Characterization and crystallography of recombinant 7S globulins of Adzuki bean and structure-function relationships with 7S globulins of various crops

The recombinant proteins Adzuki 7S1, Adzuki 7S2, and Adzuki 7S3 were prepared through the Escherichia coli expression systems of three kinds of adzuki bean cDNAs. The recombinant proteins exhibited intrinsic thermal stabilities, surface hydrophobicities, and solubilities, although the homology of their amino acid sequences ranged from 95-98%. To understand why these individual proteins exhibited different properties, their three-dimensional structures were elucidated. The three proteins were successfully crystallized, and the three-dimensional structures of Adzuki 7S1 and Adzuki 7S3 were determined. The properties and structures of these two proteins were comprehensively compared with those of recombinant 7S globulins (soybean beta-conglycinins beta and alpha'c and mungbean 8Salpha) reported previously. It was likely that cavity sizes, hydrogen bonds, salt bridges, hydrophobic interactions, and lengths of loops determine the thermal stabilities of 7S globulins, and results indicated that cavity sizes strongly contribute to such stability. Surface hydrophobicity was also found to be determined not only by distributions of hydrophobic residues on the molecular surface. Furthermore, solubility at neutral and weak alkaline pH values at mu = 0.08 was found to be dominantly influenced by the electrostatic surface potentials.     

Comparison of protein chemical and physicochemical properties of rapeseed cruciferin with those of soybean glycinin

Rapeseeds contain cruciferin (11S globulin), napin (2S albumin), and oleosin (oil body protein) as major seed proteins. The effects of oil expression and drying conditions on the extraction of these proteins from rapeseed meal were examined. The conditions strongly affected the extraction of oleosin and only weakly affected the extraction of cruciferin and napin. The protein chemical and physicochemical properties of cruciferin, the major protein present, were compared with those of glycinin (soybean 11S globulin) under various conditions. In general, cruciferin exhibited higher surface hydrophobicity, lower thermal stability, and lower and higher solubility at mu= 0.5 and mu = 0.08, respectively, than did glycinin. At the pHs (6.0, 7.6, and 9.0) and ionic strengths (mu= 0.08 and 0.5) examined, the emulsifying ability of cruciferin was worse than that of glycinin, except at mu= 0.08 and pH 7.6. The emulsifying abilities of cruciferin and glycinin did not correlate with thermal stability and surface hydrophobicity. Higher protein concentration, higher heating temperature, higher pH, and lower ionic strength were observed to produce harder gels from cruciferin. Gel hardness partly correlated with the structural stability of cruciferin.     

蚕豆蛋白的提取及NaCl浓度和pH值对其溶解性和乳化性的影响

蚕豆蛋白富含微量元素和人体必需的8种氨基酸,具有较高的开发价值。采用超声提取和水提取的方法提取蚕豆蛋白,研究了NaCl浓度和pH对蚕豆蛋白提取率、沉淀、功能性的影响。在pH值为8～12,超声提取和水提取均有较高的提取率。蚕豆蛋白的等电点在pH4.0～4.2之间。在pH4溶解性和乳化性最低,pH值在4～12时溶解性和乳化性随pH值的升高而升高, pH值为12时溶解性和乳化性最高。NaCl浓度从0 到 1.0 mol/L,溶解性和乳化性升高;当NaCl浓度继续增加,在pH 4、pH 7溶解性和乳化性随之下降。在相同的NaCl浓度和pH值时,超声提取比水提取蛋白的溶解性和乳化性高。该研究为蚕豆蛋白的提取工艺的确定及其在食品中的应用提供依据。     

Physicochemical properties of native and recombinant mungbean (Vigna radiata L. Wilczek) 8S globulins and the effects of the N-linked glycans

We have previously cloned and characterized the cDNAs of three isoforms of the 8S globulin of mungbean, expressed the major 8Salpha isoform in Escherichia coli, and purified and successfully crystallized it (Bernardo, A. E. N.; Garcia, R. N.; Adachi, M.; Angeles, J. G. C.; Kaga, A; Ishimoto, M.; Utsumi, S.; Tecson-Mendoza, E. M. J. Agric. Food Chem. 2004, 52, 2552-2560). Herein, we report the physicochemical and emulsifying properties of the native 8S and recombinant 8Salpha globulin or vicilin. The circular dichroism spectra analysis of the native 8S and recombinant 8Salpha globulins revealed that the recombinant 8Salpha formed a secondary structure close to that of the native 8S. Further, gel filtration analysis showed that 8Salpha was able to assemble into trimers. The native 8S and recombinant 8Salpha globulins were soluble at pH 3.4 and at pH 7.4-9.0 at low ionic strength, mu = 0.08. Interestingly, the native 8S was more soluble at pH 7.0 and pH 7.4 than the recombinant 8Salpha at mu = 0.08. Both forms were very soluble at pH 3.4-9.0 at high ionic strength, mu = 0.50. The native form exhibited a higher T(m) (69.2, 79.5, and 83.8 degrees C) than the recombinant form (65.6, 71.6, 77.5 degrees C) at mu = 0.1, 0.2, and 0.5, respectively. The recombinant form was found to have greater surface hydrophobicity than the native form. There was little difference in the emulsifying ability between the native 8S and 8Salpha at pH 3.4 and pH 7.6. The results indicate that the presence of N-linked glycans is not essential in the assembly and stable conformation of the mungbean vicilin. However, the N-linked glycans might have contributed to the higher solubility at low ionic strength, greater thermal stability, and decreased surface hydrophobicity of the native vicilin as compared to the recombinant 8Salpha. On the other hand, the N-linked glycans showed little effect on the emulsifying ability of the protein.     

Functional Properties of 7s Globulin Extracted from Cowpea Vicilins

The seed of cowpea (Vigna unguiculata L.) is rich in protein and the amino acid profiles of the meal are suitable for human dietary products, but little is known about the structure and chemical properties of the protein extracted from this legume. This study determined the functional properties of two selected cowpea cultivars and their solubility, emulsifying capacity, surface hydrophobicity, and thermal stability. Seeds of red and black cowpea were surface sterilized and the 7s globulin was isolated and purified using column chromatography with Sephacryl S‐300 (Hi‐Prep 26/60) gel column. Also, SDS‐PAGE and protein structure were analyzed using biochemical procedures. At high ionic strength (μ = 0.5), cowpea 7s globulin fraction exhibited better solubility for a wide range of pH levels, higher emulsifying capacities, and greater thermal stability than those obtained at low ionic strength (μ = 0.08). The lowest solubility was observed at pH 5.3–6.4 at the low ionic strength. Emulsifying capacities at high protein concentration were greater when compared with low protein concentration. T_m values of black cowpea globulin fraction were higher than those of red cowpea globulin fraction, whereas the surface hydrophobicity of the globulin fraction in red cowpea was larger than that in black cowpea.     

Physicochemical and functional properties of buckwheat protein product

This study was conducted to compare the physicochemical and functional properties of buckwheat protein product (BWP), soy protein isolate (SPI), and casein. BWP was prepared from buckwheat flour by the method including alkaline extraction and isoelectric precipitation. The amino acid composition of BWP was very similar to that of buckwheat flour. The protein solubility (PS) of BWP was much greater than that of SPI at all pH levels (pH 2-10) but lower than that of casein at pH 7-10. The isoelectric point of BWP was around pH 4. The higher aromatic hydrophobicities (ARH) of BWP, SPI, and casein were obtained at lower pH levels (pH 2-3). The emulsifying stability (ES) of BWP was lower than those of SPI and casein at high pH levels (pH 7-10). At all pH levels, BWP formed a thin emulsion. Regression analysis showed that the ARH of BWP was significantly associated with the ES. Although the water holding capacity of BWP was quite lower than that of SPI, its fat absorption capacity was slightly higher than those of SPI and casein. These results indicated that the physicochemical properties of BWP were different from those of SPI or casein. Thus, BWP is a potential source of functional protein for possible food application.     

In vitro protein digestibility and physicochemical properties of dry red bean (Phaseolus vulgaris) flour: effect of processing and incorporation of soybean and cowpea flour

A study was carried out to determine the effect of germination and drying temperature on the in vitro protein digestibility and physicochemical properties of dry red bean flours. A 2 x 3 factorial experiment with two treatments (germination and nongermination) and three drying temperatures was used for this purpose. The effect of particle size on water absorption capacity of bean flour was investigated. In addition, the effect of incorporating soybean and cowpea into the red bean flour on functional properties was equally investigated. Results reveal that protein digestibility increased with germination and also with drying temperature. Drying at 60 degrees C produced flours of optimum functional characteristics, although the hydrophilic/lipophilic index was high and the solubility index reduced. Germination and particle size as well as drying temperature all affected the water uptake properties of bean flours. Incorporation of soybean and cowpea flour into germinated bean flour at levels of 10 and 30%, respectively, produced a composite with higher functional properties.     

Some nutritional and functional properties of defatted wheat germ protein

Defatted wheat germ protein (DWGP) was isolated by alkaline extraction at pH 9.5 and subsequent isoelectric precipitation at pH 4.0, and its nutritional and functional properties were studied. The results showed that the amino acid content of defatted wheat germ was as high as 26.793 g/100 g, and the contents of eight essential amino acids were all relatively high. The isoelectric point of DWGP was 4.0. When pH >6.0, the DWGP had high solubility with a nitrogen solubility index of 70%. The emulsifying activity and emulsifying stability of DWGP were similar to those of bovine serum albumin and a little higher than those of casein. DWGP had good foaming capacity, but its foaming stability (FS) was not very good. However, the FS of DWGP can be improved through physical, chemical, or enzymatic methods. Moreover, DWGP had excellent water retention (WR); especially at pH 8.0 and a temperature of 70 degrees C, the WR of DWGP was the highest at 229.4%. DWGP offers is a potential source of functional protein isolate for possible food applications.     

Characterization of yam bean (Pachyrhizus erosus) proteins

Seed proteins from Mexican yam bean seeds (Pachyrhizus erosus L.) were sequentially extracted according to the Osborne classification. Albumins were the major fraction (52.1-31.0%), followed by globulins (30.7-27.5%). The minor protein fraction was prolamins (0.8%). Defatting with chloroform/methanol remarkably affected the distribution of protein solubility classes; albumins were the most affected fraction (4.3-17.5%). Electrophoretic patterns of albumins showed bands at 55, 40, 35, and 31 kDa. After reduction of the globulin fraction exhibited two triplets, one from 35 to 31 kDa and the second from 19 to 21 kDa, these could be compared to the acid and basic polypeptides of 11S-like proteins. Prolamins showed one band at 31 kDa, and glutelins after reduction showed three main bands at 52, 27, and 14 kDa. Trypsin inhibitors were assayed in saline extracts; the values found (1232-2608 IU/g of meal) were lower than those of other legumes. In general, yam bean seed proteins showed an excellent balance of all essential amino acids; albumins contain the highest amount of essential amino acids.     

Effect of sucrose on functional properties of soy globulins: adsorption and foam characteristics

In this contribution, we have analyzed the effect of sucrose on dynamic interfacial (dynamic surface pressure and surface dilatational properties) and foaming (foam capacity and foam stability) characteristics of soy globulins (7S and 11S). The protein (at 1 x 10(-3), 1 x 10(-2), 0.1, and 1 wt %) and sucrose (at 0, 0.25, 0.5, and 1.0 M) concentrations in aqueous solution and the pH (at 5 and 7), and ionic strength (at 0.05 and 0.5 M) were analyzed as variables. The temperature was maintained constant at 20 degrees C. We have observed the following. (i) The dynamics of adsorption (presence of a lag period, diffusion, and penetration at the air-water interface) of soy globulins depend on the peculiar molecular features of proteins (7S or 11S soy globulin) and the level of association/dissociation of these proteins by varying the pH and ionic strength, as well as the effect of sucrose in the aqueous phase on the unfolding of the protein. The rate of adsorption increases with the protein concentration in solution, at pH 7 compared to pH 5, at high ionic strength, and in the absence of sucrose. (ii) The surface dilatational properties reflect the fact that soy globulin adsorbed films exhibit viscoelastic behavior. The surface dilatational modulus increases at pH 7 compared to pH 5, but decreases with the addition of sucrose into the aqueous phase. (iii) The rate of adsorption and surface dilatational properties (surface dilatational modulus and phase angle) during adsorption at the air-water interface play an important role in the formation of foams generated from aqueous solutions of soy globulins. (iv) The increased interfacial adsorption (at high surface pressures) and the combined effects of interfacial adsorption and interfacial interactions between adsorbed soy globulin molecules (at high surface dilatational modulus) can explain the higher stability of the foam, with few exceptions.     

Study on furundu, a traditional Sudanese fermented roselle (Hibiscus sabdariffa L.) seed: effect on in vitro protein digestibility, chemical composition, and functional properties of the total proteins

Furundu, a meat substitute, is traditionally prepared by cooking the karkade (Hibiscus sabdariffa L.) seed and then fermenting it for 9 days. Physicochemical and functional properties of raw and cooked seed and of furundu ferments were analyzed. Furundu preparation resulted in significant changes in karkade seed major nutrients. Total polyphenols and phytic acid were also reduced. The increase in total acidity and fat acidity coupled with a decrease in pH indicates microbial hydrolysis of the major nutrients; proteins, carbohydrates, and fats. In vitro digestibility of the seed proteins reached the maximum value (82.7%) at the sixth day of fermentation, but thereafter it significantly decreased. The effect of furundu preparation on N solubility profiles and functional properties, such as emulsification and foaming properties and other related parameters, is investigated in water and in 1 M NaCl extracts from defatted flour samples. The results show that cooking followed by fermentation affects proteins solubility in water and 1 M NaCl. The foaming capacity (FC) from the flour of raw seed decreased as a result of cooking. Fermentation for 9 days significantly increased the FC of the cooked seed, restoring the inherent value. Foam from fermented samples collapsed more rapidly during a period of 120 min as compared to the foam from raw and cooked karkade seeds; stability in 1 M NaCl was lower as compared to those in water. In water, the emulsion stability (ES) from the fermented samples was significantly higher than that of the raw seed flour. Addition of 1 M NaCl significantly decreased the ES of the fermented samples.     

Evaluation of the solubility and emulsifying property of soybean proglycinin and rapeseed procruciferin in relation to structure modified by protein engineering

The presence or absence of a highly negatively charged extension region in beta-conglycinin (J. Agric. Food Chem. 1999, 47, 5278) and the length of a highly negatively charged variable region IV in glycinin (J. Agric. Food Chem. 2004, 52, 8197) are important determinants of solubility and emulsifying property. To examine the effects of the variable region IV from proglycinin A1aB1b and A3B4 and of the extension region from beta-conglycinin alpha' (alpha'ext) on solubility and emulsifying properties in detail, several mutants of proglycinin, procruciferin, and beta-conglycinin were designed and prepared in Escherichia coli. Nine out of 10 mutants were expressed at high levels in E. coli and shown to be homotrimer similar to the wild types as assessed by gel filtration. The position of the introduced negatively charged region as well as the amino acid composition were demonstrated to affect solubility at mu = 0.08. All of the proglycinin, procruciferin, and beta-conglycinin mutants with the alpha'ext in the C-terminus, especially the proglycinin mutant, exhibited excellent emulsifying ability and emulsion stability. These indicate that improvement of emulsifying properties by insertion of the alpha'ext in the C-terminus may be generally applicable to seed globulins.     

Formulation engineering can improve the interfacial and foaming properties of soy globulins

In this contribution, we have analyzed the effect of different strategies, such as change of pH (5 or 7) or ionic strength (at 0.05 and 0.5 M), and addition of sucrose (at 1 M) and Tween 20 (at 1 x 10(-4) M) on interfacial characteristics (adsorption, structure, dynamics of adsorption, and surface dilatational properties) and foam properties (foam capacity and stability) of soy globulins (7S and 11S at 0.1 wt %). We have observed that (1) the adsorption (presence of a lag period, diffusion, and penetration at the air-water interface) of soy globulins depends on the modification in the 11S/7S ratio and on the level of association/dissociation of these proteins by varying the pH and ionic strength (I), the effect of sucrose on the unfolding of the protein, and the competitive adsorption between protein and Tween 20 in the aqueous phase. The rate of adsorption increases at pH 7, at high ionic strength, and in the presence of sucrose. (2) The surface dilatational properties reflect the fact that soy globulin adsorbed films exhibit viscoelastic behavior but do not have the capacity to form a gel-like elastic film. The surface dilatational modulus increases at pH 7 and at high ionic strength but decreases with the addition of sucrose or Tween 20 into the aqueous phase. (3) The rate of adsorption and surface dilatational properties (surface dilatational modulus and phase angle) during adsorption at the air-water interface plays an important role in the formation of foams generated from aqueous solutions of soy globulins. However, the dynamic surface pressure and dilatational modulus are not enough to explain the stability of the foam.     

Effect of low and high pH treatment on the functional properties of cod muscle proteins

The functional properties of cod myosin and washed cod mince (myofibrillar protein fraction) treated at high (11) and low (2.5) pH were investigated after pH readjustment to 7.5. The solubility of refolded myosin was essentially the same as the native myosin. The pH-treated myofibrillar proteins had increased solubility over the whole ionic strength range studied. Acid and alkali treatment gave myosin and myofibrillar proteins improved emulsification properties, which were correlated with an increase in surface hydrophobicity and surface/interfacial activity. Enhanced gel strength was observed with acid- and alkali-treated myosin compared to native myosin, while the same treatment did not significantly improve the gel strength of acid- and alkali-treated myofibrillar proteins. The acid- and alkali-treated protein samples unfolded and gelled at a lower temperature than did the native proteins, suggesting a less conformationally stable structure of the refolded proteins. Functional studies show that acid and alkali treatment, which leads to partial unfolding of myosin may improve functional properties of cod myosin and myofibrillar proteins, with the greatest improvement being from the alkali treatment. The results also show that improvements in functionality were directly linked to the extent of partial unfolding of myosin on acid and alkali unfolding and refolding.     

Functional properties of guava seed glutelins

Five guava seed glutelin extracts were obtained with different buffer solutions: Na(2)B(4)O(7) alone (Glut.Bo) or containing SDS (Glut.BoSDS), 2-mercaptoethanol (Glut.Bo2-ME), or a combination of both (Glut.BoSDS2-ME) and NaOH (Glut.Na). All borate buffer solutions were at pH 10. The higher yield of glutelins corresponded to the Glut.BoSDS extract (81.9% dry basis) and the lower to Glut.Bo (6.8%). The functional properties of the five guava seed glutelin extracts were determined. Glut.BoSDS, Glut.BoSDS2-ME, and Glut.Na showed high values for several properties, including surface hydrophobicity (7.7, 10.8, and 0.6, respectively), solubility at pH 10 (91.1, 77.9, and 96.7, respectively), water-holding capacity at pH 3.6 (1.7, 2.5, and 2.8, respectively), emulsifying activity index (pH 10; 503.5, 238.2, and 838.0, respectively), and foaming properties (pH 10; V(0) = 0.14, 0.25, and 0.19, respectively; V(max) = 6.1, 5.59, and 4.51, respectively; t(1/2) = 266, 255.3, and 94 s, respectively). These results suggest that the denaturing reagent (SDS or NaOH) during extraction conferred on the proteins a structure that facilitated the development of their functional properties.     

Salts and pH Induced Changes in Functional Properties of Amaranth (Amaranthus tricolor L.) Seed Meal

Amaranth meal is a rich source of proteins, carbohydrates, and minerals with a low amount of anti‐nutritional factors. It exhibits good functional properties. The effect of NaCl and NaHCO₃ salts and pH level on the functional properties of amaranth meal was studied. The water absorption capacity and protein solubility were improved in the presence of the salts. Protein solubility was high at extreme pH values and minimum at pH 4. Foaming capacity was poor in the presence of the two salts, while foam stability was better at lower concentrations of NaCl (0.2–0.6M). Changes in pH had a pronounced effect on the foaming properties of amaranth meal. Salts did not change the emulsification properties but significantly increased the relative viscosity of amaranth seed meal at higher concentrations of NaCl and NaHCO₃ (0.6–1.0M). Relative viscosity was highest at pH 10 and lowest at pH 4.     

Modulation of physicochemical and conformational properties of kidney bean vicilin (phaseolin) by glycation with glucose: implications for structure-function relationships of legume vicilins

The structure-function relationships of plant oligomeric globulins are still not fully recognized. The present work investigated the influence of glycation with glucose (at 1:50 and 1:100 protein/sugar molar ratios; incubation periods of 2.5, 5.0, and 10.0 h) on the physicochemical and conformational properties of kidney bean vicilin (phaseolin), with the aim of understanding the structure-function relationships of legume vicilins. Protein solubility (PS), surface charge (isoelectric point) and hydrophobicity (H0), and secondary, tertiary, and/or quaternary conformations, as well as the emulsifying activities (emulsifying activity and emulsion stability indices, EAI and ESI) were evaluated. The 2.5 h incubation period of glycation led to least PS and highest H0, and after that, the PS and H0, on the contrary, gradually changed with increasing incubation period. The glycation increased the α-helix content and highly ordered secondary structures (α-helix+β-strand), as evidenced by far-UV circular dichroism (CD) spectroscopy. Combined analyses of differential scanning calorimetry, intrinsic emission fluorescence, and near-UV CD spectroscopy indicated that phaseolin underwent a tertiary conformation unfolding and subsequent rearrangement process (to form a new tertiary conformation), whereas the quaternary conformational flexibility progressively increased upon increasing degree of glycation. The conformation rearrangement was more distinct at the 1:100 molar ratio than at the 1:50 counterpart. The glycation at 5.0 and 10.0 h periods considerably increased the EAI, but only at the 1:50 molar ratio was the ESI progressively increased with the incubation period. These results confirmed that besides surface properties (e.g., PS and H0), the flexibility in tertiary and/or quaternary conformations played a major role in the emulsifying properties of glycated vicilins. The findings would have important implications for understanding the structure-function relationships of legume oligomeric globulins, thus providing a direction to further improve the surface-related functional properties of these proteins.     

Effects of protein engineering of canola procruciferin on its physicochemical and functional properties

The primary structure of Brassica napus procruciferin 2/3a was engineered to elucidate structure-function relationships and to improve the functionality of cruciferin. The following mutants were constructed: (1) C287T, (2) DeltaII, variable region II was deleted; (3) C287T/DeltaII, mutation involving (1) and (2); (4) DeltaIV + A1aIV; and (5) DeltaIV + A3IV, variable region IV was replaced with variable region IV containing many charged residues from soybean glycinin A1aB1b and A3B4 subunits. Differential scanning calorimetry analysis revealed that the A1aIV region has a more favorable interaction with the procruciferin molecule than does A3IV as well as the original regions. On the basis of heat-induced precipitation analysis, it was concluded that replacement of the free cysteine residue with threonine (C287T) and insertion of charged regions (DeltaIV + A1aIV and DeltaIV + A3IV) could lead procruciferin to form soluble aggregates after heating. Low solubility was observed in mutants DeltaIV + A3IV, DeltaII, and C287T/DeltaII, especially between pH 4 and 6 at mu = 0.08, but not in DeltaIV + A1aIV, indicating that the number of acidic amino acid residues and the high number of glutamine residues are important factors for solubility at mu = 0.08. None of the mutants showed any improvements in emulsifying ability, indicating that destabilization and addition of the hydrophilic region are not effective for emulsification. The insertion of the A1aIV region in procruciferin made the molecule more susceptible to alpha-chymotrypsin.     

Physicochemical and functional properties of hemp (Cannabis sativa L.) protein isolate

The amino acid composition and physicochemical and functional properties of hemp (Cannabis sativa L.) protein isolate (HPI) were evaluated and compared with those of soy protein isolate (SPI). Edestin, a kind of hexameric legumin, was the major protein component. HPI had similar or higher levels of essential amino acids (except lysine), in comparison to those amino acids of SPI. The essential amino acids in HPI (except lysine and sulfur-containing amino acids) are sufficient for the FAO/WHO suggested requirements for 2-5 year old children. The protein solubility (PS) of HPI was lower than that of SPI at pH less than 8.0 but similar at above pH 8.0. HPI contained much higher free sulfhydryl (SH) content than SPI. Differential scanning calorimetry analysis showed that HPI had only one endothermic peak with denaturation temperature (T(d)) of about 95.0 degrees C, attributed to the edestin component. The T(d) of the endotherm was nearly unaffected by 20-40 mM sodium dodecyl sulfate but significantly decreased by 20 mM dithiothreitol (P < 0.05). The emulsifying activity index, emulsion stability index, and water-holding capacity of HPI were much lower than those of SPI, and the fat adsorption capacity was similar. The data suggest that HPI can be used as a valuable source of nutrition for infants and children but has poor functional properties when compared with SPI. The poor functional properties of HPI have been largely attributed to the formation of covalent disulfide bonds between individual proteins and subsequent aggregation at neutral or acidic pH, due to its high free sulfhydryl content from sulfur-containing amino acids.