Soybean 11S Research Articles

Mild method of simultaneous methionine grafting and phosphorylation of soybean globulins improves their functional properties

Soybean 7S and 11S globulins were phosphorylated to different extent as a function of the molar ratios of POCl 3 used (75, 150 and 300 mol POCl 3 /mol protein) in the presence of equivalent amounts of triethylamine. Simultaneous addition of L-methionine during the phosphorylation yielded methionylated globulins. In some experiments, the basic amino acid arginine was used as the sole base of the reaction, instead of triethylamine, at a ratio of 6 mol arginine/mol POCl 3 . Adding L-methionine in the course of the phosphorylation reaction reduced slightly the extent of protein phosphorylation, with increasing L-methionine content in the phosphorylated globulin up to about 280% of its original level. The use of L-arginine as the reaction base led to lower phosphorylation yields but could raise simultaneously both L-methionine and L-arginine to about 143% and 155% of their original levels, respectively. Isofocusing electrophoresis showed lower isoelectric points of the phosphorylated samples, compared to the starting aliquots. SDS-PAGE did not show major changes in the subunit distribution of the modified soybean globulins. Phosphorylation and amino acylation changed the solubility of the modified samples and also other related functional properties e.g. foaming and emulsifying which were remarkably improved, especially for the samples with grafted L-methionine.

Properties of Films Produced by Cross‐linking Whey Proteins and 11S Globulin Using Transglutaminase

ABSTRACTTransglutaminase (TG) was used to produce films from whey protein isolate, soybean 11S globulin and a mixture of the two (1:1, wt/wt). Solubility of TG cross‐linked films was lower than that of control films at pH 3, 4, 6 and 8. Solubility of control films in 6.6M urea and in 10% SDS was higher than that of TG cross‐linked films. Hydrolysis of control and TG cross‐linked films by trypsin and α‐chymotrypsin was similar after 24h incubation. Mean thickness of films ranged from 69 to 77 μm and there were no differences among thicknesses. Average tensile strength values of TG cross‐linked films were two times greater than those of the homologous controls.

Biopolymers Produced by Cross‐linking Soybean 11S Globulin with Whey Proteins using Transglutaminase

ABSTRACTHeterogeneity of biopolymers was determined by cross‐linking acetylated‐11S acidic subunits (Ac‐11S) of soy protein with α‐lactalbumin and β‐lactoglobulin. The extent of polymerization was determined by electrophoresis and HPLC. Differential scanning calorimetry (DSC) was used to determine thermal properties of starting proteins and biopolymers. HPLC data demonstrated the absence of biopolymers from Ac‐11S, acetylated α‐lactalbumin and acetylated β‐lactoglobulin when each was incubated separately with transglutaminase (TG). However, Ac‐11S formed biopolymers with α‐lactalbumin and β‐lactoglobulin when TG was added. TG catalyzed the formation of heterologous and homologous biopolymers from whey protein isolate (WPI) and soybean 11S globulin (11S). Cross‐linking WPI and 11S provided biopolymers with improved heat stability which may be useful to provide functionality to food products.

Analysis of Aggregate Structure in Food Protein Gels with the Concept of Fractal.

The fractal structure of the aggregates in food protein gels was analyzed. Three kinds of food protein gels were prepared: (1) β-lactoglobulin (β-LG) gel; (2) 11S soybean globulin gel; and (3) caseinate gel. From the concentration dependence of the gel elasticity, the fractal dimensions Df of the aggregates in the gels were evaluated, according to the theory of Shih et al. These gels showed the weak-link behavior described in the theory of Shih et al The values obtained for Df were 2.6-2.7, which were larger than those predicted by the cluster-cluster aggregation model for a dilute system. In addition, for the β-LG gels, the fractal dimension was also evaluated from the analysis of the gel image obtained with a confocal scanning laser microscopy, the value being close to that evaluated from the concentration dependence of the gel elasticity. These results indicate that the elastic behavior of the aggregate gels is a reflection of fractal structure of the aggregates in the gels.

Properties of Biopolymers from Cross‐linking Whey Protein Isolate and Soybean 11S Globulin

ABSTRACTBiopolymers were prepared by cross‐linking whey protein isolate (WPI) and soybean 11S using transglutaminase. Electrophoretic pattern, solubility, emulsification, hydrophobicity and foaming properties of the biopolymers were determined. SDS‐PAGE showed bands corresponding to high‐molecular‐weight components (MW>200 kDa). Biopolymer solubility was > 90% at pH 3 and below, and at pH 7 and above. Emulsifying properties of biopolymers were lower than those of WPI. The foaming capacity of the biopolymers (23.6 mL) and WPI/11S mixture (22.7 mL) were similar to that of egg albumin (20.3 mL). The foaming stability of the biopolymers (122 min) was higher than that of WPI/11S mixture (33.7 min), and was similar to that of egg albumin (132 min).

RHEOLOGICAL PROPERTIES OF HEATED CORN STARCH + SOYBEAN 7S AND 11S GLOBULIN DISPERSIONS

ABSTRACTAt 10% solids content, magnitudes of G′ and G″ of soybean 7S dispersion were substantially lower than those of corn starch dispersion, while those of soybean 11S dispersion were much higher than those of starch. Profiles of G′ at 30 Hz as a function of starch concentration of starch + 7S and starch + 11S gels were asymmetrical reflecting phase separation due to thermodynamic incompatibility. However, the starch concentration corresponding to the critical point below which no phase separation occurs for the starch + 7S dispersion was about 7% and for the starch + 11S dispersion it was about 3%. At equal starch fractions, values of G′ at 30 rad s‐1 of corn starch + 7S dispersions were nearly equal to those of only corn starch. In contrast, at starch concentrations between 6–8%, G′ values of the corn starch + 11S dispersions were much higher than those of only starch and a minimum value occurred at a starch concentration of about 5%. Based on frequency dependence of G′ values, heated corn starch + 7S dispersions may be classified as weak gels, while heated corn starch + 11S dispersions may be classified as true gels.

Physical Properties of Soy Bean and Broad Bean 11S Globulin Gels Formed by Transglutaminase Reaction

ABSTRACTPhysical properties of transglutaminase‐induced glycinin and legumin gels were compared with thermally induced gels. Results of deformation tests showed that transglutaminase‐induced gels were more rigid and elastic than thermally induced gels. From creep compliance tests, all elastic moduli and viscosities except Newtonian viscosity were higher for transglutaminase‐induced gels. Electron micrographs revealed that network structures of transglutaminase‐induced gels were composed of larger unit particles forming more developed strands and clusters. More rigid and elastic gels were formed from glycinin as compared to legumin by both gelling methods.

Diamine Oxidase and Transglutaminase Activities in White Lupine Seedlings with Respect to Crosslinking of Proteins

Enzymes extracted from 10-d-old Lupinus albus seedlings were able to (1) polymerize casein and (2) incorporate [ 14 C]putrescine into dimethylcasein (modified casein). High molecular weight polymers formed were visualized by SDS-polyacrylamide gel electrophoresis. [ 14 C]Putrescine incorporation was not only due to transglutaminase activity but also due to diamine oxidase. The use of diamine oxidase inhibitor allowed us to localize a transglutaminase activity in the pellet after centrifugation at 41400g of the filtered homogenate of seedlings and a diamine oxidase activity mainly in the supernatant. Covalent conjugation of monodansylcadaverine and [ 14 C]putrescine to dimethylcasein by enzymes contained in the 41400g pellet was vizualized by fluorescent detection or by autoradiography in SDS-polyacrylamide gel electrophoresis. The enzyme contained in the pellet fraction was able to polymerize not only casein but also spinach ribulose-1,5-bisphosphate carboxylase/oxygenase and 7S soybean globulins. As a control we used purified diamine oxidase from porcine kidney to check the inability of this enzyme to catalyze casein polymerization. Moreover, 54% of transglutaminase activity contained in the pellet has been solubilized by 5% (v/v) detergent (Triton X-100) contrary to high concentrated salts. Transglutaminase was recovered in the 108000g supernatant. This suggests that this enzyme was an integral membrane protein.

Effects of Protein Composition on Gelation of Mixtures Containing Soybean 7S and 11S Globulins

Soybean 7S and 11S globulins, which are two major components of soybean protein, were mixed in various ratios. Gelation of the protein blend after the addition of glucono-δ-lactone (GDL) or calcium sulfate was studied to analyze the gelation process of tofu (soybean curd). Dynamic viscoelasticity measurements at a constant temperature as a function of time were done. Faster gelation was observed in mixtures containing 11S in a higher proportion for both the GDL and calcium systems. The value of the storage modulus of the 5:5 blend system of 7S and 11S globulins was minimum when the GDL concentration was adjusted to 17 mM, at which level the gels consisting of 7S globulin alone and 11S globulin alone had a similar storage modulus. Under the same conditions, although the gel of the soybean protein isolate showed almost the same value for the storage modulus as a mixture of 7S: 11S in the same ratio, the gelation was slower than that expected from the 7S and 11S blend. Since the use of calcium sulfate led to...

Rheological study on gelation of soybean 11S protein by glucono-.delta.-lactone. [Erratum to document cited in CA116:234084

Rheological study on gelation of soybean 11S protein by glucono-.delta.-lactone. [Erratum to document cited in CA116:234084

Genotypic and developmental regulation of transient expression of a reporter gene in soybean zygotic cotyledons

Processes involved in transformation of regenerable soybean (Glycine max (L.) Merrill) immature zygotic cotyledons were studied by assaying the transient expression of the β-glucuronidase gene driven by the 35S promoter and terminated at the 3' end by the soybean 7S storage protein gene. The plasmid containing the chimeric gene was delivered to the cotyledons via particle bombardment 900 PSI. Zygotic cotyledons from six soybean varieties were tested for transient expression of the β-glucuronidase gene. The level of reporter gene expression differed between genotypes. The genotypes could be classified as high, fair or poor expressors. Cotyledons from different genotypes were then bombarded at 650, 900 or 1100 PSI. GUS expression varied among genotypes independently of the pressure of bombardment. Finally, the ability of cotyledons to express the reporter gene depended on the developmental stage of the seed from which it was excised with the younger stage being the least responsive. However, genotypic specific expression remained after controlling for developmental stage of the cotyledons.

Isolation of Soybean 11S Globulin by Isoelectric Precipitation and Sephacryl S-300 Gel Filtration Chromatography: A New Purification Technique

This study outlines a simple alternative technique for the purification of the 11S soybean globulin from crude globulin using a single Sephacryl S-300 gel filtration chromatographic technique. All supporting data demonstrated that the 11S globulin was purified to homogeneity with no contamination from the 7S soybean globulin.

Tissue-specific and temporal regulation of a ?-conglycinin gene: roles of the RY repeat and other cis-acting elements

Upstream regulatory sequences (URS) of the gene that encodes the alpha' subunit of beta-conglycinin, the 7S soybean seed storage protein, includes two RY repeat elements. The role of RY elements and sequences that bind soybean embryo factors 3 and 4 (SEF3 and SEF4; Allen et al., Plant Cell 1 (1989) 623-631) in regulating expression of the promoter was studied following site directed mutagenesis. Specific mutations introduced into these sequences abolished the in vitro binding activities of SEF3 and SEF4. The biological activities resulting from the mutations were determined in transgenic plants using two chimeric promoters comprising sequences from the CaMV 35S promoter and the alpha' subunit promoter. The uidA reporter gene was used to assess the levels of gene expression in transgenic plants. The mutations in the RY element and SEF3 and SEF4 binding sites had little effect on expression of the alpha' promoter. By contrast, mutations in the RY element had significant effect on gene expression when the URS from the alpha' promoter was ligated upstream of the core 35S promoter. Mutations in the RY element abolished the seed specific enhancing activity of the alpha' URS and caused expression of the chimeric promoter in leaves. These results indicate that the RY element plays a key role in seed-specific gene regulation in coordination with other cis-acting elements.

Thermal densturation of glycinin as a function of hydration

AbstractHydration effects on the thermal stability of glycinin (soybean 11S protein) were examined by differential scanning calorimetry (DSC). In a model system with pure glycinin, the denaturation temperature (Td) decreased with increasing moisture. Between 22 and 44% moisture, two endotherms were observed, where the lower‐temperature endotherm became progressively reduced in magnitude with a concomitant increase in a higher‐temperature transition. At 45.5% moisture, only a single endotherm was observed. The regression curves over the entire moisture range from 2 to 66% were derived as asymptote functions, where M equals the percentage total moisture. Equations were developed from the curves, and the relationship between Td and moisture were: Td=92.4+196.5e−0.068M and, for the low‐temperature endotherm, 82.4+144.3e−0.068M. By interaction of 11S protein with either ethanol, a neutral detergent (Triton X‐100) or 40% sucrose, both one‐ and two‐endotherm curves were generated. Such calorimetric behavior is indicative of nonequilibrium denaturation and supports the notion that structure reorganization during DSC is water content‐dependent. Our findings suggest that either glycinin’s acidic/basic subunits or a change in secondary protein structure may give rise to two endotherms.

Rheological studies on the gelation process of soybean 7 S and 11 S proteins in the presence of glucono-.delta.-lactone

Dynamic viscoelastic study and compression testing on gelation of soybean 7S and 11S proteins by glucono-6-lactone (GDL) were done to elucidate the gelation process of tofu (soybean curd). Gelation curves at constant temperatures observed in dynamic viscoelastic measurement were approximated by first-order reaction kinetics. Both storage and loss moduli at the final stage of the gelation for 7S-GDL gels showed almost the same value as those of 11S gels when concentrations of protein and GDL were the same

The effect of glucono-δ-lactone on the gelation time of soybean 11S protein: concentration dependence

Gelation kinetics of soybean 11S protein by glucono-δ-lactone (GDL) was investigated by dynamic viscoelastic measurements. The gelation time decreased with increasing GDL concentration at a constant 11S concentration (4%). The mechanical loss tangent at the final stage of the gelation decreased with increasing protein concentration, but was independent of the GDL concentration. The gelation time of systems which consist of 11S and GDL at a fixed ratio decreased with increasing 11S concentration. However, the time at which the gelation curve reached saturation did not decrease so much with increasing GDL concentration at higher concentrations of the protein. It was found that the decrease of pH by addition of GDL greatly contributes to the gelation time. The relationship between the gelation time and 11S concentration at low concentrations suggested that the minimum 11S concentration for gelation is 1.03% and the junction zones of the systems are formed by 2.6 single polymer chains.

Rheological study on gelation of soybean 11S protein by glucono-.delta.-lactone

Dynamic viscoelasticity studies on gelation of soybean 11S protein by glucono-δ-lactone have been done to analyze the gelation process of tofu. Observed gelation curves at constant temperatures were well approximated by first-order reaction kinetics. The saturated storage modulus depended mainly on the concentration of 11S protein. The saturated modulus was proportional to 3.4 th power of 11S concentrations. The rate constant of the gelation increased with increasing gelling temperature and was mainly governed by the concentration of glucono-δ-lactone

Gelation Properties of Soymilk and Soybean 11S Globulin from Japanese-grown Soybeans.

A new method to evaluate soybeans for making tofu (soybean curd) is proposed. Dynamic viscoelasticity measurements were carried out to examine the gelation process of soymilk, storage and loss moduli being observed as a function of time after adding glucono-δ-lactone. The gelation curves fitted well with first-order reaction kinetics. The saturated value of the storage modulus correlated well with gel hardness by a curdmeter. The saturated value of the storage modulus was mainly dependent on the concentration of 11S globulin, while the gelation rate was an increasing function of the concentration of glucono-δ-lactone at a fixed 11S globulin concentration.

Dynamic Viscoelasticity of Soybean 11S Globulin Under Shear Force at Different Temperatures.

Dynamic Viscoelasticity of Soybean 11S Globulin Under Shear Force at Different Temperatures.

Electroporated protoplasts express seed specific gene promoters

The soybean 7S seed storage protein, β-conglycinin, is comprised of three major subunits, α', α, and β. Chimeric genes having β-conglycinin α' and β subunit promoters and the β-glucuronidase gene coding sequence were constructed and electroporated into protoplasts prepared from three cultured cell lines and from tobacco mesophyll cells. The β-conglycinin promoters were active in all protoplasts examined, and their activities were 10-60% of that of the cauliflower mosaic virus 35S promoter. In electroporated protoplasts isolated from tobacco suspension cultures the time course of expression and the pattern of cell cycle dependency of the β-conglycinin promoters were similar to those of the 35S promoter. The responses to exogenously added L-methionine and abscisic acid, which are known to have differential effects on the expression of β-conglycinin promoters in cultured soybean cotyledons, were essentially the same among the promoters used. The results indicate that β-conglycinin promoters are expressed in electroporated protoplasts, but their regulation is relaxed.