Chymotrypsin Inhibitory Activity Research Articles

Effects of extrusion and traditional processing methods on antinutrients and in vitro digestibility of protein and starch in faba and kidney beans

Comparative effects of extrusion cooking and conventional processing methods on protein content and reduction of antinutritional factor (phytic acid, condensed tannins, polyphenols, trypsin, chymotrypsin, α-amylase inhibitors and haemagglutinating activity) levels in Vicia faba and Phaseolus vulgaris seeds were studied. In vitro protein and starch digestibilities were assessed. P. vulgaris seeds showed highest levels of condensed tannins, chymotrypsin and α-amylase inhibitory activities and haemagglutinating activity. Dehulling significantly increased protein content and greatly reduced condensed tannin and polyphenol levels in both legumes. Extrusion was the best method to abolish trypsin, chymotrypsin, α-amylase inhibitors and haemagglutinating activity without modifying protein content. Furthermore, this thermal treatment was most effective in improving protein and starch digestibilities when compared with dehulling, soaking and germination.

Conformation of the Primary Binding Loop Folded through an Intramolecular Interaction Contributes to the Strong Chymotrypsin Inhibitory Activity of the Chymotrypsin Inhibitor from Erythrina variegata Seeds

We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (Ki, 5.6 x 10(-7) M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the "scaffold." A chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (Ki, 1.3 x 10(-6) M) than ECI (Ki, 9.8 x 10(-8) M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (Ki, 5.7 x 10(-7) M) than ETIa (Ki, 1.3 x 10(-6) M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (kon) and a decrease in the dissociation rate constant (koff) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant.

Nutritive value and effect of blanching on the trypsin and chymotrypsin inhibitor activities of selected leafy vegetables.

Proximate composition, energy, mineral and vitamin contents and the effect of blanching methods and times on the trypsin and chymotrypsin inhibitor activities were studied using cabbage, collard, turnip, peanut, and sweet potato leaves. Results of this study indicated that, crude protein, crude fat, carbohydrate and ash contents were in the range of 15.5-25.6%, 1.4-6.5%, 60.4-73.1% and 6.8-7.5%, respectively. Total dietary fiber was lowest in cabbage (28.2 g/100 g) and highest in the collard leaves (43.1%) while energy content per 100 g of vegetables was highest in sweet potato leaves (402 kcal) and lowest in cabbage (379 kcal). The mineral content per 100 g of vegetables were in the range of 33.4-249.8 mg, 241.2-471.2 mg, 12.1-75.1 mg, 14.9-98.9 mg, 0.5-3.5 mg and 0.9-3.1 mg for Ca, K, Na, Mg, Fe and Zn, respectively. For ascorbic acid, riboflavin, thiamin and total carotenoids, concentrations in 100 g of vegetables were in the range of 45.1-112.7 mg, 0.2-0.3 mg, 0.3-0.8 mg and 2.0-7.3 mg, respectively. The trypsin inhibitory activity per gram of the vegetables was highest in collard (60.1 TIU/g) and lowest in peanut leaves (41.0 TIU/g). Chymotrypsin inhibitor activity was highest in the peanut (69.6 CIU/g) but lowest in the collard leaves (48.0 CIU/g). Both trypsin and chymotrypsin inhibitor activities were significantly (p < 0.05) reduced by most of the treatments in either the conventional or microwave blanching methods. In the conventional blanching method, trypsin inhibitor activity was reduced by 0.5, 6.8, 11.9, 9.0 and 19.3 percent in cabbage, collard, turnip, sweet potato and peanut leaves, respectively, when the vegetables were blanched for 2.5 minutes but after blanching for 10 minutes, the trypsin inhibitor activity was reduced by 29.7, 34.9, 54.3, 52.3 and 65.6 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively. For the microwave oven blanching, trypsin inhibitor activity was reduced by 3.8, 3.3, 32.7, 5.0 and 9.5 percent in cabbage, collard, turnip, sweet potato and peanut leaves, respectively when the vegetables were blanched for 30 seconds. When blanched for 60 seconds, trypsin inhibitor activity was reduced by 16.2, 45.8, 46.2, 51.0 and 42.4 percent in cabbage, collard, turnip, sweet potato and peanut greens, respectively. Similar trends in the reduction of chymotrypsin inhibitor activity were observed when the vegetables were conventionally blanched for 2.5, 5 and 10 minutes and when blanched by microwave oven for 30, 45 and 60 seconds. Based on the results of this study, the vegetables were good dietary sources of minerals, vitamins, carbohydrate and proteins. Also, blanching was an effective method for reducing the trypsin and chymotripsin inhibitor activities in the leafy vegetables, however, further investigation on the heating times for both conventional and microwave blanching methods is suggested.

Effects of extrusion and conventional processing methods on protein and antinutritional factor contents in pea seeds

The effects of high temperature short time (HTST) treatment compared with other conventional processes on protein, phytic acid, condensed tannins, polyphenols, trypsin, chymotrypsin and α-amylase inhibitor activities and haemagglutinating activities in Renata, Solara and Ballet pea seeds were investigated. Ballet cultivar showed highest protein, phytic acid, tannin, polyphenol contents and trypsin and chymotrypsin inhibitory activities. All pea cultivars contained trypsin- and chymotrypsin-inhibiting activity and lectins but only Solara had α-amylase inhibitory activity. Under extrusion conditions (148°C, 25% moisture and 100 rpm) this thermal processing method was the most effective in condensed tannin, trypsin, chymotrypsin, α-amylase inhibitors and haemagglutinating activity reduction, without modifying protein content as occurs by dehulling, soaking and germination treatments. Trypsin and chymotrypsin inhibitors and haemagglutinating activities in peas were more readily abolished by extrusion treatment than was chymotrypsin inhibitory activity.

EFFECT OF PROCESSING ON AMYLASE AND PROTEASE INHIBITOR ACTIVITY IN TROPICAL TUBERS

The effect of various processing methods on amylase, trypsin and chymo-trypsin inhibitory activities in tubers of Colocasia esculenta (taro), Xanthosoma sagittifolium (tannia), Solenostemon rotundifolius (Coleus, Chinese potato), Dioscorea esculenta (lesser yam) and Amorphophallus paeoniifolius (elephant foot yam) was studied. Among the different methods, frying was the most effective method of eliminating enzyme inhibitors from tubers. When cooked by boiling, partial retention (30 to 60%) of enzyme inhibitory activity was observed in certain tubers. Further reduction in inhibitory activity was observed in baked/pressure cooked tubers. The study indicated that processing decreased the inhibitor content significantly and the reduction of inhibitory activity was brought about by heat inactivation.

Protease inhibitors and lectins in soya beans and effects of aqueous heat-treatment

Protease inhibitor and lectin concentrations and N and lipid levels were evaluated in 27 soya bean samples. All contained trypsin and chymotrypsin inhibiting activity and lectin. Shinano midori, Shinano wasediro, Shinano-oomame and Tsuronoku had high protein contents and average lipid levels, and their lectin and trypsin and chymotrypsin inhibitory activities were not unduly high. These lines, in particular, merit further nutritional evaluation. Food intake, feed conversion efficiency and growth by rats was reduced when soya bean was included in their diet, but the extent to which these parameters were affected did not appear to depend only on the protease inhibitor or lectin content. In contrast, pancreatic and small intestinal growth did seem to reflect the levels of these factors in the diet. Chymotrypsin inhibitor activity in soya bean was more readily abolished by heat-treatment than was trypsin inhibitory activity. Lectin activity was also relatively heat-resistant. However, all of these activities could be abolished by aqueous heat-treatment of fully imbibed seeds at 100°C for 10 min. The effects of a soya diet on pancreas and small intestine weights were also abolished by this pre-treatment, and food intake and nutrient utilisation by rats was greatly improved. © 1998 Society of Chemical Industry

Protease inhibitors and lectins in soya beans and effects of aqueous heat‐treatment

Protease inhibitor and lectin concentrations and N and lipid levels were evaluated in 27 soya bean samples. All contained trypsin and chymotrypsin inhibiting activity and lectin. Shinano midori, Shinano wasediro, Shinano-oomame and Tsuronoku had high protein contents and average lipid levels, and their lectin and trypsin and chymotrypsin inhibitory activities were not unduly high. These lines, in particular, merit further nutritional evaluation. Food intake, feed conversion efficiency and growth by rats was reduced when soya bean was included in their diet, but the extent to which these parameters were affected did not appear to depend only on the protease inhibitor or lectin content. In contrast, pancreatic and small intestinal growth did seem to reflect the levels of these factors in the diet. Chymotrypsin inhibitor activity in soya bean was more readily abolished by heat-treatment than was trypsin inhibitory activity. Lectin activity was also relatively heat-resistant. However, all of these activities could be abolished by aqueous heat-treatment of fully imbibed seeds at 100°C for 10 min. The effects of a soya diet on pancreas and small intestine weights were also abolished by this pre-treatment, and food intake and nutrient utilisation by rats was greatly improved. © 1998 Society of Chemical Industry

Irradiation Inactivation of Some Antinutritional Factors in Plant Seeds

Effects of γ-irradiation (1.0−10 kGy) on trypsin, chymotrypsin, and α-amylase inhibitors of soybean and Moringa peregrina seeds on tannin of sorghum, gossypol of cottonseed, and in vitro digestibility of soybean were investigated. A dose of 10.0 kGy caused decreases in trypsin (by 34.9%) and chymotrypsin (by 71.4%) inhibitor activities in soybean defatted flour, whereas its in vitro digestibility increased from 79.8 to 84.2%. The α-amylase inhibitor activity of Al-Yassar (M. peregrina) was decreased by 43.6 and 47.8% upon treatment of 7.0 and 10.0 kGy, respectively. Doses of 10.0 and 7.0 kGy significantly reduced the tannin content in Shahlla sorghum but not in Hemaira sorghum. Total and free gossypol contents were slightly reduced by irradiation. Keywords: Irradiation; proteinase inhibitors; tannin; gossypol; Moringa peregrina; soybean; sorghum; cottonseed

A succinylanthranilic acid ester and other bioactive constituents of Jolyna laminarioides

Methyl 2-[propanamide-2′-methoxycarbonyl]benzoate, fucosterol, trans-phytol and p-formylphenol were isolated for the first time from a methanolic extract of Jolyna laminarioides. Methyl 2-[propanamide-2′-methoxycarbonyl]-benzoate exhibited chymotrypsin inhibitory activity and also found to be active against Escherichia coli and Shigella boydii. Fucosterol exhibited antifungal activity against Curvularia lunata, Stachybotrys atra and Microsporum canis.

A succinylanthranilic acid ester and other bioactive constituents of Jolyna laminarioides

Methyl 2-[propanamide-2′-methoxycarbonyl]benzoate, fucosterol, trans-phytol and p-formylphenol were isolated for the first time from a methanolic extract of Jolyna laminarioides. Methyl 2-[propanamide-2′-methoxycarbonyl]-benzoate exhibited chymotrypsin inhibitory activity and also found to be active against Escherichia coli and Shigella boydii. Fucosterol exhibited antifungal activity against Curvularia lunata, Stachybotrys atra and Microsporum canis.

Protease inhibitors and in vitro digestibility of karanja (Pongamia glabra) oil seed residue. A comparative study of various treatments

AbstractTrypsin and chymotrypsin inhibitor activities (TIA and CIA) were assayed before and after processing of Karanja oil seed residue. Upon treatment, the loss of TIA and CIA varied with the type of processing or extraction: the reduction after solvent extraction ranged between 34 and 15%, respectively. These activities were completely removed in 2.4% HCl; 83.35 and 54.86% were removed on autoclaving; 33.86 and 15.30% on fermentation; and removal was 4.36–83.69% and 3.66–77.59% after exposure to different doses of gamma radiation (1, 5, 10, and 50 KGy). An in vitro digestibility study was carried out to confirm the inactivation of the inhibitors and showed improvements from 45 to 81%, with the maximum observed in 2.4% HCl solvent‐refluxed residue. A linear relationship was observed between the reduction in protease inhibitor activities and the improvement in digestibility.

Two Reactive Site Locations and Structure−Function Study of the Arrowhead Proteinase Inhibitors, A and B, Using Mutagenesis

The arrowhead (Sagittaria sagittifolia, Linn.) proteinase inhibitor A and B are double-headed and multifunctional, consisting of 179 amino acid residues with three disulfide bridges. Both their primary structures and cDNA sequences have been elucidated [Yang, H. L., Luo, R. S., Wang, L. X., Zhu, D. X., & Chi, C. W. (1992) J. Biochem. 111, 537; Xu, W. F., Tao, W. K., Gong, Z. Z., & Chi, C. W. (1993) J. Biochem. 113, 153; Luo, M. J., Lu, W. Y., & Chi, C. W. (1997) J. Biochem. (in press)]. Though they share 91% homology, they are different in inhibitory activities. Sequence analysis of their full-length cDNAs showed that there are seven extra residues in the C-terminal part which might be cleaved off by proteinase post-processing. To locate the reactive sites and study the structure-function relationship of the two forms A and B, the genes coding for the mature inhibitor B and its extended form were respectively cloned into the secretion expression vector, pVT102U/alpha, and expressed in Saccharomyces cerevisiae strain S-78. Both of the gene products were purified and characterized to have the same inhibitory activities as the natural one. The gene product of the extended form was a mixture with the extended C-terminal part of the inhibitor either completely or partially removed. The two previously predicted reactive site residues, Lys-44 and Arg-76 of inhibitor B, were then respectively substituted with Ala by site-directed mutagenesis and expressed. As compared with the natural inhibitor, each of the mutants could only inhibit one molecule, instead of two molecules of trypsin, and displayed an inhibitory activity against elastase, thus confirming the location of the two reactive sites in the inhibitors. The gene coding for inhibitor A, which for some reason could not be expressed in S. cereviciae, was successfully expressed in the reconstructed plasmid pET-1522bx in Escherichia coli strain BL21 with the expressed product existing in the inclusion body. After denaturation and renaturation, the active inhibitor A was obtained and purified by anhydrotrypsin affinity chromatography. Using site-directed mutagenesis, two residues of inhibitor A, namely, Ser-82 and Leu-87, prominently different from Leu-82 and Arg-87 in inhibitor B, were replaced by these two corresponding residues, respectively. As compared with the natural inhibitor A, its S82L mutant showed a lower inhibitory activity toward trypsin, whereas a higher activity was found in the L87R mutant. Meanwhile, both of their chymotrypsin inhibitory activities became weaker than the natural one. The important accessary role of the residue of position 87 in causing the difference in inhibitory properties between inhibitor A and B was discussed.

Chymotrypsin inhibitors in mosquitoes: activity profile during development and after blood feeding.

Chymotrypsin and trypsin inhibitors persist throughout all developmental instars of Aedes aegypti. After a blood meal, inhibitor activity against chymotrypsin was more than double that of sugar-fed females, but only weak activity was detected in midguts where proteinase inhibitors has been thought to regulate proteinases during blood digestion. A fourfold increase in the ratio of abdominal/thoracic inhibitor activity after the blood meal strongly suggested that fat body, or other abdominal tissues, represent the major source of inhibitor. Chymotrypsin inhibitor activity was deposited in maturing oocytes. Similar results were obtained with blood-fed Anopheles albimanus. Chymotrypsin inhibitor was active against different mosquito proteinases and against bovine alpha-chymotrypsin and trypsin, but not against subtilisin, pancreatic elastase, or fungal proteases; chymotrypsin inhibitors did not interfere with bacterial growth. The hypothesis on the regulation of blood digestion through the action of proteinase inhibitors during the gonotrophic cycle was abandoned and its involvement in the phenoloxidase cascade in the mosquito egg chorion is suggested instead.

Studies on karkade ( Hibiscus sabdariffa): protease inhibitors, phytate, in vitro protein digestibility and gossypol content

Karkade defatted flour extract had chymotrypsin inhibitor activity almost half that of trypsin (21.8 and 40.5 enzyme inhibitor activity units/mg protein, respectively). The protein isolate had a weak chymotrypsin inhibitor activity compared to that of tryprin (4.9–28.3 enzyme inhibitor activity units/mg protein, respectively). The trypsin inhibitor activity of the protein isolate was 70% of that of the defatted flour extracts. Extraction of trypsin inhibitor by citrate buffer (pH 4.6) gave a higher specific activity of trypsin than by phosphate buffer (pH 7.6), Tris buffer (pH 9.0) or distilled water. Heating the extract in boiling water for 10 min destroyed 66.1% of the trypsin inhibitor activity. Karkade protein isolate had lower free and total gossypol than did the whole seed and defatted flour. The in vitro protein digestibility of karkade defatted flour and protein isolate was lower than that of casein. Phytic acid was higher in karkade defatted flour than in soybean defatted flour.

Genotypic Variation in Cowpea (Vigna unguiculata) Cultivars in Relation to Insect Resistance

Six Cowpea genotypes namely TVX-7, V-154, DPLC-216, C-152, GC 82-7 and C-190 were selected for the study of the biochemical basis for insect-pest resistance. The total tannin, phenolic and protein contents for these genotypes were in the range 0.11–0.95, 0.01–0.05 and 17.41–19.60 per cent of defatted meal, respectively. The highest trypsin inhibitor and chymotrypsin inhibitor activities were found in GC 82-7, whereas highest protease inhibitor and α-amylase inhibitor activities were observed in TVX-7, α-amylase inhibitor activity was however, absent in V-154 and DPLC-216. Zymogram pattern for trypsin inhibitor activity showed considerable variations among the genotypes. The SDS-PAGE pattern indicated the major variations in the protein bands of molecular weights above 24 kD in all the six Cowpea varieties.

Effect of cooking on proteinase inhibitors of Dolichos lablab bean (Dolichos lablab perpureus L.).

Proteinase inhibitory activity in ten different varieties of Dolichos lablab perpureus. L. was determined. All the varieties tested exhibited appreciable level of proteinase inhibitory activity (PIA). The trypsin inhibitory activity (TIA) (Mean: 20170 TIU/g) was relatively higher than the chymotrypsin inhibitory activity (CIA) (Mean: 15380 CIU/g). Effect of temperature and cooking on PIA was studied. The nature of cooking medium and duration of cooking had profound effect on the PIA. The dry fried seeds lost their PIA very rapidly (91% in 20 min). Seeds cooked in slightly alkaline medium lost their PIA quickly (89% in 30 min) compared to those cooked in acidic (80% in 30 min) and neutral pH (83% in 30 min). The PIA in green pods was also determined and they had only one third of the PIA (8200 TIU/g and 8125 CIU/g) found in the dry seeds.

Changes in trypsin and chymotrypsin inhibitory activity on soaking of redgram (Cajanus cajan L.).

Changes in trypsin and chymotrypsin inhibitory activity of redgram seeds on soaking in distilled water, different salt solutions and mixed salt solution were investigated. Reduction in trypsin and chymotrypsin inhibitory activity was observed on soaking in salt solutions in comparison to soaking in distilled water. Maximum loss of trypsin and chymotrypsin inhibitory activity was observed on soaking the seeds in mixed salt solution.

Role of disulfide linkages in structure and activity of proteinase inhibitor from horsegram ( Dolichos biflorus)

Proteinase inhibitor isolated from horsegram ( Dolichos biflorus or Macrotyloma uniflorum) inhibited specifically the enzymes trypsin and chymotrypsin. The inhibitor contained seven disulfide linkages and was free from thiol groups. The inhibitor is resistant to denaturation by urea, guanidine hydrochloride or sodium dodecyl sulfate. Reduction of the inhibitor with dithiothreitol abolished both trypsin and chymotrypsin inhibitory activities. The kinetic plots of the reduction as followed by activity and loss in structure as reflected in the 257 nm CD band could be superposed; loss in the activity paralleled the loss in structure. The kinetics of the reduction process was complex; reduction of the inhibitor was slow and depended on the concentration of DTT. Reduction of the disulfide linkages with DTT affected the tertiary structure significantly and secondary structure was not affected considerably. Fluorescence quenching by acrylamide and potassium iodide suggested the unfolding of the molecule due to reduction. Thus, disulfide linkages play a predominant role in maintaining the three-dimensional structure of the inhibitor.

Determination of Pea Proteinase Inhibitors Using ELISA Based on Monoclonal Antibodies

Abstract An immunoassay method for analysis of trypsin and chymotrypsin inhibitor activity in seeds from different pea cultivars has been developed. This method is fast, cheap and well suited for screening of large numbers of seed samples. The technique is based on enzyme linked immunoassay (ELISA) using monoclonal antibodies produced against pea (Pisum sativum L.) proteintype trypsin inhibitors (PPI). The results obtained by ELISA have been compared with results achieved by using traditional enzymatic analyses for determination of both trypsin and chymotrypsin inhibitor contents, and these methods showed good agreement. Great differences in PPI levels have been found between various pea cultivars grown in Finland and Denmark, and these differences comprised both the total amount of PPI and the relative composition of individual PPI. Several proteins occurring in pea exhibited inhibitor activity, and at least 10 PPI inhibitors with p/ values 4.9–7.8 were detected. Pea cultivars with low PPI levels had a P...

Serpins from wheat grain

Wheat serpin genes have been identified by Southern blot hybridization with three distinct barley protein Z probes. Immunoblot analysis with a monoclonal antibody towards barley protein Z confirmed expression of related M r ~ 40 kDa proteins in wheat grain. The wheat serpins were extracted under reducing conditions and separated from β-amylase and other seed proteins by thiophilic adsorption and anion-exchange chromatography. One molecular form possessing chymotrypsin inhibitory activity was isolated in a reactive site cleaved form on a chymotrypsin affinity column. N-terminal amino acid sequences of a CNBr fragment and of the C-terminal peptide from the cleaved inhibitor (M r 4574 ± 4 Da) verified homology with barley protein Z and mammalian serpins. The native inhibitory serpin was demonstrated to form an SDS-stable complex with α-chymotrypsin.