Soybean Glycinin Research Articles

Molecular design of soybean glycinins with enhanced food qualities and development of crops producing such glycinins.

Soybean (Glycine max L.) protein has a function to lower cholesterol level in human serum (Kito et al., 1993) and is one of the best plant food proteins in terms of nutritional and organoleptic qualities. However, it is usually inferior in these respects to animal proteins. For example, the amino acid composition of soybean protein does not satisfy infant requirement: sulfur containing amino acids are deficient. Improvement of nutritional value and functional properties of soybean proteins is one of major objectives in the food industry (Kinsella, 1979). Soybean proteins consist of two major components, glycinin and β-conglycinin (Derbyshire et al., 1976). Of these two proteins, glycinin is superior to β-conglycinin with regard to nutritional value (Millerd, 1975) as well as functional properties (Kinsella, 1979). Five subunits are identified as constituent subunits of glycinin and classified into two groups: group I (A1aB1b, A1bB2, A2B1a) and group II (A3B4, A5A4B3) (Utsumi, 1992). The subunits belonging to group I have better nutritional value than those of group II. Therefore, a group I subunit of glycinin is a suitable target for such improvement to create an ideal food protein. Protein engineering appears to be a promising method in achieving improvement of glycinin qualities because the primary sequence of glycinin can be modified consciously and systematically.

Effect of pH and temperature on the immunogenicity of glycinin (Glycine max L.)

Sensitive immunologic techniques for the detection of alterations that occur in protein antigens were used to evaluate the immunogenicity of soybean glycinin after isolation, heat denaturation and pH alteration. The objective was to determine the effect of these agents on the immunogenic ability of this protein fraction. Immunologic assays performed on heat-denatured glycinin up to 80 degrees C in the presence of antinative glycinin serum demonstrated that glycinin retains its immunogenic properties. Above 90 degrees C this biological property begins to disappear, with protein insolubilization and epitope modification due to the conformational changes imposed by temperature. A reduction in immunogenicity also occurred when glycinin was taken to pH 2.0 (below its pl) and pH 11.00 (above its pl) and exposed to high temperatures in the presence of native antiglycinin serum. From these data one can conclude that, at extreme pH values, intramolecular reactions may occur which, in combination with the structural disorganization caused by high temperatures, may contribute to the reduction of immunogenicity.

Limited proteolysis of beta-conglycinin and glycinin, the 7S and 11S storage globulins from soybean [Glycine max (L.) Merr.]. Structural and evolutionary implications.

The G2 (A2B1a) glycinin subunit from soybean (Glycine max L. Merr.) was purified and renatured to the homohexameric holoprotein. This protein along with purified beta-conglycinin were subjected to limited proteolysis by trypsin. The generated polypeptide fragments were separated via SDS/PAGE and the amino acid sequence of the N-terminals was determined. Four cleavage points were detected in the alpha-chain A2 of glycinin as well as in the alpha'-chain of beta-conglycinin. From the known three-dimensional structure of 7S globulin and the hypothetical model of 7S globulin-like 11S globulin structure, it was possible to draw the conclusion that two distinct types of susceptible sites for proteolytic cleavage are characteristic of the subunits of both globulins. The first includes the sequences linking N- and C-terminal domains of both globulins and the sequence of N-terminal extensions of 70-kDa subunits from the vicilin-like 7S globulins. The second type includes the loop between beta-strands E and F of the N-terminal domain of 11S globulins and of the C-terminal domain of 7S globulins. A statistically significant similarity was found between the N-terminal extension of the alpha'-chain of beta-conglycinin and the interdomain linker regions of soybean glycinin and pea legumin. It is proposed that the three sequence regions which form the first type of susceptible sites are of similar structural function and might have evolved from the N-terminal segment of a putative single-domain ancestor.

High level accumulation of soybean glycinin in vacuole-derived protein bodies in the endosperm tissue of transgenic tobacco seed

Soybean glycinin genes are expressed specifically in the cotyledon and embryo of maturing soybean seed, from which the endosperm tissue is degenerated. To examine whether glycinin could be stably accumulated in endosperm tissue, the glycinin cDNA was transcriptionally fused to an endosperm-specific promoter of the rice storage protein glutelin gene and then introduced into tobacco genome via Agrobacterium-mediated transformation. Consequently the glycinin gene was expressed in a seed- and developmentally-specific manner in transgenic tobacco seeds. Glycinins were targeted to vacuole-derived protein bodies in the endosperm tissue and highly accumulated in the matrix region of many transgenic plants (1–4% of total seed proteins). Synthesized glycinin was processed into mature form, and assembled into a hexamer in a similar manner as the glycinin in soybean seed. However, it was noteworthy that about half of the synthesized glycinin was susceptible to limited degradation and that assembly into a hexamer was insufficient. Modified glycinins, in which 4 contiguous methionine residues were inserted at the variable regions corresponding to the C-terminal regions of the acidic and basic polypeptides, were also found to be accumulated similarly as in the normal glycinin. There was no apparent difference in the expression level, processing and targeting to protein bodies, or accumulation level between normal and modified glycinins.

Positive and negative cis-regulatory regions in the soybean glycinin promoter identified by quantitative transient gene expression

The 5' and 3' flanking regions of the soybean glycinin gene, Gy1, responsible for expression in seeds, were analyzed by quantitative transient expression assay. The construct containing the β-glucuronidase (uidA) reporter gene under the control of the 1.12 kb Gy1 promoter and 0.74 kb Gy1 terminator was introduced into immature soybean seeds and leaves by particle bombardment. To normalize the variability of introduction efficiency, a second reporter gene, firefly luciferase, was cobombarded as an internal standard, and relative activities (GUS/luciferase) were measured. There was a seed-specific β-glucuronidase (GUS) expression, as observed by X-Gluc staining. Compared with the nopaline synthase gene (nos) terminator, the Gy1 terminator enhanced the level of expression in immature seeds, indicating that the terminator region of the glycinin gene is involved in the activation of the gene expression in these seeds. To identify cis-regulatory elements in the glycinin gene upstream sequence, deleted derivatives of the promoter were fused to the luciferase reporter gene. The expression could be measured with a higher accuracy, and constructs were introduced with the internal reporter uidA gene into immature seeds. The results suggest the presence of a positive regulatory element in the -620 to --380 region of the Gy1 promoter. A deletion which eliminates the legumin box with its RY element led to increased relative activity, suggesting that this box is negatively regulating expression of the seed storage protein gene. Analysis of mutant promoters also suggest that the RY element involves negative regulation in seeds. This quantitative transient expression assay using particle bombardment provides a reliable system for the study of seed-specific gene expression in soybeans.

Expression and accumulation of normal and modified soybean glycinins in potato tubers

Chimeric genes composed of the class I patatin promoter, A 1aB 1b preproglycinin cDNA and its modified versions (IV+4Met and V+4Met) containing four contiguous methionine codons and nopaline synthase gene terminator were inserted into the genome of potato by Agrobacterium-mediated transformation. Transcripts of the chimeric genes were detected specifically in tubers. Translational products were observed in parenchyma cells of the tubers of potato plants transformed with the normal and modified glycinin cDNAs. This distribution is consistent with the expression specificity of the patatin promoter. The expression levels of the normal and the modified glycinins were 0.2–1.0% of the total soluble tuber proteins. Both the normal and the modified glycinins expressed in the tubers were present as proglycinin trimers. These results indicate that the modifications in IV+4Met and V+4Met did not disrupt any stage in the biosynthetic process of glycinin in potato tubers.

Stimulation of Insulin-dependent Glucose Transport and Lipogenesis in Isolated Rat Adipocytes by Trypsinized Soybean Glycinin Acidic Subunit A1a

Besides the stimulating effect of trypsinized soybean glycinin acidic subunit A1a on insulin-mediated antilipolysis, the trypsinized A1a was found to enhance insulin-mediated glucose transport and lipogenesis in the presence of cAMP stimulators such as isoproterenol and 1-methyl-3-isobutylxanthine in isolated rat adipocytes. Furthermore, it was indicated that insulin and the cAMP stimulators, which generally exert counter-regulatory effects, act synerigistically on the in vitro lipogenesis.

Gelation and gel properties of soybean glycinin in a transglutaminase-catalyzed system

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTGelation and gel properties of soybean glycinin in a transglutaminase-catalyzed systemIl Jun Kang, Yasuki Matsumura, Koji Ikura, Masao Motoki, Hiroko Sakamoto, and Tomohiko MoriCite this: J. Agric. Food Chem. 1994, 42, 1, 159–165Publication Date (Print):January 1, 1994Publication History Published online1 May 2002Published inissue 1 January 1994https://doi.org/10.1021/jf00037a028RIGHTS & PERMISSIONSArticle Views268Altmetric-Citations46LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Digestion of soybean globulins, glycinin, α-conglycinin and β-conglycinin in the preruminant and the ruminant calf

Two experiments involving either preruminant (exp. 1) or ruminant (exp. 2) fistulated calves were conducted to study the in vivo digestion of glycinin, α-conglycinin and β-conglycinin from soybean. It was incorporated as a flour (product A, antigenic in vitro) or a protein concentrate (product B, non antigenic in vitro) in milk replacers (exp. 1), or as a meal (product C, antigenic in vitro) in a weaning starter (exp. 2). ELISA detection of residual globulin immunoreactivity was determined on ileal digesta in exp. 1, and on duodenal, ileal and faecal digesta in exp. 2. Ileal flow of glycinin, α-conglycinin and β-conglycinin represented 10.3, 1.2 and 0.9% of corresponding globulin amounts ingested, in the case of product A (exp. 1). Immunoreactive α-conglycinin could only be detected in ileal digesta (1.3% of intake) of calves fed the diet containing product B. In exp. 2, immunoreactive globulins entered the duodenum in low amounts (below 1% of respective intake), especially after weaning. Accordingly, their flow at the ileum and in feces, although measurable, had no meaning from the nutritional point of view. Fermentation in the rumen of weaned calves appeared to be efficient in inactivating most of the potentially harmful dietary constituents. Key words: Soybean, protein, digestion, calf, weaning, glycinin

Interaction of Nuclear Factor with the 5’ Upstream Region of Soybean Glycinin Gene

Interaction of Nuclear Factor with the 5’ Upstream Region of Soybean Glycinin Gene

Cis-acting regulatory regions of the soybean seed storage 11S globulin gene and their interactions with seed embryo factors.

A 2.2 kb fragment containing the 5'-flanking region of the soybean glycinin A2B1a gene and its successive deletions with a shorter 5'-flanking sequence were fused, in frame, to the beta-glucuronidase (GUS) reporter gene. The resultant fusions were introduced into tobacco plants via Agrobacterium tumefaciens. Assays of the GUS activity in seeds of transgenic tobacco showed that the upstream region, -657 to -327 (relative to the transcription initiation site [+1]), of the glycinin gene is required for optimal expression of the transformed gene. Interactions between embryo nuclear factors and DNA fragments covering the downstream region of -326, in which are included the TATA box and legumin boxes, were not apparent. The embryo factors capable of binding specifically to three subregions, -653 to -527, -526 to -422, and -427 to -321, of the upstream regulatory region were detected. Such factors appeared to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowering). Evidence obtained by characterizing the nature of the binding proteins and by gel mobility shift assays established that the same factor does interact with a consensus motif 5'-ATA/TATTTCN-/CTA-3' which occurs four times in the cis-acting regulatory region between -657 and -327. Moreover, this conserved motif could also be found in the 5' regulatory region of another glycinin A1aB1b gene. Thus it is likely that the observed interaction between the nuclear factor and the conserved motifs would lead to activation of transcription from the glycinin genes in maturing soybean seeds.

Enzyme-linked immunosorbent assay and immunoblotting using IgY antibodies against soybean glycinin A

Abstract Using IgY antibodies obtained from egg yold of immunized chickens, an enzyme-linked immunoassay (ELISA) and an immunoblotting procedure has been developed for specific determination of soy protein. The procedure for antigen preparation provides a rapid method for the isolation of SDS-denatured glycinin A from polyacrylamide gels that could be used directly for immunization. ELISA with SDS-denatured soy protein isolate was linear in the range 0·5–256 μg/ml and covers the expected levels of soy protein in simulated milk and milk products. Immunostaining of SDS-PAGE blots allowed the detection of glycinin A at nanogram levels. It is claimed that the antigenic status of SDS-denatured glycinin A is independent of food processing conditions and that the specific immunoreaction is controlled by high affinity IgY antibodies against the SDS-denatured protein conformation.

Promoter Sequence of Soybean Glycinin Gene Regulates Seed-Specific Expression in Transgenic Tobacco Plant

Promoter Sequence of Soybean Glycinin Gene Regulates Seed-Specific Expression in Transgenic Tobacco Plant

Determination of the binding of hexanal to soy glycinin and .beta.-conglycinin in an aqueous model system using a headspace technique

The equilibrium binding of hexanal to soybean glycinin and beta-conglycinin has been evaluated under several conditions. Binding of the flavor ligand was allowed to approach saturation. In 0.3 M Tris, pH 8.0, glycinin and beta-conglycinin had similar affinities, 270 and 303 M-1, respectively. The numbers of binding sites for hexanal for glycinin and beta-conglycinin were 108 and 26, respectively. Different buffer conditions, 0.5 M NaCl with 0.02% NaN3 or 10 mM beta-mercaptoethanol, changed the number of binding sites and affinity for hexanal for these proteins. Positive cooperativity was observed for the binding of hexanal to both proteins. ANS surface hydrophobicity and turbidity increases suggested that structural changes may occur as a result of hexanal binding.

Temperature Effect on Binding of Volatile Flavor Compounds to Soy Protein in Aqueous Model Systems

ABSTRACTThe thermodynamics of binding was determined for soybean glycinin and β‐conglycinin and flavor ligands butanal, pentanal, hexanal, octanal, 2‐ and 3‐hexanone, 2‐ and 5‐nonanone, hexanol, and hexane. Hexane had affinity for these proteins only at 5oC. Affinities of binding for all flavor ligands were greater for glycinin than β‐conglycinin. Affinity for aldehydes increased with increasing chain length for glycinin, but remained constant for β‐conglycinin. ΔGo, ΔHo, and ΔSo for binding were determined. ΔGo was negative for all ligands and ranged from −1.74 to −4.16 Kcal/mol. All enthalpies of binding were positive except butanal and hexanol. Change in free energy of binding per CH2 in homologous aldehydes was greater for glycinin than β‐conglycinin.

ダイズグリシニン遺伝子の遺伝子工学的研究の現状

ダイズグリシニン遺伝子の遺伝子工学的研究の現状

Improvement of nutritional value and functional properties of soybean glycinin by protein engineering

Glycinin is one of the predominant storage proteins of soybean. To improve its functional properties (heat-induced gelation and emulsification) and/or nutritional value, the A1aB1b proglycinin subunit was modified on the basis of genetically variable domains suggested from the comparison of amino acid sequences of glycinin-type globulins from various legumes and nonlegumes and the relationships between the structure and the functional properties of glycinin. Thus, nucleotide sequences corresponding to each of the variable domains were deleted from the cDNA encoding the A1aB1b proglycinin, and a synthetic DNA encoding four continuous methionines was inserted into the cDNA region corresponding to each of the variable domains. Expression plasmids carrying the modified cDNAs were constructed and expressed in Escherichia coli strain JM105. Some of the modified proteins were accumulated as soluble proteins in the cells at a high level and self-assembled. They exhibited functional properties superior to those of the native glycinin from soybean, which establishes the possibility of creating theoretically designed novel glycinins with high food qualities.

The complete nucleotide sequence of soybean glycinin A2B1agene spanning to another glycinin gene A1aB1b

The complete nucleotide sequence of soybean glycinin A₂B_1agene spanning to another glycinin gene A_1aB_1b

An attempt to elucidate the origin of cultivated soybean via comparison of nucleotide sequences encoding glycinin B4 polypeptide of cultivated soybean, Glycine max, and its presumed wild progenitor, Glycine soja

Nucleotide sequences of cDNAs encoding soybean glycinin B4 polypeptide were compared for three soybean cultivars and two introductions of wild soybean, G. soja. For three G. max cultivars, only two nucleotide substitutions were found, while G. max and G. soja nucleotide sequences had four substitutions. These data give added proof that G. max originated from G. soja. On the other hand, the time required for the accumulation of four nucleotide substitutions (calculated from the parameters of 11S globulin molecular evolution) appeared to be longer than the duration of the soybean domestication period.

Characterization of the glycinin gene family in soybean.

We characterized the structure, organization, and expression of genes that encode the soybean glycinins, a family of storage proteins synthesized exclusively in seeds during embryogenesis. Five genes encode the predominant glycinin subunits found in soybeans, and they have each been cloned, sequenced, and compared. The five genes have diverged into two subfamilies that are designated as Group-I and Group-II glycinin genes. Each glycinin gene contains four exons and three introns like genes that encode related proteins in other legumes. Two other genes have been identified and designated as "glycinin-related" because they hybridize weakly with the five glycinin genes. Although not yet characterized, glycinin-related genes could encode other glycinin subunit families whose members accumulate in minor amounts in seeds. The three Group-I glycinin genes are organized into two chromosomal domains, each about 45 kilobase pairs in length. The two domains have a high degree of homology, and contain at least five genes each that are expressed either in embryos or in mature plant leaves. Gel blot studies with embryo mRNA, as well as transcription studies with 32P-RNA synthesized in vitro from purified embryo nuclei, indicate that glycinin and glycinin-related genes become transcriptionally activated in a coordinated fashion early in embryogenesis, and are repressed coordinately late in seed development. In addition to transcriptional control processes, posttranscriptional events also are involved in regulating glycinin and glycinin-related mRNA levels during embryogenesis.