean seeds are able to store nitrogen mostly in the form of either proteins or free amino acids.     

Cosuppression of the alpha subunits of beta-conglycinin in transgenic soybean seeds induces the formation of endoplasmic reticulum-derived protein bodies

The expression of the alpha and alpha' subunits of beta-conglycinin was suppressed by sequence-mediated gene silencing in transgenic soybean seed. The resulting seeds had similar total oil and protein content and ratio compared with the parent line. The decrease in beta-conglycinin protein was apparently compensated by an increased accumulation of glycinin. In addition, proglycinin, the precursor of glycinin, was detected as a prominent polypeptide band in the protein profile of the transgenic seed extract. Electron microscopic analysis and immunocytochemistry of maturing transgenic soybean seeds indicated that the process of storage protein accumulation was altered in the transgenic line. In normal soybeans, the storage proteins are deposited in pre-existing vacuoles by Golgi-derived vesicles. In contrast, in transgenic seed with reduced beta-conglycinin levels, endoplasmic reticulum (ER)-derived vesicles were observed that resembled precursor accumulating-vesicles of pumpkin seeds and the protein bodies accumulated by cereal seeds. Their ER-derived membrane of the novel vesicles did not contain the protein storage vacuole tonoplast-specific protein alpha-TIP, and the sequestered polypeptides did not contain complex glycans, indicating a preGolgi and nonvacuolar nature. Glycinin was identified as a major component of these novel protein bodies and its diversion from normal storage protein trafficking appears to be related to the proglycinin buildup in the transgenic seed. The stable accumulation of proteins in a protein body compartment instead of vacuolar accumulation of proteins may provide an alternative intracellular site to sequester proteins when soybeans are used as protein factories.     

Disappearance of immunoreactive glycinin and beta-conglycinin in the digestive tract of piglets

Soybean allergy represents a health threat to human and animals. Glycinin and beta-conglycinin, the main storage proteins in soybean, have been identified as major food/ feed allergens. The present study was conducted to investigate the disappearance of immunoreactive glycinin and beta-conglycinin in the digestive processes of piglets. Twelve crossbred piglets, weaned at 21 days of age, were allocated to three dietary treatments in a complete block design, each treatment with four replicates (female/male = 1:1). From day 22-28, the control group was fed diets without leguminous products, while the two treatment groups received diets containing 2.2% purified glycinin or beta-conglycinin. All piglets were slaughtered at 29 days of age and digesta was sampled from stomach, middle jejunum, caecum and colon. Results indicated that immunoreactive glycinin and beta-conglycinin decreased as the digesta descended down the digestive tract to 0.12% and 0.47%, respectively. Little immunoreactive glycinin was found in the digesta of caecum and colon, while immunoreactive beta-conglycinin was detected in the colon. Along the whole digestive tract the disappearance of immunoreactive glycinin was significantly higher than beta-conglycinin (p < 0.05).     

Improved isolation method and some properties of soybean gamma-conglycinin

Soybean gamma-conglycinin was isolated by isoelectric precipitation and ammonium sulphate fractionation. The crude protein was purified by ion-exchange chromatography on DEAE-Sepharose CL-6B and gel filtration on Sepharose CL-6B. The purified gamma-conglycinin was homogeneous on two kinds of gel electrophoresis and an ultracentrifugal analysis. A subunit band, distinguishable from other subunit bands of beta-conglycinin and glycinin, was detected by sodium dodecyl sulphate electrophoresis. Amino acid composition was similar to those of the other storage proteins of soybean. Some physical properties were also studied.     

Identification of marker proteins for the adulteration of meat products with soybean proteins by multidimensional liquid chromatography-tandem mass spectrometry

Soybean proteins are frequently added to processed meat products for economic reasons and to improve their functional properties. Monitoring of the addition of soybean protein to meat products is of high interest due to the existence of regulations forbidding or limiting the amount of soybean proteins that can be added during the processing of meat products. We have used chromatographic prefractionation on the protein level by perfusion liquid chromatography to isolate peaks of interest from extracts of soybean protein isolate (SPI) and of meat products containing SPI. After enzymatic digestion using trypsin, the collected fractions were analyzed by nanoflow liquid chromatography-tandem mass spectrometry. Several variants and subunits of the major seed proteins, glycinin and beta-conglycinin, were identified in SPI, along with two other proteins. In soybean-protein-containing meat samples, different glycinin A subunits could be identified from the peak discriminating between samples with and without soybean proteins added. Among those, glycinin G4 subunit A4 was consistently found in all samples. Consequently, this protein (subunit) can be used as a target for new analytical techniques in the course of identifying the addition of soybean protein to meat products.     

The effect of pH on heat denaturation and gel forming properties of soy proteins

This study is focussed on the influence of pH on the gel forming properties of soy protein isolate and purified glycinin in relation to denaturation and aggregation. At pH 7.6 more fine-stranded gels were formed characterised by low G' values, and a smooth, slightly turbid appearance, whereas at pH 3.8 coarse gels were obtained with a high stiffness and a granulated, white appearance. Low G' values, as found at pH 7.6, correlate with a high solubility of glycinin and soy protein isolate (ca. 50%) after heating at low protein concentration. At pH 3.8 all protein precipitated upon heating, which correlates with relatively high G' values. The role of beta-conglycinin during gelation of SPI seems to be minor at pH 7.6, which is indicated by the fact that, in contrast to pH 3.8, notable gel formation did not start upon heat denaturation of beta-conglycinin. Furthermore, the mechanism of gel formation seems to be affected by pH, because at pH 7.6, in contrast to pH 3.8, the disulphide bridge between the acidic and the basic polypeptide of glycinin is broken upon heating.     

Occurrence of mRNA for storage protein in dry soybean seeds

Poly(A)-containing RNA has been isolated from the cotyledons of soybean seeds by adsorption on a poly(U)-Sepharose column. Approximately 0.15% of the total soybean RNA applied bound to the column. The bound RNA (poly(A)-containing RNA) was shown to be mRNA by its ability to serve as template in a cell-free system derived from wheat germ. Poly(A)-containing RNA was polydisperse, migrating from approximately 50,000 to 700,000 daltons with a mean of 150,000 daltons in polyacrylamide gel electrophoresis. The size of the poly(A) portion of this RNA was in the range of 55 to 290 nucleotides. The adenylic acid content of the presumed poly(A) fragment was about 95%. The radioactive products of translation directed by the poly(A)-containing RNA in the wheat germ cell-free system were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by immunoprecipitation using antisera against beta-conglycinin and glycinin. The results of this investigation show that mRNAs for the subunit proteins of the major components of a soybean storage protein exist in the poly(A)-containing RNA preparation obtained from the cotyledons of dry soybean seeds.     

Effects of mixing during acid addition on fractionally precipitated protein

Isoelectric fractionation of the two major proteins of soy is characterized. Fractions are acid precipitated and cen-trifugally collected at pH 6.0 (glycinin) and pH 4.8 (beta-conglycinin). Two extremes in the speed of acid addition (rapid, with no mixing, and slow, via acid dialysis, with complete mixing) are compared to determine their effects on the properties of the precipitate. Total protein yield, fraction composition, and aggregate microstructure do not depend significantly on the method of acid addition. Particle size distribution and hindered settling behavior do differ and are explained using a model of aggregate strength. The rapid acid addition produces larger primary particles, because of higher supersaturation, and yields larger aggregates, because of higher interparticle potential and stronger aggregates. Further aggregation in low-shear hindered settling is faster for the slowly precipitated aggregate because few of these bonding sites could survive the high-shear precipitator, whereas more can contribute to aggregation during hindered settling.     

Characterization of the antigenic specificity of soybean protein beta-conglycinin and its effects on growth and immune function in rats

The objective of the present study was to characterize the antigenic specificity of purified soybean beta-conglycinin and to investigate its effects on the growth and immune responses of rats. Thirty-two Brown Norway rats, 3 weeks of age, were randomly allotted to one of four treatments and individually fed casein-cornstarch based diets. Rats were sensitised by means of intragastric gavage with purified beta-conglycinin (0, 5, 10 or 20 mg protein/ml in phosphate buffered saline at pH 7.4) on day 0, 7, 14, and 21 (1 ml/animal). On day 28, rats received a double dose of beta-conglycinin. Blood was obtained at weekly intervals after initiation of challenge. Growth declined linearly with increasing the concentration of soybean beta-conglycinin (p < 0.05). Both the total IgE and beta-conglycinin-specific IgE levels in serum increased while passive cutaneous anaphylactic reactions were induced in the rats. Lymphocyte proliferation response to concanavalin A in plasma and spleen was increased linearly with increased levels of soybean (p < 0.01) beta-conglycinin. The percentage of CD4+ lymphocyte subset linearly increased (p < 0.001). As a result, the concentrations of cytokines in plasma and spleen, including interleukin-4 (p < 0.01), interleukin-5 (p < 0.01), and tumour necrosis factor-alpha (p < 0.01) increased linearly with increasing level of purified beta-conglycinin. Our results indicate that purified beta-conglycinin possesses intrinsic immune-stimulating capacity and can induce an allergic reaction. Therefore, dietary soybean beta-conglycinin has negative effects on growth and both cell-mediated and humoral immune function in rats.     

Molecular cloning and characterization of two soybean protein disulfide isomerases as molecular chaperones for seed storage proteins

Protein disulfide isomerase family proteins play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill. cv Jack). The cDNAs encode proteins of 525 and 551 amino acids, named GmPDIL-1 and GmPDIL-2, respectively. Recombinant versions of GmPDIL-1 and GmPDIL-2 expressed in Escherichia coli exhibited oxidative refolding activity for denatured RNaseA. Genomic sequences of both GmPDIL-1 and GmPDIL-2 were cloned and sequenced. The comparison of soybean genomic sequences with those of Arabidopsis, rice and wheat showed impressive conservation of exon-intron structure across plant species. The promoter sequences of GmPDIL-1 apparently contain a cis-acting regulatory element functionally linked to unfolded protein response. GmPDIL-1, but not GmPDIL-2, expression was induced under endoplasmic reticulum-stress conditions. GmPDIL-1 and GmPDIL-2 promoters contain some predicted regulatory motifs for seed-specific expression. Both proteins were ubiquitously expressed in soybean tissues, including cotyledon, and localized to the endoplasmic reticulum. Data from coimmunoprecipitation experiments suggested that GmPDIL-1 and GmPDIL-2 associate with proglycinin, a precursor of the seed storage protein glycinin, and the alpha'-subunit of beta-conglycinin, a seed storage protein found in cotyledon cells under conditions that disrupt the folding of glycinin or beta-conglycinin, suggesting that GmPDIL-1 and GmPDIL-2 are involved in the proper folding or quality control of such storage proteins as molecular chaperones.     

Histidine tagged protein recovery from tobacco extract by foam fractionation

Tobacco plants have the potential to be used for the production of proteins for pharmaceutical applications. This work describes a novel protein recovery strategy where the protein of interest is "tagged" with a histidine sequence, which forms a complex with cobalt ions and surfactant possessing a chelating functionality, such that the protein is recovered in the foamate of a foam fractionation step. His-gus, a histidine-tagged enzyme, was chosen as a model protein to study the feasibility of this strategy. The His-gus is recovered from spiked prefoamed tobacco extract by foam fractionation in the presence of surfactant and cobalt ions with an enrichment of 1.29 and a recovery of 21.5% in terms of an adjusted activity.     

Composition of Acid-sensitive Fraction in Soybean Proteins

An acid-sensitive fraction (ASF) was prepared from defatted soybean meals by two procedures. ASF1 was prepared by precipitation at pH 4.5 followed by removal of 1 m NaCl-soluble materials from the precipitate. ASF2 was prepared by precipitation in solution containing 1 m NaCl at pH 4.5. The protein components of the two fractions were analyzed by gel electrophoresis in a dissociating-buffer system and found to contain β-conglycinin, glycinin and whey proteins. In addition to these, several other bands appeared.

Appreciable amounts of lipid (8.2% in ASF1 and 8.8% in ASF2) were also found in the fractions. They were separated by column chromatography and thin-layer chromatography. Glycolipids were the major components of the lipids. Both glycolipid and phospholipid fractions contained slower-moving materials on thin-layer chromatography.     

Effect of temperature on the separation of soybean 11 S and 7 S protein fractions during bipolar membrane electroacidification

The purpose of this study was to evaluate the effect of temperature (10 and 27 degrees C) on the efficiency of bipolar membrane electroacidification (BMEA) to fractionate soybean proteins. BMEA is a technology derived from electrodialysis, based on the isoelectric precipitation of proteins. It appears that temperature has a significant effect on the selective precipitation of the soybean protein fractions, mainly 11 S and 7 S, during BMEA. At 27 degrees C, the precipitation profile of the four protein fractions is situated in a pH range from 6.6 to 4.4, with no possibility of separating any of theses fractions. However, at 10 degrees C, the 11 S globulin precipitates at a higher pH than at 27 degrees C, pH 6.7 vs 5.9, allowing the fractionation of 11 S from the other fractions. Using electroacidification it is possible to obtain a precipitate solution enriched in the 11 S fraction (71.8% of 11 S and 10.8% of 7 S) and a supernatant solution enriched in the 7 S fraction (46.6% of 7 S and 4.6% of 11S).     

Introduction of DPR, an enterostatin fragment peptide, into soybean beta-conglycinin alpha' subunit by site-directed mutagenesis

DPR, a fragment peptide of enterostatin (VPDPR) having hypocholesterolemic activity, was introduced into the three homologous sites, EPR, DYR, and DPI, in the soybean beta-conglycinin alpha' subunit by site-directed mutagenesis. The modified beta-conglycinin was expressed in Escherichia coli and recovered in the soluble fraction. After purification on ion-exchange HPLC, the modified beta-conglycinin was digested by trypsin to release integrated DPR. The yield of DPR from 1 mole of the modified beta-conglycinin was 1.2 mole.     

A novel plant protein disulfide isomerase family homologous to animal P5 - molecular cloning and characterization as a functional protein for folding of soybean seed-storage proteins

The protein disulfide isomerase is known to play important roles in the folding of nascent polypeptides and in the formation of disulfide bonds in the endoplasmic reticulum (ER). In this study, we cloned a gene of a novel protein disulfide isomerase family from soybean leaf (Glycine max L. Merrill. cv Jack) mRNA. The cDNA encodes a protein called GmPDIM. It is composed of 438 amino acids, and its sequence and domain structure are similar to that of animal P5. Recombinant GmPDIM expressed in Escherichia coli displayed an oxidative refolding activity on denatured RNase A. The genomic sequence of GmPDIM was also cloned and sequenced. Comparison of the soybean sequence with sequences from Arabidopsis thaliana and Oryza sativa showed significant conservation of the exon/intron structure. Consensus sequences within the promoters of the GmPDIM genes contained a cis-acting regulatory element for the unfolded protein response, and other regulatory motifs required for seed-specific expression. We observed that expression of GmPDIM was upregulated under ER-stress conditions, and was expressed ubiquitously in soybean tissues such as the cotyledon. It localized to the lumen of the ER. Data from co-immunoprecipitation experiments suggested that GmPDIM associated non-covalently with proglycinin, a precursor of the seed-storage protein glycinin. In addition, GmPDIM associated with the alpha' subunit of beta-conglycinin, a seed-storage protein in the presence of tunicamycin. These results suggest that GmPDIM may play a role in the folding of storage proteins and functions not only as a thiol-oxidoredactase, but also as molecular chaperone.