The degradation of lectins,phaseolin and trypsin inhibitors during germination of white kidney beans,Phaseolus vulgaris L.

White kidney beans (Phaseolus vulgaris), cv Processor, contain a relatively high content of phaseolin (storage protein), lectins and a special group of glycoproteins as well as a considerable amount of protein-type trypsin inhibitors. Protein digestion of raw Processor beans in monogastrics, for example pigs, is disturbed by poorly digested, phaseolin lectins, which can bind to carbohydrates in brush border membranes of the small intestinal epithelium, and trypsin inhibitors. The effect of the germination of white kidney beans on lectins, phaseolin and trypsin inhibitors was studied in order to achieve a degradation of lectins, phaseolin and trypsin inhibitors and an increase ofin vitro enzymatic hydrolysis of the protein of bean flour. Therefore, whole bean extracts were examined throughout a germination period of up to seven days for their lectin and phaseolin pattern, lectin content, binding capacities of functional lectins towards brush border membranes and trypsin inhibitor content. In addition thein vitro enzymatic hydrolysis by pepsin and pancreatin of the protein from flours of (un)germinated white kidney beans was studied. SDS-PAGE demonstrated a degradation of E-lectins and a disappearance of L-lectins and phaseolin during germination. Results indicated a decrease of the lectin content by 85%, a loss of binding capacities of functional lectins towards brush border membranes by 91%, and a decrease of trypsin inhibitors by 76%, in bean flour after germination for seven days. A maximum inin vitro enzymatic hydrolysis of protein from bean flour was already established after germination for half a day.     

Effect of a Bangladeshi home-cooking procedure on the amino acid content,trypsin inhibitor activity and in vitro digestibility of some legume seeds

Raw and cooked samples of cultivars ofLens esculenta (Lentils),Pisum sativum (peas),Phaseolus vulgaris (beans),Phaseolus aureus (navy beans)Cicer arietnum (gram), andLathyrus sativus (dhal) as well as precooked commercial products were analysed for amino acids, trypsin inhibitor activity and in vitro protein digestibility. Of the fifteen samples used in the study one lentil sample, one pea sample, two gram samples and one sample of khesari dhal were from Bangladesh, one gram sample was from Sri Lanka. The other samples were obtained either in shops in Norway or from an industrial firm. The latter were also obtained precooked and dried. The two samples obtained in shops were used with hull and dehulled.Neither cooking by a Bangladeshi household procedure nor industrial precooking and drying had any effect on the amino acid contents of the samples.Cooking and precooking reduced the trypsin inhibitor activity of the raw samples more when the original activity was high then when it was low. The inhibitor activity was similar between samples after cooking.Cooking and precooking and drying increased the in vitro protein digestibility in all samples except in the lentils in which the digestibility was reduced.In the raw samples protein digestibility was negatively correlated with the trypsin inhibitor activity.     

Involvement of carboxypeptidase in the degradation of the mung bean (Vigna radiata) trypsin inhibitor during germination and early seedling growth

Examination of the substrate specifity of the carboxypeptidase activity of ungerminated and germinated mung beans (Vigna radiata (L.) Wilczek) reveals the presence of two distinct enzymes. The first of these, carboxypeptidase I, is maximally active against carbobenzyloxy-Ala-Phe. It is present in large amounts in the cotyledons of ungerminated seeds, and declines rapidly during germination. The second, carboxypeptidase II, is most active against carbobenzyloxy-Phe-Ala. This enzyme increases in the cotyledons during germination and seedling growth.The possible involvement of one or both of these enzymes in the degradation of the native mung bean trypsin inhibitor (MBTI - F) and its proteolytically modified forms (MBTI - E, - C, and - E') during germination was also examined. The enzyme catalyzing one step in the conversion of MBTI - E to MBTI - C has been isolated with 557-fold purification from germinated mung beans. The inhibitor-degrading enzyme has a molecular weight of approximately 60,000. It is optimally active against MBTI - E at between pH 4.0 to 4.5. No activity was found with MBTI - F, - E', or - C as substrates. The enzyme has been identified as the carboxypeptidase II on the basis of (1) coelution during chromatography, (2) action on synthetic and natural substrates, (3) sensitivity to enzyme inhibitors, and (4) temporal variation during germination. The MBTI degrading carboxypeptidase removes the two carboxyl-terminal residues from MBTI - E to produce an inhibitor species that co-migrates with MBTI - C on polyacrylamide gel electrophoresis, but has not been subjected to the internal cleavage and proteolysis at the amino-terminus found in MBTI - C.Supported by National Science Foundation Grants PCM 8003854 and PCM 8301202Abbreviations used are: TNBS, 2,4,6-trinitrobenzenesulfonic acid; Cbz-, carbobenzyloxy-; PMSF, phenylmethylsulfonyl fluoride; MBTI, mung bean trypsin inhibitor; SDS, sodium dodecylsulfate; PAGE, polyacrylamide gel electrophoresis.     

Genetic control of phaseolin protein expression in seeds of common bean,Phaseolus vulgaris L.

Phaseolin, the major globulin seed storage protein of common bean,Phaseolus vulgaris L., accounts for up to 50% of the total seed protein. The rapid accumulation of phaseolin in the maturing seeds begins about 14 days after flowering and continues for some 12–14 days longer. However, the amount and rate of phaseolin accumulation, related to variation in onset, length, termination, and rate of synthesis, have been shown to vary between genotypes.Only three phaseolin electrophoretic types, designated T, S, and C after the cultivars Tendergreen, Sanilac, and Contender, respectively, have been identified among over 100 cultivated accessions. The narrow ranges of molecular weights and isoelectric points of the 14 protein polypeptides of phaseolin, as well as the homology observed from peptide mapping, suggest that the phaseolin polypeptides are similar proteins. Based on the results of crosses among cultivars having the three electrophoretic patterns, the genes controlling the polypeptides of each of the phaseolin types appear to be tightly linked, inherited in a block and the alleles are codominant.Substantial variation in phaseolin content, based on estimations using rocket immunoelectrophoresis, has been found among bean lines. Although most segregating populations show continuous distributions and quantitative inheritance, some inbred backcross lines having enhanced phaseolin accumulation appear to carry a few genes with major effects. A single gene that reduces the amount of phaseolin to less than one-half of the normal levels has been identified recently in an accession of wildP. vulgaris.     

Screening soybean (grain and vegetable) genotypes for nutrients and anti-nutritional factors

Fifty six genotypes of grain-type soybean and 17 genotypes of vegetable-type soybean collections were analyzed for protein and oil content, trypsin inhibitor, and lipoxygenase activities. The protein and oil content ranged from 36.9 to 47.9% and from 13.3 to 23.0% for different accessions in grain- and vegetable-type soybeans, respectively. Trypsin inhibitor and lipoxygenase activities ranged from 22.0 to 47.0 trypsin inhibitor units/mg meal and from 482 to 6265 lipoxygenase units/min/mg meal for grain- and vegetable-type soybeans, respectively. Significant correlations (r=–0.62 and –0.52,P<0.05) were found between protein and oil, and between protein and trypsin inhibitor. A significant positive correlation (r=0.42,P<0.05) was also calculated for oil and lipoxygenase activity. Several genotypes of soybean and vegetable soybean (plant introductions 423905, 417330, 417223, 171451, 200506, 200523, 417124, 227687, 203402, 445842, 203399, 423852, 416771, FC31927, Avoyelles, and Sooty) showed good nutritional potential and may be useful in a breeding program to improve the nutritional quality of soybean. Screening for essential amino acids, fatty acids, and trace minerals for selected genotypes is underway.     

Chemistry of legume protease inhibitors and their use in taxonomy

Protease inhibiting proteins, especially those that bind to trypsin and chymotrypsin, are widely distributed withinLeguminosae. Data on the primary structure and reactive sites known for some of them allow a classification into inhibitor families; inhibitors with the same topological molecular structures are grouped together. Evaluation of amino acid sequences based on amino acid replacements has been shown to be useful for understanding taxonomic and phylogenetic relations at the molecular level. The application of this method to theLeguminosae is demonstrated here with the example of the Bowman—Birk inhibitor family.A rapid method of obtaining inhibitor patterns from seed extracts is the use of disc electrophoresis followed by specific staining of the inhibitor bands. The types of inhibitor patterns, in combination with trypsin and chymotrypsin inhibitor activities and the quotients of these two activities, can be used for taxonomic studies. Results obtained with this method were first proven to correlate with the results of other chemotaxonomic techniques within the genusAcacia. In the case of Australian acacias, this technique was used to demonstrate closer relationships between uninervedRacemosae andBotryocephalae (Bipinnatae) and also betweenPlurinerves andPulchellae (Bipinnatae) than between the twoBipinnatae and betweenPlurinerves andJuliflorae. This technique has also been used to excludeAcacia mitchellii from the seriesPulchellae.Held at the 75th Annual Meeting of the AOCS in Dallas, TX, April 29–May 3, 1984.     

Effects of heat treatments on trypsin inhibitor and hemagglutinating activities in winged bean

The effect of different heat treatments on inactivation of trypsin inhibitor and hemagglutinin of winged bean was investigated. Trypsin inhibitor extracted from winged bean meal was stable at 60 °C for 60 min. At 80 °C, the activity of the extracted inhibitor decreased by 25% within 5 min, and continued to decline gradually to a loss of 45% of activity after 30 min. When the extracted inhibitor was incubated at 100 °C, it exhibited a triphasic pattern of inactivation. The winged bean extract incubated at 60 °C lost 60% of its hemaggluinating activity within 30 min. At 80 °C, there was a complete loss of activity within 5 min. The microwave treatment to winged bean meal had no effect on trypsin inhibitor or hemagglutinating activities in the meal. However, infrared treatment to winged bean seeds for 60 seconds was effective in destroying most of the trypsin inhibitor and hemagglutinating activities. Autoclave treatment (120 °C at 15 lb pressure) for 10 min inactivated trypsin inhibitor and hemagglutinin in winged bean meal almost completely. Cooking of presoaked beans in boiling water for 30 min was effective in destroying most of the trypsin inhibitor and hemagglutinating activities.     

Characterization of Phaseolus vulgaris L. Landraces Cultivated in Central Italy

Eight Phaseolus vulgaris L. landraces cultivated on farm in marginal areas of Central Italy (Lazio region) were investigated in order to evaluate chemical composition of storage proteins and secondary metabolites fractions. The total protein content showed some differences among landraces; the maximum value was next to 30 g for 100 g of dry weight. The seed storage proteins were screened by polyacrylamide gel electrophoresis (SDS/PAGE): seven landraces exhibited phaseolin patterns type S, one landrace showed a phaseolin pattern type T. A morphological analysis of cotyledon parenchyma performed by scanning electron microscopy (SEM) revealed differences in size of starch granules. Moreover the polyphenolic composition was investigated using HPLC-APCI; from the methanol extracts a flavonoid, kaempferol, and a coumarin, 5,7-dimethoxycoumarin, were identified. To our knowledge, this is the first time that 5,7-dimethoxycoumarin has been reported in P. vulgaris seeds.     

Gamma-rays and EMS induced pentaphyllous mutant in black gram (Vigna mungo)

Pentaphyllous mutants in black gram were isolated in M₂ generation of a segregating family, irradiated at 20kR. The genetic nature of mutants was tested by hybridizing with controls, and chi-square tests applied to the F₂ population, proved it to be a monogenic recessive. The pentaphyllous mutant had a greater number of pods and leaves per plant and larger and more root nodules. It also showed improved nutritional value with increased seed protein percentage and no increase in TIA (trypsin inhibitor activity).     

豆制品中胰蛋白酶抑制剂活性测定方法研究

利用紫外分光光度计，对豆制品中胰蛋白酶抑制剂在抑制牛胰蛋白酶活性反应前后的吸光值差异制作差值曲线，从而得出斜率与抑制剂活性之间关系的方法。并通过反复的实验对比，最终可以快速准确地测定大豆制品中胰蛋白酶抑制剂活性。     

Specific role of endopeptidases in modulating the nature of protein digestion products

The impact of various endopeptidases on the nature of protein digestion products was measured with the digestion cell technique. After a 30 min pepsin pre-digestion, casein and rapeseed concentrates were hydrolyzed with various amounts of pancreatin, trypsin and/or chymotrypsin. This hydrolysis was performed in a dialysis tube (molecular weight cut-off 1000) with continuous collection of the digested material. The addition of pure trypsin or chymotrypsin to pancreatin (Enzyme:Substrate 125) did not change the digestibility of casein. Only a higher pancreatin level (Enzyme: Substrate 112.5) increased the total protein digestibility without affecting the amino acid spectra. Rapeseed digestibility was markedly increased by the addition of pure trypsin to pancreatin. Lysine and arginine, target amino acids of trypsin, were favored at the expense of chymotrypsin and elastase target amino acids. Supplementation of pancreatin with, chymotrypsin enhanced rapeseed digestibility without affecting the relative amino acid digestibility. The impact of a higher pancreatin ratio (Enzyme: Substrate 112.5) was similar to that of enriched pancreatin but the rate of amino acid release was modified. The differences between protein sources were mainly attributed to protein structure.     

大豆胰蛋白酶抑制因子的研究进展

大豆胰蛋白酶抑制因子是大豆中的主要抗营养因子,是一种多肽类或蛋白质。该文对大豆胰蛋白酶抑制因子的种类,Kunitz型胰蛋白酶抑制因子和Bowman-Birk型胰蛋白酶抑制因子的结构和特性,大豆胰蛋白酶抑制因子的功能及提取纯化方法进行了综述,旨在为大豆胰蛋白酶抑制因子的进一步开发和利用提供参考。     

Differential cytotoxicity of the isolated protein fraction of Escumite bean (Phaseolus acutifolius)

The Escumite bean (Phaseolus acutifolius) is an edible legume which in raw form is highly toxic to rats. Proteins were separated by DEAE cellulose and affinity chromatography. Hemagglutinating activity, trypsin inhibitory effect, cytotoxicity on human peripheral lymphocytes and on intestinal epithelial cells of rats, and mitogenic activity were assayed with each protein fraction. Hemagglutinins and trypsin inhibitory fractions showed differential toxicity with lymphocytes as compared to intestinal epithelial cells. A protein fraction without the previous activities was cytotoxic mainly to intestinal epithelial cells.     

Nutrient composition and anti-nutritional factors in selected vegetable soybean (Glycine Max [L.] Merr.)

The genetic variation in the nutrient composition and anti-nutritional factors of 17 vegetable soybean genotypes were determined and a wide variation in protein %, total phosphorus (TP_i) and available phosphorus (AP) was found among these genotypes. Variations in Ca, K, Fe, Mn, and Cu were also documented. Variation was also found for trypsin inhibitor (TI) activity and Phytate (PA) content. A highly significant and negative correlation (r=–0.533,P<0.01) was observed between TI and total protein. Strong positive correlation (r=0.90) was also found between TP_i and AP. Several genotypes (Sooty, Emperor, Wilson-5, PI 416771, PI 417322) showed good nutritional potential and can be used in the breeding program. High protein %, TP_i, and minerals are desirable qualities for vegetable-type soybeans that make it as food with high nutrient density. Studies on the nutritional evaluation of immature vegetable type soybean seeds at different reproductive stages are also underway.Agricultural Research Station Journal Article Series No. 172. The use of any trade name varieties, and/or vendors does not imply the exclusion of other products or vendors that may also be suitable.     

Trypsin inhibitor in moth bean: thermal stability and changes during germination and cooking

Trypsin inhibitor from moth bean was studied for thermal stability and changes during germination and cooking. The application of dry heat did not inactivate the inhibitor. However, autoclaving at 120°C at 15lbs pressure destroyed inhibitor activity completely. The extracted inhibitor lost 70% activity in 60 min when incubated at 100°C. Soaking of moth bean seeds for 8 h decreased trypsin inhibitor activity by 20%. The germination of seeds for 24 h resulted in 70% reduction in inhibitor activity. No activity was detected in 48 h germinated seeds. Germination (for 24 h) followed by cooking of moth bean seeds destroyed the trypsin inhibitor completely.     

Studies on α-amylase and trypsin inhibitors in legume seeds using agar diffusion and isoelectric focusing techniques

Amylolytic and tryptic inhibitors of faba bean extracts were determined by an agar diffusion test. The amylolytic inhibitor had protein characters. Furthermore, water-soluble trypsin inhibitors ofCicer arietinum, Lens esculenta, Lupinus termis, Phaseolus vulgaris, Pisum sativum, Trigonella foenum-graecum andVicia faba which were separated by polyacrylamide gel isoelectric focusing (PAGIF) in thin-layers, showed species specific patterns. Negative staining showed 10 bands for French beans, 9 for fenugreek seeds, 8 for lentils and chickpeas, 7 for peas and 6 for faba beans. Lupin seeds were free from trypsin inhibitors. Treatments (soaking, germination and heat processing) of faba beans reduced the number of trypsin inhibitors in PAGIF patterns, less after soaking and germination, but more after roasting and frying. No inhibitors were detected after cooking.     

多年生野生大豆Kunitz型胰蛋白酶抑制剂基因的克隆及分析

利用RT-PCR方法,从对蚜虫高抗的多年生野生大豆短绒野大豆(G.tomentella)(2n=78)未成熟子叶中扩增到了大豆Kunitz型胰蛋白酶抑制剂同源基因单一目的片段,该基因和蛋白分别被命名为KTiPW54和KTiPW54。序列分析表明:该片段为654 bp的完整编码序列,编码218个氨基酸残基,编码的蛋白与栽培大豆(G.max)、野生大豆(G.soja)、短绒野大豆(G.tomentella)的Kunitz型胰蛋白酶抑制剂基因编码蛋白高度保守的区域一致。将KTiPW54编码区克隆到表达载体pTWIN1,在宿主菌BL21(DE3)中经IPTG诱导获得高效表达。     

Trypsin inhibitor activity of conventional foods which are part of the British diet and some soya products

The inhibition of bovine trypsin by ordinary Western foods was estimated and the daily intake of trypsin inhibitor activity (TIA) from the average British diet was calculated and shown to be 330 mg per person per day. Examination of some traditional Oriental soya foods showed that most of the TIA had been removed or inactivated during processing and the remainder was further reduced during cooking. The Japanese food, miso, was an exception and showed heat-stable inhibition of unknown biological significance, associated with the presence of free fatty acids rather than the specific soya bean trypsin inhibitors which are proteins.     

Kunitz Soybean Trypsin Inhibitor is Modified at its C-terminus by Novel Soybean Thiol Protease (Protease T1)

Abstract

Kunitz soybean trypsin inhibitor (KSTI) is hydrolyzed during seed germination to yield amino acids needed to support initial seedling growth. The type of KSTI from Glycine max (L.) Merrill cv. Toyokomachi is KSTI-Ti ^b. The KSTI-Ti ^b from 4-day-old post-germination cotyledons (KSTI-Ti ^b’) has 3 or 4 amino acid residues cleaved off at the C-terminus. This KSTI modification is important to understand the mechanism of degradation in seed reserve proteins by proteases. Protease K1 also cleaves amino acid residues at the C-terminus of KSTI but it removes 5 amino acid residues. Therefore, we presumed the KSTI-Ti ^b’ was produced by a protease other than protease K1. In this study, the protease T1 responsible for cleavage of KSTI-Ti ^b at the C-terminus was purified. The enzyme was estimated to have a molecular mass of 33 kDa from its mobility on SDS-PAGE gels. The N-terminal amino acid sequence of the purified protease T1 corresponded to amino acids Phe-73 to Phe-92 of both thiol protease isoforms A and B from the soybean leaf, and shared 83% identity with the partial amino acid sequence of the membrane-associated cysteine protease from mung bean seedlings, a protease known to perform post-translational cleavage of C-terminal peptides of target proteins. Finally, this enzyme was shown to convert KSTI-Ti ^b to KSTI-Ti ^b’.     

中国大豆种子蛋白中胰蛋白酶抑制剂等位基因的频率及分布

我国１．９万份大豆（栽、野）种质资源的胰蛋白酶抑制剂（ＳＢＴＩ）等位基因电泳分析结果指出，９８．２％的材料具有Ｔ型，１．３％的材料具有Ｔ型。在野生材料中发现有Ｔ类型。三种基因型出现的频率比例为１００：１．３：０．２。未发现不含胰蛋白酶抑制剂的ｔｉ型材料．栽培种Ｔｉ类型单一，野生种Ｔｉ类型变异较大。