| Soybean（Glycine max[L.]Merr.）is an important source of high-quality protein in the human diet,and the structure-function relationship of soybean protein is the core theory of soy-bean processing research industry.With the development of molecular separation,purification and analysis technology,the analysis of structure-function relationship of soybean protein at molecular level has become a research hot spot.The dispersion properties of protein refers to the solubility and aggregation state of protein in characteristic conditions,which is influenced by the structure of protein molecules and the conditions they are in,and also directly affects functional properties.The functional properties of protein vary greatly in different conditions,where the ionic strength and p H have an important influence on the dispersion properties of proteins.Glycinin andβ-conglycinin are important components of soybean protein,of whichβ-conglycinin consists of three subunits（α′-,α-andβ-subunits）,and the glycosyl groups of the three subunits and the extension regions ofα′-andα-subunits are important protein structures.At present,the dispersion properties of subunits that delecting glycosyls and extension region are not completely clear.Conventional separation and purification techniques for subunits can not produce them at the molecular level.Prokaryotic expression system can not only accu-rately produce subunits without extension region,but also subunits without glycosyl.In this paper,the molecular structure,thermal stability,particle size and potential,solubility,turbid-ity,surface hydrophobicity and apparent viscosity of the subunits in deionized water（μ=0）and acidic（p H 4.0）,neutral（p H 7.0）and alkaline（p H 8.0）conditions with low（μ=0.05）and high（μ=0.5）ionic strengths were tested by using the subunits prepared from the prokary-otic system with the deletion of the N-glycosyl and the extension region to analysis of the dispersion properties of the subunits,the main results of the study are as follows:（1）The cloning and expression systems were constructed for the subunits that containing the extension region（α′-andα-subunits）and lacking N-glycosyl and extension region（α′c-,αc-andβ-subunits）,and the base sequencing results showed that the amino acid sequences encoded by the target genes in the expression vectors were consistent with the theoretical sequences.The expression conditions ofα′-,α′c-,α-andαc-subunits are as follows:when the OD value of the bacterial solution was 0.6,the IPTG was added and the recombinant subunits was induced at 37°C for 9 h.The expression leveal is 20%～28%.The expression condition ofβ-subunits is as follows:when the OD value of the bacterial solution was 0.6,the IPTG was added and theβ-subunits was induced at 37°C for 3 h～5 h.The expression leveal is 24%～25%.The subunits were purified by Ni2+affinity column with high purity（>90%）.（2）The dispersion properties of the subunits in the deionized water condition were inves-tigated,and the results showed that the denaturation temperature ofα′c-subunit（80.2°C）andαc-subunit（76.1°C）was lower thanα′-（83.8°C）andα-subunit（87.3°C）,respectively,but the enthalpy showed an opposite trend,indicating that the denaturation temperature of the sub-units without glycosyl and the extension region was significantly lower.The extension region increased the disordered structure and decreased the enthalpy ofα′-andα-subunits.The aver-age particle size ofβ-subunit was the largest（3192.33 nm）,and that ofα′-andα-subunit were smaller thanα′c-subunit andαc-subunit,respectively,indicating thatα′-andα-subunit formed uniform protein particles in the deionized water condition,and their molecular structures were flexible and not easy to form large aggregates.Theζpotential ofα′-andα′c-subunit were not significantly different,but the absolute potential ofα-subunit was significantly larger than that ofαc-subunit,indicating that the extension region increased the absolute potential ofα-subunit,resulting in the difference in solubility and surface hydrophobicity ofα′-andα-subunit.（3）The dispersion properties of the subunits in acidic,neutral and basic conditions with low ionic strength were investigated,and the results showed that the trend of the thermal sta-bility of subunits was consistent with their changes in the deionized water condition and did not change by the p H value of the solution system.The dispersion behavior of the subunits in acidic conditions is different from their behavior in neutral and basic conditions.In acidic condition,the absolute potential of the subunits with the extension region decreased,the sur-face hydrophobicity increased,and the apparent viscosity increased after heat treatment（100°C,30 min）,but the solubility of the deletion of the extension region subunits did not change.In neutral and alkaline conditions,the turbidity of the subunits containing extension region decreased and solubility increased.The results of thermal stability,mean particle size,sur-face hydrophobicity,turbidity and solubility of the subunits showed that the subunits without extension region were more sensitive to p H value.（4）The dispersion properties of the subunits that deleted glycosyl and extensible were investigated in high ionic strength with acidic,neutral and alkaline conditions.The results showed that the thermal stability followed the same trend in deionized water and low ionic strength conditions,but the denaturation temperature of the subunits was higher in the high ionic strength condition than in the first two conditions.The structure ofα′-subunit that con-taining extension region is more sensitive to the p H change of the system.The average parti-cle size ofα′-andα′c-andα-andαc-subunits no significant difference due to the electrostatic shielding effect of high salt concentration,which resulting in greater differences in the molecu-lar structure and dispersion properties ofα′-andα-subunits.In neutral and alkaline conditions,the absolute potential ofα′-subunits was greater than that ofα′c-subunits,and there was no sig-nificant difference betweenα-andαc-subunits.The turbidity ofα′-andα-subunits was greater than that ofα′c-andαc-subunits in acidic conditions,respectively,with an opposite trend in neutral and basic conditions.The solubility ofα-subunits was significantly greater than that ofα′c-subunits,but the solubility ofα′-subunits did not change significantly in acidic and alkaline conditions.α′-subunit apparent viscosities did not differ in acidic and neutral conditions and decreased in alkaline conditions.α-subunit apparent viiscosity were lower thanαc-subunits and were not altered by p H.In alkaline condition,the apparent viscosity ofα’-andα-subunits decreased at low shear rates,and once the shear rate increased,the interaction between subunits is disrupted and the difference disappeared,which does not change of heating.In summary,the extension region increases the surface potential,decreases surface hy-drophobicity and increases solubility of subunits in deionized water and neutral and alkaline conditions with low ionic strength,so that the particles ofα′-andα-subunits are more uniform in the deionized water condition.Meanwhile,the effect extension region on the subunit par-ticle size and surface hydrophobicity were weakened due to electrostatic shielding inα′-andα-subunit.The absence of the extension region increased the tendency of the subunits to form aggregates and to be more sensitive to p H,as the subunits lose the protection of hydrophilic groups and acidic amino acids.The dispersion properties ofα′-andα-subunits differ under specific conditions.The difference in amino acid composition of theα′-andα-subunit ex-tension regions,the different response of extension region to the p H,and the intermolecular forces of the protein contribute to this phenomenon. |
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