大豆Kunitz胰蛋白酶抑制因子单克隆抗体的制备与鉴定

为制备免疫学特性良好的大豆Kunitz胰蛋白酶抑制因子(Kunitz trypsin inhibitor,KTI)单克隆抗体,以50μg/只的免疫剂量将KTI免疫BALB/c小鼠,利用间接ELISA和间接竞争ELISA鉴定多抗血清效价和敏感性,选择血清效价和敏感性较优的小鼠进行细胞融合,多次筛选得到稳定分泌KTI单克隆抗体(Monoclonal antibody,mAb)的杂交瘤细胞株,采用体内诱生腹水法制备mAb并对其免疫学特性进行鉴定。结果表明,1号小鼠经免疫后效价最高且敏感性最好,半数抑制浓度(IC50)为157.33ng/mL,经细胞融合筛选得到4E6-E9阳性杂交瘤细胞株,所制备的抗体效价达到11 638 400,亚型为IgG1型,亲和常数为1.45×108 L/mol,IC50为28.39ng/mL,且特异性强。说明试验所制备的mAb免疫学特性良好,能够为建立灵敏、特异的KTI免疫学检测方法提供良好的抗体保障。     

荧光法检测鱼糜制品中大豆蛋白的研究

鱼糜制品中大豆蛋白的过量添加已成为水产食品生产的一个新问题,建立有效的检测方法尤为重要.本研究通过丙酮浸泡、酸处理、硫酸铵盐析和柱层析等步骤分离纯化大豆胰蛋白酶抑制剂(Soybean trypsin inhibitor,STI)并制备抗STI单克隆抗体(A11-6).经过Protein G Sepharose柱层析分离得到高纯度的免疫球蛋白(IgG).采用SPDP(N-琥珀酰亚氨基-3-2-吡啶)和DTT(二硫苏糖醇)组合处理,成功将R-藻红蛋白偶联到抗STI单克隆抗体.利用该荧光标记的抗体建立的免疫荧光法能够检测出鱼糜制品中的STI,检测限为1.56μg/mL,灵敏度略高于传统的化学发光法.该免疫荧光法具有操作时间短、灵敏度高和使用安全等优点.     

大豆乳清蛋白的胰蛋白酶改性研究

大豆乳清蛋白虽然具有一些较好的功能特性,但由于其主要组分胰蛋白酶抑制剂存在热稳定性差,抑制血清中胰蛋白酶的活性等缺点。采用相应的改性技术对超滤提取的大豆乳清蛋白进行了胰蛋白酶改性研究,确定的改性条件为:底物浓度2%,酶用量3500 U/100 g蛋白,水解时间3h,水解温度60℃。改性后的大豆乳清蛋白起泡性和乳化稳定性、NSI值、相对抗氧化能力均得到提高,分别提高108.33%、6.29%、0.96%、23.15%。     

一种胰蛋白酶亲和材料的制备及应用

基于抑制剂与酶的特异亲和作用,将具有良好稳定性的荞麦胰蛋白酶抑制剂(Buckwheat trypsin inhibitor,BTI)固定化,制备成一种胰蛋白酶的亲和吸附剂,实现胰蛋白酶的高效纯化。通过原核表达、Ni2+-NTA亲和层析和Superdex G-25凝胶过滤技术得到电泳纯的BTI,以溴化氰活化琼脂糖凝胶(CNBr-Sepharose CL-4B)作为亲和层析载体,制备亲和吸附剂BTI-Sepharose,检测其对胰蛋白酶的特异性吸附作用,并进一步研究BTI-Sepharose对胰蛋白酶吸附及解吸附的条件。制备的BTI-Sepharose对胰蛋白酶具有特异吸附性,可用于胰蛋白酶的亲和纯化。缓冲液p H对BTI-Sepharose与胰蛋白酶的吸附及解吸附均有重要影响,二者吸附作用的最适pH为7.2,在pH为3.5时两者能够完全分离,且不影响胰蛋白酶的生物活性。试验制备的BTI-Sepharose可实现一步层析制备高纯度胰蛋白酶,为胰蛋白酶的高效纯化及新型胰蛋白酶的研究开发提供理论依据。     

大豆胰蛋白酶抑制剂的异源表达与生化特性解析

本文对一疑似大豆胰蛋白酶抑制剂(STI;sti)的开放阅读框在毕赤酵母中进行了克隆表达与生化特性分析。结果表明,在摇瓶水平上,重组菌GS115(pPIC-STI)分泌表达了30 mg/L STI;重组STI在(40~80)℃或pH 2.0~11.0孵育1 h后,仍能保持85%以上的抑制活性;K+、Zn2+和Mg2+对其胰酶抑制活性有明显的激活作用,而Cu2+、Mn2+、Ca2+、Fe2+和Fe3+则有明显的抑制作用;重组STI对胰蛋白酶具有较强的、专一性的和非竞争性的抑制作用,是一种典型的多肽类胰酶抑制剂。良好的酶学性质使STI在食品、医药等行业具有潜在的应用价值。     

基于Exo III信号放大的荧光适配体传感器检测Kunitz型大豆胰蛋白酶抑制因子

Kunitz型大豆胰蛋白酶抑制因子（Kunitz Soybean Trypsin Inhibitor，KTI）是一种很关键的抗营养因子，不仅对动物消化系统和生长发育有不良影响，还制约各个行业对大豆的利用率，因此快速有效的检测方法是非常必要的。该研究建立一种基于核酸外切酶III（Exonuclease III，Exo III）和碳纳米颗粒（Carbon Nanoparticles，CNPs）的信号辅助放大荧光传感体系用于KTI的检测。具体体系包括KTI适配体（Aptamer，APT）、互补链（Complementary DNA，cDNA）、信号探针（Signal Probe，SP）、Exo III和CNPs共5个部分。通过可行性分析和CNPs浓度优化试验，测得KTI在100~600 ng/mL范围内呈线性相关，检测限为12.59 ng/mL。以豆浆作为样品，采用加标回收测得回收率为97.42%~102.85%，RSD在0.61%~2.36%之间，该方法可对实际样品中的KTI进行测定。     

枯草杆菌蛋白酶对大豆胰蛋白酶抑制剂的水解钝化研究

研究了枯草杆菌蛋白酶(Subtilisin)对大豆胰蛋白酶抑制剂(STI)酶解活性的影响。实验在50℃,pH7.5条件下反应1h,并以BAPNA为底物,采用改进的方法测定枯草杆菌蛋白酶对大豆胰蛋白酶抑制剂的水解钝化程度,用SDS-PAGE方法和分子排阻色谱法研究其蛋白酶钝化敏感性。结果证明,枯草杆菌蛋白酶可以水解大部分的抑制剂,并通过SDS-PAGE进一步证实了比色法得出的结论。而由分子排阻色谱图可以分析得出抑制剂经枯草杆菌蛋白酶酶解后,活性降低。分析认为,可能是由于抑制剂中二硫键被打断使得其结构发生改变,同时生成大量复杂的小分子多肽物质。因此,可以推断大豆胰蛋白酶抑制剂的稳定性与二硫键的存在有关。     

重组荞麦胰蛋白酶抑制剂理化性质的研究

荞麦胰蛋白酶抑制剂(BuckwheatTrypsinInhibitor,BTI)是存在于荞麦种子中的一种抗营养因子。本文经过原核表达及两步层析纯化得到高纯度的重组荞麦胰蛋白酶抑制剂rBTI。通过分析表明,重组荞麦胰蛋白酶抑制剂与天然荞麦胰蛋白酶抑制剂的性质类似。rBTI的相对分子质量约为11kDa,等电点约为6.2。rBTI对胰蛋白酶有较强的抑制作用,抑制摩尔比(以BApNA为底物)为1:1.5。rBTI的最适pH值为8.0,在pH3.0～8.0之间有较好的热稳定性,100℃加热120min仍保持70%的胰蛋白酶抑制活性。     

固定化酶法分离纯化大豆胰蛋白酶抑制剂

大豆分离蛋白生产中的大豆乳清废水中含有多种生理活性成分,其中大豆胰蛋白酶抑制剂可作为癌症和糖尿病治疗的药物。利用固定化胰蛋白酶分离纯化大豆胰蛋白酶抑制剂可得到高纯度的大豆胰蛋白酶抑制剂。研究结果表明:大豆胰蛋白酶抑制剂通过各阶段纯化后其活性从0.95TIU/mL增高至325.5TIU/mL,纯化程度提高324.6倍,得率0.033%;电泳结果表明:利用固定化胰蛋白酶分离纯化的大豆胰蛋白酶抑制剂有单一谱带,纯化效果明显。     

罗非鱼肠道胰蛋白酶和猪胰蛋白酶性质对比研究

系统对比了罗非鱼肠道胰蛋白酶与猪胰蛋白酶的酶学特性,以揭示两种不同来源胰蛋白酶的性质异同,为水产胰蛋白酶的开发利用提供理论依据。研究发现,罗非鱼肠道胰蛋白酶和猪胰蛋白酶的分子量分别为28kDa和24kDa;以Na-苯甲酰-DL-精氨酸对硝基苯胺盐酸盐(BAPNA)为底物时,两种胰蛋白酶的最适pH分别为9.5、10.0;最适温度分别为40、50℃;罗非鱼肠道胰蛋白酶在pH4～11比较稳定,猪胰蛋白酶在pH2～6较稳定;两种胰蛋白酶的温度稳定性相似,均在55℃以下稳定;Na+、Cu2+、Ba2+对罗非鱼肠道胰蛋白酶的抑制作用强于猪胰蛋白酶,Mg2+、K+、Ca2+对罗非鱼肠道胰蛋白酶有抑制作用,而对猪胰蛋白酶有一定的激活作用。两种胰蛋白酶均被大豆胰蛋白酶抑制剂(SBTI)、甲苯磺酰氟(PMSF)强烈抑制,其中PMSF对罗非鱼肠道胰蛋白酶的抑制作用强于猪胰蛋白酶,乙二胺四乙酸(EDTA)对两种胰蛋白酶也有一定的抑制作用,但EDTA对猪胰蛋白酶的抑制作用强于罗非鱼肠道胰蛋白酶,而碘乙酸及2-巯基乙醇对两种胰蛋白酶抑制作用不强;动力学研究表明两种胰蛋白酶的Km分别为0.591mmol/L和0.9mmol/L。上述研究说明,水产胰蛋白酶和哺乳动物胰蛋白酶的酶学特性存在较明显的差异。     

蛋白含量对牦牛乳清蛋白浓缩物功能特性的影响

通过不同截留分子质量的再生纤维素膜过滤纯化牦牛原乳清液和牦牛甜乳清液,分别制取牦牛原乳清蛋白浓缩物（native whey protein concentrate,NWPC）和牦牛甜乳清蛋白浓缩物（sweet whey protein concentrate,SWPC）,研究蛋白含量不同的乳清蛋白浓缩物（whey protein concentrate,WPC）主要成分（乳糖含量、pH值和总蛋白质含量）和功能特性（溶解性、持水性、持油性、起泡性、乳化性及热稳定性）的特征。结果表明：10 000 Da再生纤维素膜透析得到的牦牛WPC中总蛋白含量达到80%以上,不含乳糖,功能特性（溶解性、持水性、持油性、起泡性、乳化性及热稳定性）均显著高于经3 500 Da卷式膜、5 000 Da再生纤维素膜透析得牦牛WPC,WPC蛋白含量越高,其功能特性越好;不同蛋白含量的牦牛SWPC起泡能力、泡沫稳定性、乳化活性和乳化稳定性均显著（P＜0.05）高于牦牛NWPC。牦牛乳WPC最不稳定温度为85 ℃,高于荷斯坦牛乳WPC的80 ℃,热处理会适当改善牦牛WPC的起泡性能、乳化性能和热稳定性。通过膜牦牛处理获取的高蛋白含量的WPC,功能特性较好,应用广泛,对解决牦牛乳清资源的利用问题、保护环境、提高企业的经济效益起到关键性作用。     

复合酶法改善大豆分离蛋白起泡性的工艺优化

运用配料试验设计解决了多酶复配的比例优化问题,采用米曲蛋白酶、木瓜蛋白酶、胰蛋白酶对大豆分离蛋白进行水解,建立复合酶配合比例与起泡性之间的数学模型。确定最佳比例为:米曲蛋白酶5.68%、胰蛋白酶39.03%、木瓜蛋白酶55.29%;最佳水解条件为:[S]=8%、[E]/[S]=2%、pH=7.0、T=40℃、t=2h。     

Preparation of trypsin‐immobilised chitosan beads and their application to the purification of soybean trypsin inhibitor

BACKGROUND: Trypsin inhibitors are among the most important antinutritional factors in legumes. Recent research has shown that soybean trypsin inhibitor (SBTI) exhibits multiple bioactivities, but very few studies on the purification of SBTI are available. Enzymes are commonly used as biospecific ligands in affinity purification of their substrates or inhibitors. The aim of the present study was to prepare trypsin (EC 3.4.21.4)‐immobilised chitosan beads and use them to purify trypsin inhibitor from soybean whey. RESULTS: Compared with free trypsin, the immobilised trypsin had higher thermal and pH stability. The adsorption ratio of SBTI from crude SBTI aqueous solution by trypsin‐immobilised chitosan beads was 33.3%. The purified SBTI obtained by affinity chromatography was characterised by sodium dodecyl sulfate polyacrylamide gel electrophoresis as a single polypeptide band with an M_r of 8.3 kDa belonging to the Bowman–Birk family. CONCLUSION: Trypsin‐immobilised chitosan beads were effectively used in the affinity separation of trypsin inhibitor from soybean seeds, thus indicating that immobilised trypsin may have practical application in the soybean‐processing industry. The results of this study provide a background for further investigation of potential applications of soybean bioactive constituents in the areas of agriculture and food. Copyright © 2008 Society of Chemical Industry     

A comparison study of the impact of boiling and high pressure steaming on the stability of soybean trypsin inhibitor

The aim of the study is to compare the effect of boiling and high pressure steaming (HPS) on the degradation, inhibitory activity reduction and gastric digestibility of soybean trypsin inhibitor (STI). Thermal stability analysis showed that HPS treatment was effective in eliminating the inhibitory activity of STI than boiling. SDS‐PAGE and Western blot analysis indicated that boiling has less impact on the gastric digestibility of STI than HPS. More importantly, boiling‐pretreated STI revealed high inhibitory activity against trypsin even after digestion by pepsin in simulated gastric fluid (SGF), while HPS treatment was more effective. SDS‐PAGE analysis further verified that after boiling, STI still revealed strong binding ability to trypsin, while STI could be completely degraded by trypsin after HPS treatment for 30 min.     

Immunoassays for Bowman-Birk and Kunitz Soybean Trypsin Inhibitors in Infant Formula

ABSTRACT: Because the consumption of soybean inhibitors of digestive enzymes in processed foods may have both beneficial and adverse health-related effects, reliable and rapid analytical methods for these inhibitors are needed. Monoclonal antibody-based sandwich enzyme-linked immunosorbent assays (ELISAs) were developed for the 2 major soybean protease inhibitors, the Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) of trypsin and chymotrypsin. The ELISAs had limits of quantification of approximately 1 and 3 ng/mL for BBI and KTI, respectively, and were used to measure active inhibitors in soy infant formulas. Results were compared with enzymatic analyses and demonstrated that most of the trypsin- and chymotrypsin-inhibitory activities of infant formula were due to constituents other than KTI and BBI. The sandwich ELISA for BBI was also effective in detecting soybean germplasm with atypically low levels of BBI.     

COMPARATIVE STUDY OF STRUCTURAL CHARACTERISTICS AND THERMAL BEHAVIOR OF WHEY AND ISOLATE SOYBEAN PROTEINS

Electrophoretic profiles, sulfhydryl(SH)/disulfide (SS) groups content, and surface hydrophobicity (H₀) values of whey soybean proteins (WSP) and native soy isolates (NSI) were determined. WSP, composed mainly by Kunitz trypsin inhibitor (KTI), and lectin (L), has a H₀ value of 24.0 ± 1.0, which is 6.8 times lower than that of NSI ones, and SH/SS groups content in the same range of NSI. The thermal behavior of WSP and NSI was studied by differential scanning calorimetry (DSC). The WSP thermogram in water, similar to NSI, showed two main peaks (Tp values: 74.0 ± 0.5 C and 90.4 ± 0.8C) attributed to thermal denaturation of KTI and L, respectively. These endotherms are slightly affected by μ, whereas those of NSI are strongly affected (Tp of 7S and 11S peaks increase 17C and 20C respectively, by increasing NaCl concentration from 0 to 1M). WSP has low Ho values not noticeably affected by ionic strength changes, whereas NSI has higher Ho, that increase in saline media and favor intermolecular hydrophobic interactions. The consequent low tendency to protein aggregation by hydrophobic interactions in WSP would explain their lower thermal stability at high μ. Control proteins of both preparations (7S and 11S enriched fractions for NSI, and purified trypsin inhibitors, urease and lectin for WSP) were use to confirm these results.     

Purification and Quantification of Kunitz Trypsin Inhibitor in Soybean Using Two-Dimensional Liquid Chromatography

Kunitz trypsin inhibitor (KTI) is one of the major antinutritional factors in soybean and results in inhibition of digestion of dietary protein. In this study, we developed a novel strategy to purify and quantify KTI from soybean using two-dimensional liquid chromatography. Lipids from ground soybean were removed using hexane after which the ground soybean was extracted with protein extraction buffer. The crude extract was first purified by weak anion exchange chromatography, and then the fraction containing KTI was further separated by size exclusion chromatography. The fraction containing KTI was collected and analyzed by SDS-PAGE and electrospray ionization mass spectrometry. Results indicated that purified KTI has a molecular mass of 20 kDa and a purity of ~98% with inhibitory activity of 2425 TIU/mg protein. This assay was used for the quantification of KTI in soybean samples. The assay showed concentrations with a range between 7.81 and 500.00 μg/mL and a limit of detection of 0.12 mg/g. The recoveries of KTI in spiked soybean samples were between 82.19% and 86.65%, and intra- and interday precisions (% CV) were less than 7.35% and 8.42%. The developed method was used to analyze soybean samples from different sources and soybean products derived from different processing techniques, which demonstrated that the developed procedure provided an accurate and sensitive tool for separation and quantification of intact KTI in soybean.     

Kunitz trypsin inhibitor in addition to Bowman-Birk inhibitor influence stability of lunasin against pepsin-pancreatin hydrolysis

Soybean contains several biologically active components and one of this belongs to the bioactive peptide group. The objectives of this study were to produce different lunasin-enriched preparations (LEP) and determine the effect of Bowman-Birk inhibitor (BBI) and Kunitz trypsin inhibitor (KTI) concentrations on the stability of lunasin against pepsin-pancreatin hydrolysis (PPH). In addition, the effect of KTI mutation on lunasin stability against PPH was determined. LEP were produced by calcium and pH precipitation methods of 30% aqueous ethanol extract from defatted soybean flour. LEP, lunasin-enriched commercially available products and KTI control and mutant flours underwent PPH and samples were taken after pepsin and pepsin-pancreatin hydrolysis. The concentrations of BBI, KTI, and lunasin all decreased after hydrolysis, but they had varying results. BBI concentration ranged from 167.5 to 655.8 μg/g pre-hydrolysis and 171.5 to 250.1 μg/g after hydrolysis. KTI concentrations ranged from 0.3 to 122.3 μg/g pre-hydrolysis and 9.0 to 18.7 μg/g after hydrolysis. Lunasin concentrations ranged from 8.5 to 71.0 μg/g pre-hydrolysis and 4.0 to 13.2 μg/g after hydrolysis. In all products tested, lunasin concentration after PPH significantly correlated with BBI and KTI concentrations. Mutation in two KTI isoforms led to a lower concentration of lunasin after PPH. This is the first report on the potential role of KTI in lunasin stability against PPH and must be considered in designing lunasin-enriched products that could potentially survive digestion after oral ingestion.     

Proteose Peptones and Physical Factors Affect Foaming Properties of Whey Protein Isolate

Effects of peptides and nonprotein components of whey on whey protein isolate (WPI) were studied using a differential pressure method. Decay of WPI foam followed biphasic first-order kinetics, but was affected by solution conditions. WPI foam stability exhibited two pH optima (5.0 and 8.5). Addition of 0.02–0.15M NaCl progressively decreased foaming capacity and foam stability. Addition of 0.01–0.2% proteose-peptones caused a sharp decrease in foam stability, but did not affect WPI foaming capacity. Foam stability was increased by addition of up to 20% lactose. Removal of proteose-peptones should greatly improve foaming properties of whey proteins.