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.     

Functional properties of purified vicilins from cowpea (Vigna unguiculata) and pea (Pisum sativum) and cowpea protein isolate

The major storage globulins (vicilins) of cowpea (Vigna unguiculata) and pea (Pisum sativum) seeds were purified by ammonium sulfate precipitation, and a semipurified cowpea protein isolate (CPI) was prepared by isoelectric precipitation. Some of the functional properties of these proteins, including solubility, foaming, and emulsifying capacities, were investigated and compared. The solubility of purified cowpea vicilin was reduced at pH 5.0, increasing markedly below and above this value. Pea vicilin exhibited poor solubility between pH 5.0 and pH 6.0, and CPI was little soluble in the pH range from 4.0 to 6.0. At neutral pH, the emulsifying activity indexes (EAI) of purified pea vicilin and CPI were 194 and 291 m(2)/g, respectively, which compare quite favorably to EAIs of 110 and 133 m(2)/g for casein and albumin, respectively. Remarkably, purified cowpea vicilin exhibited an EAI of 490 m(2)/g, indicating a very high emulsifying activity. Purified cowpea and pea vicilins exhibited lower foaming capacities and foam stablity indexes (FSI) than CPI. FSI values of 80 and 260 min were obtained for purified pea and cowpea vicilin, respectively, whereas a FSI value of 380 min was obtained for CPI. These results are discussed in terms of the possible utilization of purified vicilins or protein isolates from pea and cowpea in the food processing industry.     

Organic complexed superphosphates (CSP): physicochemical characterization and agronomical properties

A new type of superphosphate (organic complexed superphosphate (CSP)) has been developed by the introduction of organic chelating agents, preferably a humic acid (HA), into the chemical reaction of single superphosphate (SSP) production. This modification yielded a product containing monocalcium phosphate complexed by the chelating organic agent through Ca bridges. Theoretically, the presence of these monocalcium-phosphate-humic complexes (MPHC) inhibits phosphate fixation in soil, thus increasing P fertilizer efficiency. This study investigateed the structural and functional features of CSP fertilizers produced employing diverse HA with different structural features. To this end were used complementary analytical techniques: solid-phase 31P NMR, 13C NMR, laser-confocal microscopy, X-ray diffraction, and molecular modeling. Finally, the agronomical efficiency of four CSP have been compared with that of SSP as P sources for wheat plants grown in both alkaline and acidic soils in greenhouse pot trials under controlled conditions. The results obtained from the diverse analytical studies showed the formation of MPHC in CSP. Plant-soil studies showed that CSP products were more efficient than SSP in providing available phosphate for wheat plants cultivated in various soils with different physicochemical features. This fact is probably associated with the ability of CSP complexes to inhibit phosphate fixation in soil.     

Comparison of physicochemical properties of 7S and 11S globulins from pea, fava bean, cowpea, and French bean with those of soybean--French bean 7S globulin exhibits excellent properties

Legume seeds contain 7S and/or 11S globulins as major storage proteins. The amino acid sequences of them from many legumes are similar to each other in the species but different from each other, meaning that some of these proteins from some crops exhibit excellent functional properties. To demonstrate this, we compared protein chemical and functional properties (thermal stability, surface hydrophobicity, solubility as a function of pH, and emulsifying properties) of these proteins from pea, fava bean, cowpea, and French bean with those of soybean as a control at the same conditions. The comparison clearly indicated that the 7S globulin of French bean exhibited excellent solubility (100%) at pH 4.2-7.0 even at a low ionic strength condition (mu = 0.08) and excellent emulsion stability (a little phase separation after 3 days) at pH 7.6 and mu = 0.08, although the emulsions from most of the other proteins separated in 1 h. These results indicate that our assumption is correct.     

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.     

Effects of a chitin-binding vicilin from Enterolobium contortisiliquum seeds on bean bruchid pests (Callosobruchus maculatus and Zabrotes subfasciatus) and phytopathogenic fungi (Fusarium solani and Colletrichum lindemuntianum)

Chitin-binding vicilin from Enterolobium contortisiliquum seeds was purified by ammonium sulfate followed by gel filtration on Sephacryl 300-SH and on Sephacryl 200-SH. The vicilin, called EcV, is a dimeric glycoprotein composed of 1.03% carbohydrates and a Mr of 151 kDa, consisting of two subunits of Mr of 66.2 and 63.8 kDa. The EcV homogeneity was confirmed in a PAGE where it was observed to be a unique acid protein band with slow mobility in this native gel. E. contortisiliquum vicilin (EcV) was tested for anti-insect activity against C. maculatus and Zabrotes subfasciatus larvae and for phytopathogenic fungi, F. solani and C. lindemuntianum. EcV was very effective against both bruchids, producing 50% mortality for Z. subfasciatus at an LD50 of 0.43% and affected 50% of the larvae mass with an ED50 of 0.65%. In artificial diets given to C. maculatus, 50% of the larvae mass was affected with an ED50 of 1.03%, and larva mortality was 50% at LD50 of 1.11%. EcV was not digested by midgut homogenates of C. maculatus and Z. Subfasciatus until 12 h of incubation, and at 24 h EcV was more resistant to Z. subfasciatus larval proteases. The binding to chitin present in larvae gut associated to low EcV digestibility could explain its lethal effects. EcV also exerted an inhibitory effect on the germination of F. solani at concentrations of 10 and 20 microg mL-1. The effect of EcV on fungi is possibly due to binding to chitin-containing structures of the fungal cell wall.     

Sorption isotherms and kinetics of Sb(V) on several Chinese soils with different physicochemical properties

Purpose

Sorption of antimony on soils is the primary factor that influences its immobilization and migration in the environment. In the present study, the sorption of Sb(V) onto seven Chinese soils with different physicochemical properties was investigated for exploring the relationship between the sorption capacity of Sb(V) and the physicochemical properties of the soils.

Materials and methods

Sorption isotherms and kinetics experiments were performed to ascertain the sorption capacity and the kinetic rate, respectively. The relationship between the sorption capacity of Sb(V) and the physicochemical properties of the soils was analyzed by multiple linear regressions.

Results and discussion

The results showed that the sorption isotherms fitted with both the Langmuir and Freundlich equations very well (R ²?=?0.936–0.997), and the sorption kinetic of Sb(V) onto the seven Chinese soils followed a pseudo-second-order reaction. The maximum sorption capacity of Sb(V) on the soils ranged from 134 to 1,333 mg?kg^?1. Nearly 94 % of the variability in maximum sorption of Sb(V) modeled by Freundlich equation could be described by Fe_DCB (dithionite–citrate–bicarbonicum extractable), and nearly 98 % of the variability could be described by Fe_DCB and Al_DCB.

Conclusions

Multiple linear regressions can be successfully applied to analyzing the relationship between sorption capacity and soil properties. Fe_DCB and Al_DCB played important roles in Sb(V) sorption onto soils. It would be useful to understand the environmental behaviors of Sb and for the implementation of risk assessment management and remediation strategies of Sb.     

Conformational study of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) by tryptophan fluorescence and differential scanning calorimetry

Fluorescence and differential scanning calorimetry (DSC) were used to study changes in the conformation of red kidney bean (Phaseolus vulgaris L.) protein isolate (KPI) under various environmental conditions. The possible relationship between fluorescence data and DSC characteristics was also discussed. Tryptophan fluorescence and fluorescence quenching analyses indicated that the tryptophan residues in KPI, exhibiting multiple fluorophores with different accessibilities to acrylamide, are largely buried in the hydrophobic core of the protein matrix, with positively charged side chains close to at least some of the tryptophan residues. GdnHCl was more effective than urea and SDS in denaturing KPI. SDS and urea caused variable red shifts, 2-5 nm, in the emission λ(max), suggesting the conformational compactness of KPI. The result was further supported by DSC characteristics that a discernible endothermic peak was still detected up to 8 M urea or 30 mM SDS, also evidenced by the absence of any shift in emission maximum (λ(max)) at different pH conditions. Marked decreases in T(d) and enthalpy (ΔH) were observed at extreme alkaline and/or acidic pH, whereas the presence of NaCl resulted in higher T(d) and ΔH, along with greater cooperativity of the transition. Decreases in T(d) and ΔH were observed in the presence of protein perturbants, for example, SDS and urea, indicating partial denaturation and decrease in thermal stability. Dithiothreitol and N-ethylmaleimide have a slight effect on the thermal properties of KPI. Interestingly, a close linear relationship between the T(d) (or ΔH) and the λ(max) was observed for KPI in the presence of 0-6 M urea.     

Flavonol glycosides from Montcalm dark red kidney bean: implications for the genetics of seed coat color in Phaseolus vulgaris L.

Three flavonol glycosides were isolated and identified from the commercial dark red kidney bean (Phaseolus vulgaris L.) cultivar Montcalm. In order of highest to lowest concentration these compounds were 3',4',5,7-tetrahydroxyflavonol 3-O-beta-D-glucopyranosyl (2-->1) O-beta-D-xylopyranoside (compound 1), quercetin 3-O-beta-D-glucopyranoside (compound 2), and kaempferol 3-O-beta-D-glucopyranoside (compound 3). Compound 1 is a flavonol glycoside that has not been reported before in P. vulgaris L. These three flavonol glycosides were yellow compounds that do not contribute to the garnet red color of Montcalm seed coats. Red-colored compounds which tested positive for proanthocyanidins are most likely responsible for the red seed coat color of Montcalm. Previous work on the chemistry of the compounds produced from the multi-allelic seed coat gene series C-C(r)()-c(u) indicated that neither anthocyanins nor flavonol glycosides were detected from seed coat extracts in the presence of the c(u)() locus. However, the seed coat color genotype of Montcalm is c(u) J g B v rk(d) and three flavonol glycosides were found. Technological advances such as modern HPLC analysis of seed coat extracts may allow for detection of small amounts of compounds which previously could not be seen using paper chromatography. Alternatively, the change of the Rk allele to rk(d) may allow for the synthesis of flavonol glycosides in the presence of c(u).     

Effect of thiram (tetramethyl-thiuram-disulphide) application on nodulation in soybean and kidney bean plants: Observation using the root-box-culture technique

Abstract

In agricultural practice, legume seeds are often treated with fungicides to protect them from harmful soil microorganisms. Most fungicides also are toxic to rhizobial growth. Among the synthetic fungicides, thiram (TMTD: tetramethyl-thiuram-disulphide) appeared to be most suitable for leguminous seed protection, because of its low toxicity to some species of rhizobium (NifTAL Project and FAO 1984).     

Study of some physicochemical and functional properties of quinoa (chenopodium quinoa willd) protein isolates

The amino acid composition and the physicochemical and functional properties of quinoa protein isolates were evaluated. Protein isolates were prepared from quinoa seed by alkaline solubilization (at pH 9, called Q9, and at pH 11, called Q11) followed by isoelectric precipitation and spray drying. Q9 and Q11 had high levels of essential amino acids, with high levels of lysine. Both isolates showed similar patterns in native/SDS-PAGE and SEM. The pH effect on fluorescence measurements showed decreasing fluorescence intensity and a shift in the maximum of emission of both isolates. Q9 showed an endotherm with a denaturation temperature of 98.1 degrees C and a denaturation enthalpy of 12.7 J/g, while Q11 showed no endotherm. The protein solubility of Q11 was lower than that of Q9 at pH above 5.0 but similar at the pH range 3.0-4.0. The water holding capacity (WHC) was similar in both isolates and was not affected by pH. The water imbibing capacity (WIC) was double for Q11 (3.5 mL of water/g isolate). Analysis of DSC, fluorescence, and solubility data suggests that there is apparently denaturation due to pH. Some differences were found that could be attributed to the extreme pH treatments in protein isolates and the nature of quinoa proteins. Q9 and Q11 can be used as a valuable source of nutrition for infants and children. Q9 may be used as an ingredient in nutritive beverages, and Q11 may be used as an ingredient in sauces, sausages, and soups.     

Proteolytic digestive enzymes and peritrophic membranes during the development of Plodia interpunctella (Lepidoptera: Piralidae): targets for the action of soybean trypsin inhibitor (SBTI) and chitin-binding vicilin (EvV)

The digestive system of P. interpunctella was characterized during its larval development to determine possible targets for the action of proteinaceous enzyme inhibitors and chitin-binding proteins. High proteolytic activities using azocasein at pH 9.5 as substrate were found. These specific enzymatic activities (AU/mg protein) showed an increase in the homogenate of third instar larvae, and when analyzed by individual larvae (AU/gut), the increase was in sixth instar larvae. Zymograms showed two bands corresponding to those enzymatic activities, which were inhibited by TLCK and SBTI, indicating that the larvae mainly used serine proteinases at pH 9.5 in their digestive process. The presence of a peritrophic membrane in the larvae was confirmed by chemical testing and light microscopy. In a bioassay, P. interpunctella was not susceptible to the soybean trypsin inhibitor, which did not affect larval mass and mortality, likely due to the weak association with its target digestive enzyme. EvV (Erythrina velutina vicilin), when added to the diet, affected mortality (LD50 0.23%) and larval mass (ED50 0.27%). This effect was associated with EvV-binding to the peritrophic membrane, as seen by immunolocalization. EvV was susceptible to gut enzymes and after the digestion process, released an immunoreactive fragment that was bound to the peritrophic matrix, which probably was responsible for the action of EvV.     

Evidence for protein degradation by Botrytis cinerea and relationships with alteration of synthetic wine foaming properties

Botrytis cinerea is an important fungal pathogen particularly dreaded in the cool climate vineyard. It is responsible for important damage, especially the decrease in foamability of sparkling wines, such as Champagne. Different studies have shown that proteins are largely involved in the stabilization of Champagne foam despite their low concentration. Other works demonstrated changes in the electrophoretic characteristics of must proteins originating from botrytized grapes, although the cause of such alterations was never explained. In the first part of this study, results showed the release by B. cinerea of 3.5 mg/L total proteins in a synthetic liquid medium. Among these proteins, the presence of a protease activity on bovine serum albumin (BSA) and must proteins was demonstrated by using a colorimetric method and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the model wine, the Bradford method showed a BSA loss of 66% after 24 h and a loss of 96% after 120 h. In the same model wine, the soluble must protein concentration decreased by 35% after 1 week and by 53% after 2 weeks while the control showed no protein loss. B. cinerea proteases were then able to degrade BSA and must proteins and were above all active at must and wine pH and in the presence of ethanol and SO(2). The second part of this work was dedicated to the relationship between the presence of B. cinerea proteases and its effects on the synthetic wine foaming properties. The addition of a B. cinerea culture medium (1/33 v/v) to the synthetic wine containing 21 mg/L soluble grape proteins induced a decrease in foamability by 60% after 1 week. For BSA in the model wine, the foamability decreased by 32% after 24 h and by 95% after 120 h, as shown by the colorimetric method. These experiments demonstrate for the first time the relationship between B. cinerea protease activity and the decrease in wine foaming properties.     

Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity

Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.     

Modifications to physicochemical and nutritional properties of hard-To-cook beans (Phaseolus vulgaris L.) by extrusion cooking.

The objective of this work was to evaluate extrusion cooking as a means to improve the nutritional properties of Phaseolus vulgaris L. that had been stored either at 42 degrees C and 80% relative humidity for 6 weeks or for periods >1 year in cereal stores in tropical conditions. Storage under these conditions resulted in an increase in cooking time increased (7.7- and 12-fold, respectively) as a result of development of the hard-to-cook (HTC) defect. Single-screw extrusion of the milled beans was carried out at four barrel temperatures and two moisture contents. The extrudate bulk density and water solubility index decreased with increasing temperature, whereas the water absorption index increased due to the higher proportion of gelatinized starch in the extruded samples. Both fresh and HTC beans contained nutritionally significant amounts of lectins, trypsin, and alpha-amylase inhibitors, which were mostly inactivated by extrusion. Extrusion also caused a considerable redistribution of insoluble dietary fiber to soluble, although the total dietary fiber content was not affected. Changes in solubility involved pectic polysaccharides, arabinose and uronic acids being the main sugars involved. Stored beans subjected to extrusion cooking showed physical and chemical characteristics similar to those of extrudates from fresh beans.     

Wild-weed-crop complexes of common bean (Phaseolus vulgaris L., Fabaceae) in the Andes of Peru and Colombia, and their implications for conservation and breeding

During germplasm explorations carried out in Peru and Colombia, interbreeding complexes of wild and cultivated common bean were observed in both countries, eight in Apurimac and Cusco departments of Peru and eight in Cundinamarca and Boyaca´ departments of Colombia. The existence of complexes was evidenced both by segregation of wild and cultivated morphological traits in certain populations, and by the presence of genetically stabilized weedy types which were assumed to have arisen from past hybridization. Observations on phaseolin seed protein confirmed that genetic exchange was occurring. Phaseolin types introduced from other regions were in incipient stages of introgression into local populations. On the other hand, local phaseolin types were observed in all phases of the complexes from totally wild to fully cultivated beans, suggesting that the complexes had undergone a long evolution. Complexes could be an effective means to generate genetic variability, introgressing genes from wild populations into cultivated types and complementing modern plant breeding programs. The conservation of such complexes depends on the continued existence of the wild, weedy and cultivated beans in close proximity; on the maintenance of a semi-domesticated environment; and on the willingness of farmers to leave weedy types in the field.     

低温等离子快速提高糖基化花生分离蛋白溶解性及乳化性

为进一步提高花生蛋白的溶解特性,扩大花生蛋白在食品工业中的应用。采用低温等离子（Non-Thermal Plasma,NTP）诱导花生分离蛋白-葡聚糖（Peanut Protein Isolate-Dextran,PPI-Dex）湿法糖基化反应,研究NTP在处理0、0.5、1.5、2.0 、3.0 min的情况下,反应时间对花生分离蛋白与葡聚糖糖基化反应的影响。在低温等离子处理功率为70 W,反应液温度为60 ℃的状态下,随着NTP处理时间的延长,PPI-Dex的接枝度增加,在处理时间为1.5 min时,PPI-Dex接枝度达最大为21.62%,与超声波接枝PPI-Dex需要40 min,传统湿接枝需要24 h相比,缩短了接枝时间。PPI-Dex接枝后,接枝物溶解度和乳液稳定性显著增强,与未接枝相比,溶解度提高了22.28%。通过测定其分子量、氨基酸含量、红外图谱及表面疏水性变化分析NTP处理对花生分离蛋白结构影响。分析结果表明,NTP 处理1.5 min后,花生分离蛋白与葡聚糖发生糖基化反应形成偶联物,偶联物中羟基特征峰3 000～3 500 cm-1及1 000～1 260 cm-1的吸光度与未处理时相比增加,赖氨酸和苯丙氨酸相对含量显著降低（P<0.05）;同时,α-螺旋含量降低,β-折叠向β-转角转变,蛋白的有序结构被破坏,结构变松散,PPI构型向亲水型转变;接枝物的表面疏水性指数降低。花生分离蛋白与葡聚糖发生糖基化反应,反应位点可能为Lys和Phe。结果表明,低温等离子处理是一种快速促进蛋白与多糖接枝的有效方法。     

Water stability of aggregates in subtropical and tropical soils (Georgia and China) and its relationships with the mineralogy and chemical properties

Water-stable aggregates isolated from three subtropical and one tropical soil (Western Georgia and China) were studied for their organic carbon, cation exchange capacity (CEC), specific surface area, magnetic susceptibility, and total chemical elements. The soils were also studied for their particle-size distribution, mineralogy, and nonsilicate Fe and Al oxides. Describe the water stability, three indices have been used: the content of water-stable macroaggregates (>0.25 mm), the mean weighted diameter of the aggregates, and the numerical aggregation index. The yellow-cinnamonic soil (China) was neutral, and the three other soils were acid. The soils were degraded with a low content of organic matter. The yellow-cinnamonic soil was characterized by the lowest water stability due to the predominantly vermiculite composition of the clay. The high water stability of the Oxisol structure was determined by the kaolinites and high content of oxides. In three out of the four soils studied, the hierarchical levels of the soil structure organization were defined; they were identified by the content of organic matter and the Ca + Mg (in Oxisols). Iron oxides mainly participated in the formation of micro-aggregates; Al and Mn contributed to the formation of macroaggregates. The water-stable aggregates acted as sorption geochemical barriers and accumulated Pb, Zn, Cd, Cs, and other trace elements up to concentrations exceeding their levels in the soil by 5 times and more. The highest correlations were obtained with CEC, Mn, and P rather than with organic carbon and Fe.     

Determination of advanced glycation endproducts by LC-MS/MS in raw and roasted almonds (Prunus dulcis)

A sensitive and reliable LC-(ESI)MS/MS method was developed and validated for the simultaneous analysis of five common advanced glycation endproducts (AGEs) after enzymatic digestion in raw and roasted almonds. AGEs included carboxymethyl-lysine (CML), carboxyethyl-lysine (CEL), pyralline (Pyr), argpyrimidine (Arg-p), and pentosidine (Pento-s). This method allows accurate quantitation of free and AGE-protein adducts of target AGEs. Results indicate that CML and CEL are found in both raw and roasted almonds. Pyr was identified for the first time in roasted almonds and accounted for 64.4% of free plus bound measured AGEs. Arg-p and Pento-s were below the limit of detection in all almond samples tested. Free AGEs accounted for 1.3-26.8% of free plus bound measured AGEs, indicating that protein-bound forms predominate. The roasting process significantly increased CML, CEL, and Pyr formation, but no significant correlation was observed between these AGEs and roasting temperature.