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。     

豆浆中胰蛋白酶抑制剂活性测定方法的研究

目的在GB/T 21498-2008《大豆制品中胰蛋白酶抑制剂活性的测定》方法的基础上,对影响豆浆中胰蛋白酶抑制剂活性测定的一些关键因素进行研究和改进。方法豆浆样品加水提取并稀释至一定体积后,加入L-BAPA试剂和胰蛋白酶使用液,(37±0.25)℃水浴中保温10 min±5 s后,加入乙酸溶液终止酶反应,并采用分光光度计在410 nm波长下测定其吸光度值,代入公式计算胰蛋白酶抑制剂的活性。结果改进后的方法添加高、中、低3个浓度水平胰蛋白酶抑制剂的回收率分别为113.1%、114.6%、134.9%,平行测定的RSD分别为1.4%、3.6%、7.2%,方法的准确性和重复性能满足实验的需求。结论改进后的方法快速简便,节约了时间和实验成本,方法的准确性和重复性能满足实验的需求。     

加热处理对大豆乳清中胰蛋白酶抑制剂活性以及大豆乳清蛋白体外消化率的影响

采用胃蛋白酶-胰蛋白酶两步体外消化法测定了加热对大豆乳清蛋白的体外消化率的影响。与没有经过加热的对照蛋白相比,适当的加热处理可以提高蛋白的体外消化率,80℃,10min处理的体外消化率达到最高。加热对抑制剂的破坏是正相关,加热处理的程度越高,胰蛋白酶抑制剂活性降低的百分比就越大。1000℃,50min处理以及120℃,8min处理可以将其活性降低到90％以下,达到充分消除豆制品中抗营养因子的目的。     

Changes in lipoxygenase isozymes and trypsin inhibitor activity in soybean during germination at different temperatures

Influence of germination temperature on lipoxygenase isozymes and trypsin inhibitor activity, the two undesirable components in soybean for human consumption, is not yet reported in soybean sprouts. Two Indian soybean genotypes were incubated for 144 h in a seed germinator at two different temperatures (25 and 35 °C) and the activities of lipoxygenase isozymes and trypsin inhibitor were monitored in the germinating seedlings every 24 h. Lipoxygenase-I as well as lipoxygenase-II + III were degraded continuously over the 144 h and the rate of degradation, of both the classes of lipoxygenase, was faster at the higher germinating temperature (35 °C) in both the genotypes. Trypsin inhibitor was also degraded continuously during germination upto 144 h and the degradation was faster at higher germination temperature. Protein extracts of seedlings of different periods, developed at different temperatures, and analyzed using polyacrylamide gel electrophoresis, indicated that the original Kunitz inhibitor band (R_f = 0.75) declined continuously in intensity during germination at both temperatures in both genotypes, and a new band (R_f = 0.72) possessing trypsin inhibitor activity appeared at 48 h at 35 °C, while it appeared at 72 h at 25 °C. Early appearance of a modified form of Kunitz inhibitor, a degraded product of native form, at 35 °C as compared to 25 °C, confirms that the faster quantitative reduction at higher temperature is due to faster degradation of the original Kunitz inhibitor form at higher temperature.     

Alcalase蛋白酶降解大豆胰蛋白酶抑制剂的研究

研究了不同酶解条件下 (pH值、温度、时间、加酶量和添加巯基还原剂 ) ,碱性内切蛋白酶Alcalase对大豆蛋白和大豆胰蛋白酶抑制剂的降解作用。研究结果表明 ,Alcalase可同时降解大豆蛋白和胰蛋白酶抑制剂。该酶解反应的最适条件为 :pH 8 0、温度 6 0℃、最适加酶量 10 μL/g蛋白 (约 0 0 2 832AU/ g蛋白 ) ,添加Na2 SO3为ω(Na2 SO3) =0 3% ,水解时间 4h。在此条件下 ,残留胰蛋白酶抑制活性为对照的 2 0 % ,可溶性蛋白含量可达 2 7mg/mL ,游离氨基酸含量为 7 1mg/mL ,大豆蛋白的水解度为 8 9%。还讨论了Alcalase蛋白酶降解大豆蛋白生成小肽的最佳反应条件     

Trypsin inhibitor activity in vegetative tissue of sweet potato plants and its response to heat treatment

Trypsin inhibitor activity (TIA) and crude protein content of seven genotypes of sweet potato were investigated. There was considerable genotypic variation in TIA, with a four‐ to fivefold range in roots and a threefold range in stems. The mean TIA in stems at harvest time was 36% of that in roots, whilst the mean TIA in leaves was only 17% of that in roots. The TIA level in roots was correlated with that in stems (r = 0.83, p = 0.02) and leaves (r = 0.70, p = 0.08). In most genotypes the TIA level in vine‐tips was low during the early growth stage, increased from day 30 to day 110 after transplanting, then remained constant in subsequent growth stages. However, in genotype Guang 70‐9, vine‐tips had a high level of TIA at all growth stages. Sweet potato green tissue contained three‐ to fivefold more crude protein than roots. No correlation between TIA and crude protein in sweet potato roots was found across genotypes, but TIA was significantly correlated with crude protein content (r = 0.73, p = 0.06) in sweet potato vine‐tips. Moist heat treatment (MHT) was found to be effective in eliminating TIA in sweet potato. Most TIA in sweet potato green tissue and roots was eliminated by MHT at ≥80 °C, but heat stability was dependent on genotype. Guang 70‐9 had relatively highly heat‐stable trypsin inhibitor. The results suggested that screening for genotypes with high protein content and low TIA and use of an appropriate processing method could improve the utilisation of sweet potato for both human food and animal feed. © 2001 Society of Chemical Industry     

Proteins and trypsin inhibitor activity of guar (Cyamopsis tetragonoloba L. Taub) seed

Proteins soluble in tris-acetate buffer (pH9.0) were fractionated by DEAE-cellulose, DEAE-Sephadex A-50 and Sephadex G-200 column chromatography. The purified proteins which contained 5–6% carbohydrate, had molecular weights of 125 900 and 22 390 amu. The high molecular weight fraction was homogeneous by polyacrylamide gel electrophoresis. Proteins extracted in phosphate buffer (0.1M , pH7.6) when subjected to Sephadex G-200 gel chromatography were resolved into three fractions, all of which showed considerable trypsin inhibitor activity. Germination for 3 days reduced the trypsin inhibitor activity of the seed by about 30%.     

Antinutritional and/or toxic factors in soybean (Glycine max (L) Merril) seeds: comparison of different cultivars adapted to the southern region of Brazil

Soybean (Glycine max (L) Merril) seeds are known to contain different proteins displaying antinutritional and/or toxic effects, such as soybean agglutinin (an N‐acetylgalactosamine‐specific lectin), proteinase inhibitors (Kunitz‐ and Bowman–Birk‐type trypsin and chymotrypsin inhibitors) and urease (seed and tissue isoforms). Two other toxic proteins were previously isolated from soybeans, soyatoxin (21 kDa) and soybean toxin (18.4 kDa), which are immunologically related to canatoxin, a toxic protein from Canavalia ensiformis (jackbean) seeds. In this work we have screened crude extracts from seeds of six different soybean cultivars, which together represent most of the crop harvested in the southern region of Brazil, for the presence of urease, trypsin inhibitory and haemagglutination activities, intraperitoneal toxicity in mice and immunoreactivity against anti‐canatoxin antibodies. Significant differences were found in the contents of proteinase inhibitors, lectin, urease activity and lethality in mice. The relevance of these findings to the agronomic qualities and to the choice of soybean cultivars to be used as food or feed is discussed. Copyright © 2004 Society of Chemical Industry     

Effects of heating on protein quality of soybean flour devoid of Kunitz inhibitor and lectin

The effects of autoclaving on protein quality of soybean flours prepared from a conventional soybean (CSB) and an isoline lacking Kunitz inhibitor and lectin (KFLF) were studied. The heating was efficient in the urease, trypsin inhibitors and lectin inactivation, being 15 min sufficient to reduce more than 90% of these compounds and provide protein solubility over 80%. The results of PER, NPR and weight gain showed that heating equally improved the nutritional quality of both soybean flours, although higher autoclaving time was required for KFLF. No significant improvement was observed on NPU and in vivo digestibility of the diets containing KFLF at any heating time. As these later results were similar to those obtained with diets containing CSB, they show the importance of the heating to improve the nutritional value and show that other components rather than trypsin inhibitors and lectins impair the nutritive value of raw soybean.     

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。     

绿豆中胰蛋白酶抑制子的纯化及部分性质的研究

本文研究将绿豆经过粉碎、过筛和正己烷脱脂后,采用（NH4）2SO4分级沉淀、DEAE-Sepharose CL-6B离子交换、Sephacryl S-200凝胶过滤等方法,对其中的胰蛋白酶抑制因子进行了分离纯化。然后研究了所纯化出的胰蛋白酶抑制子的pH和温度稳定性,实验结果表明：在20～100℃的范围和pH 2.2～10.2范围内,胰蛋白酶抑制因子性质较稳定。     

低胰蛋白酶抑制剂活性保健豆乳的研制

采用碱性蛋白酶水解豆浆,可使其中的胰蛋白酶抑制剂活性降至生豆浆的91.9%水解时间采用6min。以水解豆浆为主要原料,添加白砂糖、姜汁、鲜桔汁和柠檬酸,制成低胰蛋白酶抑制剂活性的保健豆乳,其最佳配方为:白砂糖10%,姜汁1%,鲜桔汁8%,柠檬酸0.15%。      

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。     

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。     

Changes in inhibitory activity and secondary conformation of soybean trypsin inhibitors induced by tea polyphenol complexation

BACKGROUND: Tea polyphenol (TP) is a new food additive for antioxidant application, while soybean is an important resource for food and feed processing. It is therefore of rational and practical significance to investigate the influence of TP on soybean trypsin inhibitors (TIs). The aim of this study was to determine the effects of TP on the inhibitory activity of Kunitz (KTI) and Bowman–Birk (BBTI) TIs and to reveal the relationship between the inhibitory activity and conformation of KTI and BBTI by measurement of circular dichroism (CD) spectra. RESULTS: KTI and BBTI were found to be partially deactivated by TP. BBTI exhibited stronger resistance than KTI to TP deactivation. The unchanged K_M value of trypsin for benzoyl‐DL ‐arginine‐p‐nitroanilide hydrolysis indicated that KTI and BBTI inhibited trypsin in a non‐competitive pattern when complexed with TP. As the TP/TI ratio was increased and the inhibitory activity of KTI and BBTI decreased, the conformation of KTI and BBTI showed relevant changes and the major CD negative bands shifted progressively towards the near‐UV region. CONCLUSION: These results show the deactivation effects of TP on KTI and BBTI and reveal preliminarily the relationship between the inhibitory activity and secondary structure of KTI and BBTI. Copyright © 2009 Society of Chemical Industry