Trypsin and chymotrypsin inhibitor activities in plant foods from Vietnam and Hungary

A method for the determination of chymotrypsin inhibitor activity and a screening test for evaluating higher activities of trypsin inhibitor were elaborated. The protease inhibitor activities of the most important plant foods from Vietnam and Hungary were determined. In cereals and legume seeds the activity of chymotrypsin inhibitors is generally lower than that of the trypsin inhibitors. In various soy products (isolates, texturates) the extent of lowering of chymotrypsin inhibitor activity was less than that of trypsin inhibitor related to raw soy bean. The cooking process according to the usual kitchen technique of Vietnam has more effect on the higher trypsin inhibitor level of soy bean than on other legumes of average activity.     

Trypsin inhibitors in leaf protein concentrate

A study has been made of the tryptic inhibitory activity of leaf protein concentrate (LPC) prepared from several crops. The highest level of inhibition was found in LPC extracted from lucerne (50%), compared with that from fescue, Italian ryegrass and quinoa, which contained about 20% tryptic inhibitory activity. No inhibitory activity was found in kale LPC when measured under the defined conditions. Aqueous extraction of LPC decreased inhibitory activity, but solvents (propan-2-ol and acetone) were ineffective. The inhibitory activity in lucerne LPC was reduced by autoclaving, but the inhibitors in fescue, Italian ryegrass and quinoa were heat stable. Phenolic compounds were implicated in the inhibitory activity of fescue and Italian ryegrass LPC. Kinetic studies of the inhibitory activity of standard LPC samples prepared from Italian ryegrass, fescue and quinoa indicated that, in each instance, there was mixed inhibition.     

Trypsin inhibitors in some lesser known seeds in Nigeria

Trypsin inhibitors (TIs) were extracted and determined spectrophotometrically from seed meals of African breadfruit (Treculia africana), African walnuts (Coula edulis Bail), Calabash (Lagenaria sicerania) and Castor (Ricinus communis). The TI activities were for Castor 1319 × 10⁻⁴, African breadfruit 620 × 10⁻⁴, and Calabash 28·4 × 10⁻⁴ and for African walnuts only a trace of TI units per milligram protein of the extract. The total protein content and in vitro protein digestibility for the four seed meals were also determined and observed to be quite high for Calabash seeds.     

Trypsin and chymotrypsin distribution between the fractions of the duodenal contents and the parietal mucosal deposits

An attempt has been made to assay the distribution of the typical representatives of pancreatic enzymes such as trypsin and chymotrypsin between dense and liquid fractions of the duodenal contents on an empty stomach and parietal mucous overlaps. The mass of the dense fraction being formed and specific activity of the enzymes under study are affected depending on the duodenal contents pH (4.5, 4.0, 3.5). Within the pH ranges indicated, specific activity of trypsin expressed in per cent of the total enzyme activity in the duodenal contents changed from 2-41 to 79-97%. It has been also shown that sorption of the enzymes is possible as well on the structures of the parietal mucous overlaps. A definite correlation has been demonstrated to exist between the activity of the enzymes under study in mucous overlaps and duodenal contents. Thus, the content of two endopeptidases of the pancreatic origin has been measured in the dense fraction of the duodenal contents on an empty stomach and mucous overlaps which are regarded as potential zones of heterophasic digestion in the intestinal cavity.     

Trypsin inhibitors from Pisum sativum L exhibit identical epitopes

Pea (Pisum sativum L) trypsin inhibitor, known to be a mixture of at least eight different peptides exhibiting different charges as shown by electrophoresis, was subjected to an immunochemical analysis. By PAGE-SDS analysis, only one large diffuse band was detected showing that pea trypsin inhibitor peptides have a molecular weight between 12000 and 15000 amu. After preparative non-denaturing electrophoresis, four major bands, as judged by Coomassie blue staining, were purified and each of them was used to raise specific antibodies in rabbits. By ELISA test, immunoelectrophoresis and absorption on an immunoaffinity column, it was shown that each antiserum directed against any one of the four bands completely cross-reacted against each other. Thus, it can be concluded that each component of the pea trypsin inhibitor should exhibit a very strong sequence homology.     

Isolation and properties of trypsin and chymotrypsin inhibitors from barley grits after storage

The occurrence of antiproteolytic activity in albumins of barley grits has been shown. As a result of storage lasting 6 months at a temperature of ?10 °C the freeze-dried albumin of barley grits lost its antiproteolytic activity and a proteolytic activity appeared. The antiproteolytic activity was partially regained after dissociation of the enzyme–inhibitor complex which had formed. From the reactivated freeze-dried albumins of stored barley grits two thermolabile protease inhibitors were isolated and partially purified. The first trypsin–chymotrypsin inhibitor B_7.1 inhibits, at a concentration of 2.5 μg, 1 μg of crystalline trypsin and 1.9 μg of this inhibitor inhibits 1 μg of crystalline alpha-chymotrypsin. Lower activity was demonstrated for the second inhibitor of chymotrypsin B_7.4; 4.8 μg of this inhibitor is needed to inhibit the activity of 1 μg of crystalline alpha-chymotrypsin. The occurrence of both inhibitors has been confirmed by means of starchgel electrophoresis. Their specific activity was small in comparison with that of some commercial crystalline preparations of inhibitors. The capacity to inhibit native proteases shows their important physiological function, based mainly on the control of proteolytic activity of endogenous enzymes. However it seems unlikely that they could have a negative influence on the nutritive value of proteins in barley grains or grits.     

Tolerance of growing pigs to trypsin and chymotrypsin inhibitors in chickpeas (Cicer arietinum) and pigeonpeas (Cajanus cajan)

The threshold level of growing pigs to trypsin and chymotrypsin inhibitors was investigated by adding graded levels of meals rich in these inhibitors to diets and recording responses. Diets were formulated to contain either 250, 500 or 750 g kg^?1 of Opal chickpea, dehulled Tyson chickpea or dehulled pigeonpea meals and pig response compared to that of pigs given a wheat and soya-bean meal control. Trypsin inhibitor levels (mg g^?1) of the diets were, respectively, control, 0.2; chickpea meal 1, 1.2.32; chickpea meal 2, 1.7–4.7; pigeonpea meal, 1.4–3.6. Chymotrypsin inhibitor levels (mg g^?1) of the diets were, respectively, control, 0.2; chickpea meal 1. 0.9–2.2; chickpea meal 2, 1.6–4.5; pigeonpea meal. 0.8–2.1. The diets were offered ad libitum over the 20–50 kg growth phase. Growth responses of the pigs fed the two chickpea meals were similar to those of the pigs fed the control soya-bean meal diet (P>0.05). In contrast, the addition of pigeonpea meal linearly depressed growth rate (P<0.001), feed intake (P<0.05) and increased the feed conversion ratio (P<0.05), inclusion levels of the chickpea meals had no effect on organ weights, whereas the inclusion of pigeonpea meal significantly affected the weights of the liver and pancreas (P<0.05), indicating the presence of other anti-nutritional factors. The results indicate that the growing pig can tolerate dietary levels of at least 4.7 and 4.5 mg g^?1 of trypsin and chymotrypsin inhibitors, respectively. These threshold levels are unlikely to be exceeded in conventional diets containing the majority of grain legumes. The results also indicate that dehulled pigeonpea meal contains an anti-nutritional factor(s) for growing pigs.     

Urea and Heat Unfolding of Cold-Adapted Atlantic Cod(Gadusmorhua)Trypsin and Bovine Trypsin

The reversible unfolding reactions for phenylmethylsulphonyl fluoride (PMSF)-modified trypins from Atlantic cod (cod PMS-trypsin) and cattle (bovine PMS-trypsin) were monitored by fluorescence spectrophotometry as a function of urea concentration and temperature. For urea unfolding at 25°C, the free energy change at zero concentration of urea (ΔG(H₂O)) for cod PMS-trypsin was 11(±4·4) kJ mol⁻¹ compared with 18(±1·14) kJ mol⁻¹ for bovine PMS-trypsin, while the mid-point concentration for urea unfolding curve ([urea]_1/2) was 3·0(±0·57) M and 4·1(±0·16) M, respectively. From studies of enzyme heat unfolding, the mid point temperature of the thermal unfolding curve ( T _m ) was 46(±1·4)°C for cod PMS-trypsin compared with 57(±2)°C for bovine PMS-trypsin. The standard free energy change (Δ G° ) for reversible thermal unfolding of cod PMS-trypsin was 9(±1) kJ mol⁻¹ compared with 19(±1) kJ mol⁻¹ for bovine PMS-trypsin. Values for the enthalpy (Δ H _m ), entropy (Δ S _m ) and heat capacity (Δ C _p ) for heat unfolding are compared. Results from urea and thermal unfolding studies show that cod PMS-trypsin has a significantly lower conformational stability than bovine PMS-trypsin.     

可溶性固定化胰蛋白酶的制备及性质研究

将胰蛋白酶偶联在亲水性高分子聚合物N-琥珀酰壳聚糖上,制备得到水溶性固定化胰蛋白酶,并对其部分酶学性质进行了研究。结果表明,可溶性固定化胰蛋白酶的最适pH为9·0,最适温度为60℃,Km值为0·28mg/mL;该酶在pH6·0以上能够完全溶解,在pH4·0左右基本不能溶解;可溶性固定化胰蛋白酶在60℃条件下,保温8h,仍有55%的活性;在4℃条件下,30d后活性保留达98%;在4℃下保存72h,活性没有明显降低。pH8·0~10·0稳定性最好(相对活性>90%)。     

豆浆豆腐中胰蛋白酶抑制因子活性的研究

豆制品中含有丰富的营养物质,但其中的抗营养因子阻碍人体对营养物质的吸收。分别采用6种不同制浆工艺将5种原料豆进行豆浆、豆腐的制备,测定大豆和豆浆中蛋白含量以及豆浆、豆腐的产量,通过分析不同大豆品种及制浆工艺与豆制品产量及蛋白含量之间的关系,以产量为指标,研究不同大豆品种及其加工工艺对豆制品产量及蛋白含量的影响,选取高产量、高蛋白的豆制品。并采用紫外分光光度计法分别对原料豆、豆浆、豆腐等进行大豆胰蛋白酶抑制因子活性的检测,得出低大豆胰蛋白酶抑制因子活性的品种及加工豆制品的制浆工艺。结果表明,豆制品产量与蛋白含量成正比,不同大豆品种采用不同制浆工艺所制得的豆浆、豆腐中胰蛋白酶抑制因子活性具有显著性差异,为不同大豆品种适用性加工及工业化生产提供理论依据。     

大豆胰蛋白酶抑制剂研究概况

该文介绍国内外研究大豆胰蛋白酶抑制剂概况,并对其失活和测定方法进行初步探讨。     

大豆胰蛋白酶抑制剂的制备及性质

采用硫酸钠盐析法从大豆乳清废水中选择性回收大豆胰蛋白酶抑制剂(soybean trypsin inhibitor,STI),且以商品化的Kunitz型胰蛋白酶抑制剂(soybean Kunitz trypsin inhibitor,KTI)为对照表征STI的理化性质和界面性质。结果表明,STI提取优化条件为:乳清溶液固形物含量13%、pH 4、加盐量9 g/100 m L,此条件下STI的得率为20.54%;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳图谱显示,其主要成分为KTI,以苯甲酰-DL-精氨酸-p-硝基酰替苯胺盐酸盐为底物的胰蛋白酶抑制活力为2 135.00 TIU/mg,且具有良好的温度和pH值稳定性(80℃加热30 min后仍保持73.19%的抑制活力,在pH 2~11范围内抑制活力无明显变化);傅里叶变换红外光谱和圆二色性结果显示,其与KTI(Sigma T9218)的结构类似,二级结构主要是β-折叠和无规卷曲;界面性质数据表明,STI分子能很快吸附到气水界面形成高弹性界面,从而使其具有良好的起泡性和泡沫稳定性。因此,简单的硫酸钠盐析法是大规模制备高纯度且功能性质良好的STI的有效方法,所获得的STI在医药及功能性食品领域有潜在的应用价值。     

Natural plant enzyme inhibitors. Isolation and characterisation of a trypsin/chymotrypsin inhibitor from indian red wood (Adenanthera pavonia) seeds

A trypsin/chymotrypsin inhibitor was isolated from Indian red wood seeds by extraction with 0.01m HCl, chromatography on diethyl amino ethyl-cellulose, ammonium sulphate fractionation and gel chromatography on Sephadex G-100. The homogeneity of the final product was ascertained by affinity chromatography on trypsin sepharose and chromatography on phenyl sepharose CL-4B columns. During all stages of purification and characterisation the ratio of activities against trypsin and chymotrypsin remained constant at about 1.1:1 indicating that the same factor is responsible for both activities. The size of the inhibitor was found to be 24 000 daltons based on gel chromatographic studies on Sephadex G-100 and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The molar ratio of interaction between the inhibitor and bovine trypsin for complete inactivation of the enzyme was found to be 1.04:1. Electrophoretic and gel chromatographic studies indicated that the purified inhibitor is capable of undergoing aggregation to form dimers and trimers. Even in the presence of sodium dodecyl sulphate and sodium dodecyl sulphateurea, this phenomenon was discernible. The binding sites on the inhibitor for trypsin and chymotrypsin were not mutually exclusive, based on the data from mixed enzyme studies and on analysis of the inhibitor-enzyme complexes by gel chromatography on Sephadex G-100. Modification of the arginyl residues of the inhibitor resulted in the loss of more of the antitryptic activity than of antichymotryptic activity. Conversely, modification of amino groups resulted in the loss of more of the antichymotryptic activity. The inhibitor was stable to exposure to a wide range of pH (1.0–12.0), but it was completely inactivated on heat-treatment at 100°C for 15 min. The mode of inhibition of trypsin as well as chymotrypsin was non-competitive and K_i values for the inhibitor were 2.92 × 10-¹⁰M and 4.46 × 10-¹⁰M , respectively, for the two enzymes.     

Presence of Nonprotein Trypsin Inhibitor in Soy and Winged Beans

The total trypsin inhibitor was determined in extracts of 11 cultivars of soybeans and 11 strains of winged beans. The proteins in the extracts were precipitated by 16% trichloroacetic acid (w/v), and nonprotein trypsin inhibitor activities were determined in the protein-free supernatants. The nonprotein trypsin inhibitor activities varied between 10.32 ± 1.39 and 24.41 ± 1.04 units/mg defatted soybean samples and in winged beans ranged between 2.65 ± 0.62 and 6.79 ± 0.27 units/mg defatted samples. The nonprotein trypsin inhibitor composed 27–55% of the total trypsin inhibitor activity in the soybeans, and 5–14% of the total activity in winged beans.     

Amino acid analyses of germinating seeds of some members of the leguminosae

The free and bound amino acid contents of soaked seeds and 7- and 14-day old seedlings of Pisum sativum, Phaseolus vulgaris and Canavalia eniformis were determined. It was found that on germination the free amino acid content of the plants increased, while the total amount of amino acid decreased. Most of the essential amino acids were shown to be present in the plants.     

紫甘薯花色素与胰蛋白酶相互作用特性

测定紫甘薯花色素与胰蛋白酶反应前后酶的催化活性、催化反应动力学并采用紫外光谱法、荧光光谱法和红外光谱法研究紫甘薯花色素与胰蛋白酶相互作用特性。结果表明：紫甘薯花色素对胰蛋白酶催化活性有明显的抑制作用,抑制类型为可逆的竞争性抑制,抑制常数Ki＝6.16×10－4 mmol/L,当紫甘薯花色素与胰蛋白酶的物质的量比为140∶1,在 37 ℃反应 15 min,抑制率达到38.61%,而反应时间对催化活性的影响不明显;紫甘薯花色素可使胰蛋白酶的内源荧光猝灭,猝灭类型为静态猝灭,室温下猝灭常数Kq为1.73×1012 L/（mol·s）,结合常数KA为3.88×104 L/mol,结合位点数n为0.86;热力学参数确定两者之间的作用力主要为氢键和范德华力;根据Förster能量转移理论得出它们的结合距离为3.56 nm;红外光谱经过去卷积、二阶导数处理得知与紫甘薯花色素作用后胰蛋白酶的α-螺旋含量降低,β-折叠含量升高。     

磁性亲合分离法纯化胰蛋白酶

采用壳聚糖包埋纳米钡铁氧体,经环氧氯丙烷交联制得磁性亲和载体,应用激光粒度分析仪和振动样品磁性测定仪对磁性亲和载体进行表征,动态激光散射分析结果表明,磁性粒子的平均直径为401纳米.磁性测量表明,超细磁粉具有较高的磁性.然后利用碳二酰亚胺活化法,将抑肽酶与此微粒共价结合得到磁性亲和吸附剂,应用于胰蛋白酶的亲和纯化.论文详细讨论胰蛋白酶亲和纯化条件,将磁性亲合分离法成功地应用于亲和纯化胰蛋白酶.