β-伴大豆球蛋白天然活性亚基的分离纯化

本研究将离子交换层析和金属螯合亲和层析方法相结合,建立了系统的分离纯化β-伴大豆球蛋白天然状态亚基的方法,通过梯度脲透析复性,使纯化亚基恢复天然构象。利用β-伴大豆球蛋白免疫动物制备抗血清对纯化亚基的免疫活性进行检测。结果表明：离子交换层析结合金属螯合亲和层析方法可以有效地纯化β-伴大豆球蛋白的α、α＇和β亚基,该方法产率高,制备的亚基纯度好,而且能与抗β伴大豆球蛋白血清特异性结合,表明提纯的亚基具有免疫活性。     

大豆球蛋白碱性亚基的分离纯化、特性及应用研究进展

大豆蛋白是一种含有低纤维和平衡氨基酸的优质植物蛋白来源,营养价值高,应用范围广。大豆球蛋白碱性亚基是大豆蛋白11S组分中的碱性多肽链。大豆球蛋白碱性亚基具有独特的溶解性、低抗原性和优异的乳化性、耐高温、易吸收及抗菌特性,将其添加到食品中既能发挥其营养功能又能提升食品的品质,延长储存期,具有广阔的开发前景。本文介绍了大豆球蛋白碱性亚基的等电点沉淀法及层析法等分离纯化方法研究及其功能特性、抗菌特性的研究进展,并指出了大豆球蛋白碱性亚基未来的研究方向及可应用的发展前景,为其进一步的深入研究和综合开发利用提供一定的参考。     

β-伴大豆球蛋白α-亚基核心区的基因克隆、表达及纯化

利用RT-PCR技术获得了齐黄34大豆种子的全长cDNA,采用自行设计的引物对目的基因αc进行扩增,并将目的基因与载体连接,构建了重组克隆载体pGEM-α_c。重组克隆载体经XhoⅠ/EcoRⅠ双限制酶酶切鉴定后,与载体pET-28a连接构建重组质粒pET-28a-α_c,经菌落PCR、双限制酶酶切及测序判定准确后,将其转入到感受态细胞E.coli BL21(DE3)中,当诱导温度为37℃、菌液OD_(600nm)值为0.8、诱导剂(IPTG)浓度为0.2mmol/L,诱导时间为9h时,重组α-亚基核心区蛋白分子质量为50kU;工程菌pET-28a-α_c-BL21经超声破裂、低温离心后,上清液利用镍离子亲和层析色谱柱经AKTA蛋白纯化系统纯化可以获得较高纯度的目的蛋白。     

大豆中β-伴球蛋白的分离与纯化

从大豆中分离制备β—伴球蛋白，对通过电泳进行分析鉴定。采用分级盐析(70％～90％)和凝胶过滤(sepharose 6B)分离纯化大豆中的β—伴球蛋白。免疫电泳的结果证实所提取的β—伴球蛋白纯度很高，大豆中的其它成分与β—伴球蛋白抗血清无交叉免疫反应。SDS—PAGE的结果表明本实验所提取的β—伴球蛋白是由不同形式单体组成的复合物，共有6种亚基。     

大豆球蛋白和β-伴大豆球蛋白的富集分离研究

以低温脱脂豆粕为原料,采用优化Nagano法分离大豆球蛋白和β-伴大豆球蛋白。本文系统考察提取过程中多个单因素对分离大豆球蛋白和β-伴大豆球蛋白的蛋白质含量和提取率的影响。并根据单因素试验结果设计响应面试验,对浸提温度、浸提pH、沉淀剂用量进行优化,分别确定高提取率大豆球蛋白和β-伴大豆球蛋白的分离条件。试验结果表明:大豆球蛋白的最佳提取条件为浸提温度44.4℃,浸提pH 8.5,CaCl220 mmol/L,提取率64.14%,纯度83.6%;β-伴大豆球蛋白的最佳提取条件为浸提温度49.5℃,浸提pH 8.6,CaCl_2 0.00 mmol/L,提取率40.15%,纯度82.9%。     

分离β-伴大豆球蛋白和大豆球蛋白工艺研究

采用大容量(250×4mL)低速(5300×g)离心机分离β-伴大豆球蛋白和大豆球蛋白,进行碱溶条件、酸沉条件等条件的研究。结果表明:提取蛋白的碱性溶液(pH8.5的Tris-HCl溶液)离心效果较佳,沉淀大豆球蛋白pH6.4为佳,沉淀β-伴大豆球蛋白pH4.8为佳,用pH5.0沉淀杂蛋白离心分离效果较好;每一个待离心的浊液,在离心之前4℃冷藏2h以上,对离心效果的提高十分有效;采用最佳条件,从300g脱脂豆片分离大豆球蛋白和β-伴大豆球蛋白,收率分别为6.8%和2.2%,用SDS-PAGE电泳实验方法测定大豆球蛋白和β-伴大豆球蛋白样品纯度分别为89.1%和78.9%。因此该低速离心法较适合数十克级别的β-伴大豆球蛋白和大豆球蛋白的分离。     

大豆伴球蛋白组成亚基热稳定性及其相关影响因素分析

为了明确大豆伴球蛋白良好热稳定性的根源,以大豆伴球蛋白及其组成亚基为研究对象,从组分的角度分析探讨了表面疏水性和表面电荷对热稳定性的影响,探求热稳定性与表面性质之间的关系。实验结果表明,在低离子强度下,三种亚基都具有良好的热稳定性,粒度分布显示,β亚基生成的聚集体最大,达到100nm;在高离子强度下,三种亚基的粒度分布皆呈增大趋势,β亚基增幅最大,致使部分β亚基絮凝沉淀;大豆伴球蛋白亚基组分的热稳定性与表面疏水性及Zeta-电位有紧密的关系,通过直接控制三者的热聚集行为,从而影响体系的热稳定性。     

大豆伴球蛋白组成亚基热稳定性及其相关影响因素分析

为了明确大豆伴球蛋白良好热稳定性的根源,以大豆伴球蛋白及其组成亚基为研究对象,从组分的角度分析探讨了表面疏水性和表面电荷对热稳定性的影响,探求热稳定性与表面性质之间的关系。实验结果表明,在低离子强度下,三种亚基都具有良好的热稳定性,粒度分布显示,β亚基生成的聚集体最大,达到100nm;在高离子强度下,三种亚基的粒度分布皆呈增大趋势,β亚基增幅最大,致使部分β亚基絮凝沉淀;大豆伴球蛋白亚基组分的热稳定性与表面疏水性及Zeta-电位有紧密的关系,通过直接控制三者的热聚集行为,从而影响体系的热稳定性。      

β-伴大豆球蛋白中α、α′亚基对大豆分离蛋白薄膜微观结构和功能特性的影响

为了研究β-伴大豆球蛋白中不同亚基组成对大豆分离蛋白薄膜微观结构和功能特性的影响,采用东农47(Wild对照)、α亚基缺失型(α-lack)、α′亚基缺失型(α′-lack)以及α、α′双亚基缺失型[(α+α′)-lack]的大豆为原料提取大豆分离蛋白。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析4种大豆分离蛋白的亚基组成,采用溶液流延法制备不同亚基组成的大豆分离蛋白薄膜,探究不同亚基组成对薄膜流变行为、微观结构、机械强度、耐水性、屏障特性、热稳定性及光学性能的影响。结果表明,不同亚基组成对大豆分离蛋白膜的微观结构和功能特性均有不同影响,与α亚基缺失和α、α′双亚基缺失相比,α′亚基缺失对薄膜结构和功能特性影响最为明显。红外光谱分析结果显示,相较于Wild薄膜,α′亚基缺失加强了蛋白质分子间氢键的相互作用,α′-lack薄膜β折叠含量减少到27.84%,α螺旋和无规则卷曲含量增加到16.83%和13.84%。扫描电镜分析结果显示,α-lack和(α+α′)-lack薄膜纹理形貌较散乱疏松,而α′-lack薄膜无明显气泡且具有平整致密的网络结构。流变特性分析结果发现,α亚基缺失会降低成膜强度,而α′亚基缺失会明显提高膜液的G′,使α′-lack具有更好的潜在成膜能力。与其他3种薄膜相比,α′-lack薄膜具有最大的抗拉伸强度2.31MPa,最小的水分含量(9.54%)和水溶性(30.81%),最低的水蒸气透过系数[21.89g·mm/(d·m²·kPa)],表现出优异的热稳定性和可见光阻隔特性。α′亚基的缺失能显著改善大豆分离蛋白薄膜微观结构和功能特性,研究结果旨在为生产高成膜性大豆蛋白系列产品提供理论参考。     

大豆球蛋白的纤维聚集体行为及其稳定性研究

为了明确蛋白质的纤维聚集行为,本研究以大豆球蛋白(soy globulin,11S)为原料,从亚基层面对酸性条件下热诱导的11S纤维聚集过程进行跟踪,监测蛋白及其亚基的水解过程、结构变化及其稳定性。结果表明,11S的纤维化是一个多步骤的过程,包括多肽链的水解、自组装成淀粉样纤维聚集结构及逐渐生长成宏观可见的具有扭曲螺旋结构的纤维聚集体。与11S纤维化过程的单指数增长相比,酸性亚基的纤维化过程存在迟滞期。酸性亚基在纤维化聚集的初期主要贡献于纤维聚集的成核过程,碱性亚基的加入改变其纤维聚集进程。蛋白质的纤维化过程会增加11S在等电点处的溶解度,降低中性和酸性pH下的溶解度。此外,碱性环境(pH值10.0)会导致11S纤维聚集体全部溶解、宏观纤维长度变小、结构发生改变。以上研究结果旨在为合理利用蛋白纤维化聚集体作为新的功能性食品配料提供理论依据。     

Controlled enzymatic hydrolysis of glycinin: Susceptibility of acidic and basic subunits to proteolytic enzymes

Glycinin, the major storage protein of soyabeans was enzymatically modified using papain, alcalase and fungal protease. The degree of hydrolysis (DH) was monitored by using trinitrobenzene sulphonic acid reaction with liberated α-amino groups. The DH could be varied by varying the ratio of enzyme to substrate, time and temperature of hydrolysis. The measured K_m and V_max values of glycinin with different proteases suggested that the susceptibility for hydrolytic cleavage of glycinin followed the order fungal protease>alcalase>papain. Electrophoretic analysis of cleaved glycinin suggested that acidic subunits of glycinin were cleaved preferentially over basic subunits. The measured K_m and V_max with acidic and basic subunits with fungal protease correlated with cleavage susceptibility. The functional properties of glycinin could be tailored by controlling the DH and using appropriate protease. Modified glycinin had better functional characteristics compared to glycinin.     

Structure of soya glycinin and conglycinin gels

Gels of glycinin and conglycinin formed at various heating temperatures, in the absence and presence of 0.2M sodium chloride were characterised by transmission electron microscopy. In distilled water both proteins formed gels consisting of strands with a thickness of 10–15 nm. The strands of glycinin were very regular and cross sections of strands showed a hollow cylindrical structure. In the presence of sodium chloride, glycinin formed an aggregated gel structure at 85°C, but at 95°C an ordered strand structure was formed. Dissociation of the quaternary structure on heating and reassociation of subunits into regular strands were considered the most probable mechanisms for strand formation from glycinin. The aggregated structure at 85°C was interpreted as a transient state prior to dissociation. Conglycinin rich gels were less regular and more crosslinked than gels of glycinin. Also, the strands of conglycinin showed a complex mode of aggregation possibly in the form of double spirals. The addition of sodium chloride caused a denser and more aggregated structure at 75 and 85°C, but the effects were not as drastic as in the case of glycinin. Heating temperature had only minor effects on the gel structure in the range studied.     

The interaction of zinc(II) and phytic acid with soya bean glycinin

Binding of phytic acid to Zn(II) free glycinin was not observed in 0.5 M KCl at pH 6.2. The addition of varying quantities of phytic acid to a Zn(II)- glycinin system at pH 6.2 ([KCl]=0.5 M ) initially resulted in binding of phytate and increased binding of Zn(II) to glycinin probably as a phytate-Zn(II)-glycinin complex. Further addition of phytate resulted in precipitation of zinc and protein. Bovine serum albumin also showed increased affinity for Zn(II) owing to the presence of phytic acid. Phytic acid-Zn(II) precipitates have a capacity of removing glycinin from solution, presumably by surface adsorption. The presence of soluble Zn(II) inhibits this adsorption. Bovine serum albumin is removed from solution by phytic acid-Zn(II) precipitates only when soluble zinc is present.     

Tryptic hydrolysis of glycinin and its subunits

Glycinin, the major storage protein of soybeans, was modified by alkali (pH 12.0) and acid (pH 2.0) induced denaturation, and by unfolding in 6 M urea followed by cleavage of the disulphide (SS) bonds and blockage of the sulphydryl (SH) groups with iodoacetoamide to produce carboxyamidemethyl (CAM)-glycinin. The CAM-acidic and CAM-basic subunits of the protein were also isolated. All the above samples were used to investigate their rate (pH-stat method) and extent (gel filtration analysis) of hydrolysis by trypsin at pH 8.0, 25°C. Native and acid-denatured glycinin and the CAM-basic subunits were hydrolysed slowly over a period of several hours whereas the CAM-acidic subunits, alkali-denatured glycinin, and CAM-glycinin were attacked at a very fast rate by the enzyme. The molecular weight distribution of the peptide fragments varied in all samples. Characteristic peaks of molecular weight above 30 000, around 7000 and below 5000 daltons were observed. In modifications where unfolding of the protein and SS reduction was implicated the peptide fragments were shifted toward the lowest peak(s) i.e. below 5000 daltons.     

The effects of ultra-high-pressure treatments combined with heat treatments on the antigenicity and structure of soy glycinin

Glycinin is one of the important allergens found in soybeans, which can potentially cause severe allergic reactions. Therefore, reducing the antigenicity of glycinin is of major significance to the research of soybean allergies. In order to detect the relationships between the antigenicity and structure of glycinin, samples were extracted from defatted soybean and then processed by ultra-high-pressure combined heat treatments. The processed proteins were determined using SDS-PAGE, enzyme-linked immunosorbent assay (ELISA), immunoblotting, exogenous fluorescence, free sulfhydryl groups and Fourier transform methods. The results revealed that the antigenicity of the processed soy glycinin had decreased. In addition, the content levels of the hydrophobic groups, free sulfhydryl groups, α-helix, β-turn and random coils had increased, and the content of β-sheets had decreased. The results indicated that the reduction in the antigenicity of the glycinin was due to the processing treatments, which effectively destroyed the spatial structure of the glycinin.     

Effects of limited proteolysis and high-pressure homogenisation on structural and functional characteristics of glycinin

Glycinin was treated by limited proteolysis and/or high-pressure homogenisation in this work. The combined treatments significantly improved the solubility of glycinin, especially in acidic aqueous solution. Moreover, the modified glycinin had better emulsification capability and lower surface hydrophobicity. Electrophoresis analysis indicated more aggregates were formed after above treatments. But the laser scanning test showed the particle size of glycinin decreased. Compared with limited proteolysis, the combined treatments further increased the storage modulus and postponed the onset of glycinin gelation. The results of scanning electron microscopy showed that a fibrous curly structure was present in native glycinin. It was not found in modified glycinins. High-pressure homogenisation slightly shortened the onset of glycinin gelation, while limited proteolysis apparently postponed this behaviour. All the treatments formed weaker gels than native glycinin.     

Formation and dynamic interfacial adsorption of glycinin/chitosan soluble complex at acidic pH: Relationship to mixed emulsion stability

The formation and interfacial adsorption of glycinin/chitosan (CS) soluble complex were investigated at acidic pH. The stability of the mixed emulsion stabilized by the complex was also evaluated at pH 4.5. Turbidimetric analysis, isothermal titration calorimetry (ITC) and dynamic light scattering were used to characterize the dynamic formation of the complex. The results showed that soluble complexes were formed mainly at pHs between 4.0 and 6.0, depending on CS/Glycinin mixing ratio. At pH 4.5, soluble complex was formed and saturated at mixing ratio = 0.1, showing a maximum size distribution at 164.2 nm. We found that the glycinin/CS soluble complex showed improved interfacial adsorption than glycinin at pH 4.5. In detail, dynamic interfacial adsorption data showed the coefficient of diffusion (K_diff), unfolding (K₁) and rearrangement (K₂) for soluble complex (K_diff, K₁ and K₂: 0.58 mNm⁻¹ s^−0.5, 2.23 E−4 s⁻¹ and 5.78 E−4 s⁻¹) were higher than those of the glycinin (K_diff, K₁ and K₂: 0.32 mNm⁻¹ s^−0.5, 1.72 E−4 s⁻¹ and 4.63 E−4 s⁻¹). The droplet size and confocal observation of the mixed emulsion fabricated with glycinin/CS soluble complex displayed improved stability at mixing ratios of 0.1 to/and 0.2, suggesting the synergistic effect of the two molecules. We concluded that interfacial and emulsifying properties of glycinin could be improved by formation of glycinin/CS soluble complex at acidic pH.     

Molecular weight and amino acid composition of glycinin subunits

Glycinin, the major soyabean globulin, is composed of subunits having molecular weights of about 22,300, and 37,200. On the basis of amino acid analysis, the six submits of glycinin isolated by isoelectric focusing are all different. The ‘acidic’ subunits have higher content of glutamic acid and proline, whereas the ‘basic’ subunits are higher in the hydrophobic amino acids leucine, tyrosine, phenylalanine, valine and alanine.     

商品普鲁兰粗酶的分离纯化

对商品普鲁兰酶进行分离纯化。用已购得的商品普鲁兰酶,制成酶液,采用硫酸铵进行分级盐析,通过脱盐、离子交换层析和凝胶色谱,分离得到较纯的普鲁兰酶。通过SDS-PAGE电泳分析,获得了单一条带,表明所分离的酶已经达到电泳纯,该酶的分子量约为82kDa。     

Extracellular protease from Mucor pusillus: purification and characterization

Extracellular protease from Mucor pusillus was purified 18-fold with 7.56% recovery by ion-exchange chromatography and gel filtration. The enzyme was found to be monomeric in nature, having a molecular mass of 49 kDa. The enzyme acted optimally at 50°C and was stable in the temperature range 30–50°C. It was completely inactivated by heating for 30 min at 65°C. The optimum of activity for the purified extract was observed at milk CaCl₂ concentration of 0.02  m and at milk pH of 5. These properties, except for temperature, were similar to those of rennet.