Polymerization of Several Proteins by Ca2+-Independent Transglutaminase Derived from Microorganisms

α_s1-Casein and soybean globulins were polymerized and gelatinized by Ca²⁺-independent transglutaminase that was isolated from the culture filtrate of a microorganism thought to belong to Streptoverticillium sp. of actinomycetes. This enzyme polymerized such albumins as bovine serum albumin, human serum albumin and conalbumin in the presence of dithiothreitol. Rabbit myosin was polymerized by the present emzyme but actin was not. An RP-HPLC analysis after enzymic digestion of the polymerized a_sl -casein showed existence of the £-(y-Glu)Lys bond. Thus, it was confirmed that the polymerization was formed by a catalytic reaction of the transglutaminase.     

Precursors to Nitrosopyrrolidine and Nitrosopiperidine in Black Pepper Treated with Nitrous Acid

The following properties of food proteins polymerized by guinea pig liver transglutaminase were investigated: (1) solubility, (2) emulsifying activity and emulsion stability, and (3) unfrozen water content by pulsed NMR. Several food proteins (α_sl- and k-caseins, and soybean 7S and 11S globulins) were polymerized by this enzyme. Solubility and emulsifying activity of polymerized α_sl-casein were higher than those of the native protein in the range of pH 4~6. Unfrozen water contents of polymerized soybean globulins were much higher than those of the native proteins. These results suggest that transglutaminase treatment may be used for the production of new food protein material with higher hydration ability.     

Enzymatic Cross-Linking of Casein Facilitates Gel Structure Weakening Induced by Overacidification

Casein (α_S1, α_S2, β, κ) is the major protein fraction in milk and, together with heat denatured whey proteins, responsible for gel network formation induced by acidification. Rheological measurements during gelation typically reveal a maximum storage modulus (G') at a pH close to the isoelectric point (pI) of casein (~4.6). With further decreasing pH gel stiffness decreases because of increased electrostatic repulsion, which is referred to as overacidification. In this study we investigated the effect of casein cross-linking with microbial transglutaminase on gel structure weakening induced by acidification to pH below the pI. Although enzymatic cross-linking increased the maximum stiffness (G' _MAX ) of casein gels the reduction of G' during overacidification, expressed as ratio of the plateau value (G' _FINAL ) to G' _MAX , was more pronounced. Almost no soluble protein was detected in the serum of gels from cross-linked casein, whereas considerable amounts of α_S- and κ-casein were released from reference gels below the pI. This suggests that covalent cross-linking of casein retains charged molecules within the gel network and therefore causes a higher reduction of protein-protein interactions because of higher electrostatic repulsion. Furthermore, higher amounts of uncross-linked β-casein, which was the only casein type not found in the serum, resulted in higher G' _FINAL to G' _MAX ratios, underlining the important contribution of β-casein to acid gel formation and prevention of gel structure weakening.     

Simultaneous Determination of Patulin and Penicillic Acid in Grains by Gas Chromatographic Method

Changes in aggregation and properties of alkali-treated soybean 7S and 11S globulins depend upon protein concentration during alkali-treatment. Such variables were investigated by viscosity, electrophoresis, circular dichroism, pulsed NMR, emulsion capacity and CaCl₂ precipitation measurements. In lower protein concentrations, the intrinsic viscosity decreased and the penetrative fractions into electrophoresis gel increased. The reduced contacts of proteins during neutralization resulted in smaller aggregates. Also specific fractions which were more sensitive to protein concentration on aggregation were observed for 11S globulin. The quantity of bound water depended only on pH at 7% concentration treatment. When the gel was formed, the bound water of protein increased, e.g., 0.085 g and 0.135 g/g protein at pH 10.6 and 13.2 treatment, respectively, whereas at 1% treatment, bound water showed almost no pH dependence (about 0.13 g/g protein). Furthermore, proteins prepared at higher protein concentrations were characterized by higher emulsion capacity and CaCI₂ precipitation ability. However, no protein concentration dependence was seen in the secondary structure of the aggregates.     

Crosslinking of Soybean 7S and 11S Proteins by Transglutaminase

Transglutaminase catalyzes the formation of intermolecular and intramolecular ε-(γ-glutamyl)lysyl crosslinks in proteins. The study here examined the substrate effectiveness of soybean 7S and 11S proteins in the intermolecular-crosslinking reaction catalyzed by guinea pig liver transglutaminase.

Both 7S and 11S proteins could act as the substrate for the transglutaminase reaction. The reaction with 11S protein was faster than that of 7S protein. Analyses of the reaction products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that three main subunit groups of 7S protein and two acidic subunit groups of 11S protein were polymerized through the formation of intermolecular crosslinks by transglutaminase. Interestingly enough, no intermolecular crosslink was formed between the basic subunits of 11S protein. The possible significance of the intermolecular crosslinking catalyzed by transglutaminase is discussed, including the use of this enzyme reaction to improve the properties of food protein.     

Formation of Pseudo- and Hybrid-11S Globulins from Subunits of Soybean and Broad Bean 11S Globulins

Pseudo- and hybrid-11S globulins were reconstituted from native acidic and basic subunits of soybean and broad bean 11S globulins. The subunit structures of these two globulins are known to be similar to each other. Pseudo-11S globulins were formed in combinations between glycinin acidic subunit (G-AS_{1 + 2}) and glycinin basic subunit (G-BS) and between legumin acidic subunit (L-ASII) and legumin basic subunit (L-BS). Hybrid-11S globulins were formed in combinations between G-AS_{1 + 2} and L-BS and between L-ASII and G-BS. The yields of the reconstituted 11S components of G-AS_{1 +2} + G-BS and G-AS_{1 + 2} + L-BS were lower than those of L-ASII + G-BS and L-ASII + L-BS. These pseudo- and hybrid-11S globulins were similar to native 11S globulins; they all consisted of reconstituted intermediary subunits which were composed of acidic and basic subunits linked by disufide bridges and had molecular weights similar to those of native 11S globulins. However, the dissociation-association behaviors of pseudo-glycinin and hybrid-11S globulins were different from those of native 11S globulins.     

Relationship between Gelation and Aggregation of Alkali-treated Soybean 7S and 11S Globulins

Gel was obtained when alkaline dope solutions of the 7S and 11S globulins (8% protein concentration) prepared at pH above 11 were dialyzed against phosphate buffer, pH 7.6, µ= 0.3. To make clear the mechanism of gelation, the relationship between changes in viscosity and aggregation phenomena of the neutralized dope solutions was investigated by means of viscosity measurement, disc electrophoresis and gel filtration, comparing the 7S and 11S fractions. In conclusion, it is revealed that the gel is constituted with macromolecule aggregates, and to form the aggregates which are suitable for gelation, all of the following conditions must be satisfied at least : 1); Unfolding and dissociation into subunits once (above pH 11), 2); High ionic strength in the media (µ=0.3), 3); Formation of hydrogen, hydrophobic and disulfide bonds, 4); High protein concentration (above 8%).     

Textural and Rheological Properties of Soy Protein Isolate Tofu-Type Emulsion Gels: Influence of Soybean Variety and Coagulant Type

The contribution of soybean variety and coagulant type to the textural and rheological properties of soy protein isolate (SPI) tofu-type emulsion gels was studied. SPIs from eight soybean varieties were subjected to amino acid and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and results showed that the 11S fraction proteins (r?=?0.833, p?<?0.05) and the ratio of 11S to 7S (r =?0.920, p <?0.01) were positively correlated with the hardness of CaSO₄-induced emulsion gels and glucono-δ-lactone (GDL)-induced gels, with the correlation coefficients of 0.827 (p <?0.05) and 0.893 (p <?0.01), respectively. In the case of microbial transglutaminase (MTGase), strong relations between the content of glutamate (r =?0.886, p?<?0.01) and lysine (r =?0.810, p <?0.05) and gel hardness were found. Rheological data demonstrated that CaSO₄-induced emulsion gel was stiffer with high rigidity but gel induced by MTGase performed better elasticity. The findings of this study are of great importance to further understand the gelation mechanisms of different coagulants and provide useful information for the development of SPI-based filled tofu.     

Influence of Ionic Strength on Conformation Changes of Soybean Proteins Caused by Heating,and Relationship of Its Conformation Changes to Gel Formation

Changes of disc electrophoretic and ultracentrifugal patterns of soybean protein by heating were different, depending on whether the protein is in soybean milk or in acid precipitated protein solution. It was revealed that ionic strength has definite effects on these changes, and this is why the changes are different between soybean milk and acid precipitated protein solution. 7S protein is sensitive to heating at higher ionic strengths, forming aggregates directly, whereas 11S protein is sensitive at lower ionic strengths, dissociating to subunits which form aggregates partly. The fact that 7S protein cannot form firm gel by glucono-delta-lactone after heating and 11S protein can form firm gel when reasonably heated is supposed to be attributed to the difference of the process of aggregates formation during heating between the two proteins.     

A Comparison of Soybean Globulins and the Protein Bodies in the Protein Composition

The protein compositions between soybean globulins and the protein bodies were compared by gel filtration with Sephadex G-200, sedimentation analyses and disc isoelectric focusing.

From the results of the three comparisons, it was difficult to find an essential difference in the protein compositions of the both. And, the 7S and the 11S globulins were the main components in the both. This fact was more strongly suggested that these globulins were the typical reserve proteins.     

Gelation Phenomena Induced by Alkali-alcohol Treatment of 7S and 11S Components in Soybean Globulins

Transparent gels containing about 2% protein were obtained by mixing alkaline dope solution of 7S or 11S soybean proteins with alcohol. The 7S component showed the ability to form a stronger gel than the 11S. This phenomenon depended on pH and alcohol concentration. In 66 % ethanol, the viscosity of the 7S and 11S reached maxima at pH 11.4 and 11.2, respectively. Above these pH levels where further unfolding and dissociation into subunits of the protein molecules occur, the viscosity decreased rather. The effectiveness of alcohol to increase viscosity increased in the order; n-butanol < tert-butanol < n-propanol < iso-propanol < ethanol < methanol. Alcohols having minor hydrophobicity were more effective for increasing viscosity, but ethylene glycol was ineffective. The addition of NaCl or 2-mercaptoethanol to ethanol-mixed alkaline dope solutions resulted in the remarkable increment of the viscosity, especially for the 7S.     

The chaperone action of bovine milk αS1- and αS2-caseins and their associated form αS-casein

α_S-Casein, the major milk protein, comprises α_S1- and α_S2-casein and acts as a molecular chaperone, stabilizing an array of stressed target proteins against precipitation. Here, we report that α_S-casein acts in a similar manner to the unrelated small heat-shock proteins (sHsps) and clusterin in that it does not preserve the activity of stressed target enzymes. However, in contrast to sHsps and clusterin, α-casein does not bind target proteins in a state that facilitates refolding by Hsp70. α_S-Casein was also separated into α- and α-casein, and the chaperone abilities of each of these proteins were assessed with amorphously aggregating and fibril-forming target proteins. Under reduction stress, all α-casein species exhibited similar chaperone ability, whereas under heat stress, α-casein was a poorer chaperone. Conversely, α_S2-casein was less effective at preventing fibril formation by modified κ-casein, whereas α- and α_S1-casein were comparably potent inhibitors. In the presence of added salt and heat stress, α_S1-, α- and α_S-casein were all significantly less effective. We conclude that α_S1- and α-casein stabilise each other to facilitate optimal chaperone activity of α_S-casein. This work highlights the interdependency of casein proteins for their structural stability.     

Production of Yeast Cells from Methanol

Using immunochemical technique thermal denaturation of soybean 11S globulin, dissolved in different ionic strength solutions (µ=0~4.0) and heated at 100°C for 5 min, has been quantitatively studied. The curves of the percentage of antigenicity remaining were obtained as a function of salt concentration. The 11S globulin became strongly resistant to thermal denaturation with increasing both KCl and potassium phosphate. The stabilizing effect (in terms of percent antigenicity) was separated into three regions. At ionic strength below 0.7, potassium phosphate had no stabilizing effect while KCl had aslightly effect. The rise in stabilizing effect up to about 50%, near 1.0~1.5 µ, represented a second transition to a different denatured state which retains undissociated molecule. At rises up to 75~95%, near 2.5~3.5µ, a different conformational state resulted in which thermally denatured 11S globulin maintained almost intact native conformation after heating. The selection of an adequate ionic strength of protein solution has enabled preparation of thermally denatured 11S globulins which have desired-residual amounts of structured regions.     

Cold-Set Whey Protein Gels with Addition of Polysaccharides

Cold-set whey protein (WP) gels with addition of xanthan or guar were evaluated by mechanical properties and scanning electron microscopy. Gels were formed after the addition of different amounts of glucono-δ-lactone to thermally denatured WP solutions, leading to different acidification rates and final pH values. At lower acidification rates and higher final pH, gels showed more discontinuous structure and weaker and less elastic network, which was attributed to a predominance of phase separation during gel formation due to slower gelation kinetics. In contrast, at higher acidification rates and lower final pHs, gelation prevailed over phase separation, favoring the formation of less porous structures, resulting in stronger and more elastic gels. The gels’ fractal dimension (D _f; structure complexity) and lacunarity were also influenced by the simultaneous effects of gelation and phase separation. For systems where phase separation was the prevailing mechanism, greater lacunarity parameters were usually observed, describing the heterogeneity of pore distribution, while the opposite occurred at prevailing gelation conditions. Increase in guar concentration or lower final pH of xanthan gels entailed in D _f reduction, while the increase in xanthan concentration resulted in higher D _f. Such a result suggests that the network contour length was rugged, but this pattern was reduced by the increase of electrostatic interactions among WP and xanthan. Guar addition caused the formation of gel network with smoother surfaces, which could be attributed to the guar–protein excluded volume effects leading to an increase in protein–protein interactions.     

Instability of Pressure-Treated Reconstituted Skim Milk to Acidification

When high-pressure (HP)-treated reconstituted skim milks (200–600 MPa/5–30 min) were acidified with glucono-δ-lactone, the elastic modulus (G′) displayed atypical behaviour with pH, increasing as the pH decreased between 6.0 and 5.3, indicating that a weak gel had formed as soon as the pH of the milk decreased. The formation of a weak gel at pH 6.0 to 5.3 indicates that HP milks are more unstable to acidification than untreated or heated milks; this effect has not been previously reported. Untreated and heated (90 °C/30 min) milks did not show an increase in G′ until the pH was below 4.9 and 5.3, respectively. Frequency sweeps confirmed that the HP-treated milks formed weak gels at pH much higher than where typical acid gelation of milk occurs. Microstructural and particle size analyses indicated that the HP-treated milks started aggregating as soon as the pH declined whereas the heated milks did not aggregate until the pH was below 5.5. Heat treatment of milk either before or after HP treatment completely eliminated the weak gelation as these samples did not form gels until the pH decreased below pH 5.3. It is apparent that the restructured colloidal particles formed when milk is HP treated are unstable to acidification, and it is proposed that the redistributed κ-casein cannot stabilize these particles when the milk is acidified. The role of denatured whey proteins in the weak gelation phenomenon is unclear.     

Genetic mapping and validation of the loci controlling 7S α′ and 11S A-type storage protein subunits in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

Key message

Four soybean storage protein subunit QTLs were mapped using bulked segregant analysis and an F₂ population, which were validated with an F₅ RIL population.

Abstract

The storage protein globulins β-conglycinin (7S subunit) and glycinin (11S subunits) can affect the quantity and quality of proteins found in soybean seeds and account for more than 70% of the total soybean protein. Manipulating the storage protein subunits to enhance soymeal nutrition and for desirable tofu manufacturing characteristics are two end-use quality goals in soybean breeding programs. To aid in developing soybean cultivars with desired seed composition, an F₂ mapping population (n = 448) and an F₅ RIL population (n = 180) were developed by crossing high protein cultivar ‘Harovinton’ with the breeding line SQ97-0263_3-1a, which lacks the 7S α′, 11S A₁, 11S A₂, 11S A₃ and 11S A₄ subunits. The storage protein composition of each individual in the F₂ and F₅ populations were profiled using SDS-PAGE. Based on the presence/absence of the subunits, genomic DNA bulks were formed among the F₂ plants to identify genomic regions controlling the 7S α′ and 11S protein subunits. By utilizing polymorphic SNPs between the bulks characterized with Illumina SoySNP50K iSelect BeadChips at targeted genomic regions, KASP assays were designed and used to map QTLs causing the loss of the subunits. Soybean storage protein QTLs were identified on Chromosome 3 (11S A₁), Chromosome 10 (7S α′ and 11S A₄), and Chromosome 13 (11S A₃), which were also validated in the F₅ RIL population. The results of this research could allow for the deployment of marker-assisted selection for desired storage protein subunits by screening breeding populations using the SNPs linked with the subunits of interest.

     

Production of S-Methyl Thioacetate from Methyl Mercaptan by Saccharomyces cerevisiae

Physicochemical properties of human к-casein were studied by ultracentrifugal analysis and circular dichroism (CD) measurement. The result of sedimentation velocity analysis in 50mm imidazole-HCl buffer at pH 7.0 showed that human к-casein was present in a monomerie form with S^°_20.w of 2.7S. The molecular weight of this protein was estimated to be 38,000 by a short column method. The molecular shape was considered to be a flat ellipsoid with the shape factor of 16.74 and with the frictional coefficient of 2.17. From the result of CD measurement, human к-casein was computed to have 2% α-helix, 43% α-sheet and 26% α-turn structures. Interaction of human к-casein with human к-casein was observed by sedimentation velocity analysis and discpolyacrylamide gel electrophoresis, but no association occurred between human к-casein and human lactoferrin under the conditions we studied.     

The Glycoproteins of Plant Seeds : ANALYSIS BY TWO-DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS AND BY THEIR LECTIN-BINDING PROPERTIES

Protein from the jack bean, peanut, soybean and kidney bean seeds were extracted with a solution containing 9.3 molar urea, 5 millimolar K₂CO₃, 0.5% dithiothreitol and 2% Nonidet P-40 and then subjected to two-dimensional gel electrophoresis. After electrophoresis, the slab gels were stained with a variety of ¹²⁵I-labeled lectins and the lectin-binding proteins were identified after autoradiography. Incubation of slab gels of jack bean with concanavalin A, peanut with peanut agglutinin, soybean with soybean agglutinin, and kidney bean with phytohemagglutinin showed that the majority of the polypeptides in each seed type were able to bind to their homologous lectins. Control slab gels in which incubations were carried out with identical amounts of proteins, ¹²⁵I-lectin and an appropriate sugar inhibitor showed little or no lectin binding to the polypeptides. Additionally, incubation of slab gels of peanut proteins with ¹²⁵I-ricin, ¹²⁵I-wheat germ agglutinin, ¹²⁵I-concanavalin A, and ¹²⁵I-soybean agglutinin each revealed a clearly distinct binding pattern compared to the one observed with the peanut agglutinin. The results demonstrate that a large number of legume seed polypeptides are glycoproteins and that the carbohydrate groups within a seed species are heterogeneous in structure, thus indicating the existence of complex glycosylating enzyme systems in legume seeds. It is suggested that the high degree of binding between seed proteins and their homologous lectins might have some functional significance in maintaining large aggregates of protein in compact, insoluble form.     

Studies on Molecular Properties of αs1-κ-Casein Complex by the Hydrodynamical Methods

Some molecular properties of α_s1-κ-casein complex, α_s1- and κ-casein polymers were examined by gel filtration, ultracentrifugation, and viscometry at pH 7.1. The Stoke’s radii of α_s1-κ-casein complex, α_s1- and κ-casein polymers were 99, 44 and 108 Å, respectively. The molecular weight of the above proteins were approximately 45 × 10⁴, 10 × 10⁴ and 80 × 10⁴, respectively. The stokes radius of α_s1-κ casein complex reduced compared with that of κ-casein polymer and the molecular weight of the complex was about half that of κ-casein polymer. These results suggest that κ-casein polymer dissociates into 4 smaller particles when α_s1-κ-casein complex is formed. The frictional coefficient and Scheraga-Mandelkern constants for each protein suggest that the molecular shape of α_s1-casein polymer is globular and that of α_s1-κ-casein complex and κ-casein polymer is rod-like.     

Mixed Function Oxidase Inhibitors Protect Plants from Ozone Injury

κ-Casein and α_s1-κ-casein complex with a weight ratio of unity were dissolved in 50mm cacodylate-HCl-70 mm KC1 buffer containing 0.02% of sodium azide (pH 7.1), and their size and shape in the absence and/or presence of calcium ions were observed with the electron microscope. In the absence of calcium ions, both κ-casein and α_s1-κ-casein complex were spherical particles. However, the mean length of α_s1-κ-casein complex (12 nm) was smaller than that of κ-casein (17 nm), which suggested that complex formation led to dissociation of the κ-casein polymer. The addition of calcium ions to the complex led to the formation of bent chains, though micelle-like aggregates were not observed even at 20 nm calcium. Comparison of the frequency distributions of α_s1-κ-casein complex at 0, 5, 10, 15 and 20 mm of calcium with the calculated probability distributions suggested that most α_s1-κ-casein complexes had two binding sites above 10 mm of calcium, which seemed to be essential for the stability of casein micelle.