Chymotrypsin Inhibitory Activity Research Articles

Phytochemical analysis, antimicrobial, antioxidant and enzyme inhibitory activities of ethanolic extract ofCentaureasolstitialis L. and its different fractions

Resistance to conventional antimicrobial regimes is one of the issues of concern in healthcare and it drives the need for development of new antimicrobial agents. Medicinal plants, as rich source of biochemical and bioactive compounds, serve as potential source for new drugs. Here, we evaluated the ethanolic extract of Centaureasolstitialis L.and its different fractions (n-hexane, choloroform and n-butanol soluble fraction) for antimicrobial, antioxidant and enzymes inhibitory activities. Antibacterial activity against two gram-positive and three gram-negative bacteria species were determined by using agar well diffusion and 96-wells microplate methods. Similarly, antifungal activity against two fungal strains was also evaluated by agar well diffusion method. Antioxidant activity analyzed by measuring the scavenging activity of DPPH radicals and acetylcholinesterase, butrylcholinesterase and chymotrypsin inhibitory activity was determined at 10 µg/mL and 1.0 mg/mL concentrations. Results revealed that the ethanolic extract of C. solstitialis and its different fractions possesses significant ( p <0.05) antibacterial activity and effective against fungi Aspergillus niger and Macrophominaphaseolina . Significant ( p <0.05) DPPH scavenging activity (88.52±0.23%) among all fractions was noted. n-butanol fraction showed significant acetyl-cholinesterase (78.55±0.76%) and butrylcholinesterase inhibitory activity (78.1±0.41%) with IC 50 values of 54.6±0.39 µg/mL and 211.9±0.15 µg/mL, respectively. Maximum chymotrypsin inhibition activity was shown by crude ethanolic extract (87.76±1.17) with IC 50 valueof 38.23±0.75 µg/mL. It is concluded that C. solstitialis extract and its fractions possess significant antimicrobial, antioxidant and enzyme inhibitory activity

Nutritional properties of pea protein‐enriched flour treated with different proteases to varying degrees of hydrolysis

AbstractBackground and objectivesPulses are a good source of protein; however, they contain phytochemicals that can negatively affect the protein quality. Enzymatic hydrolysis could be a means to enhance the nutritional value of pea by improving the digestibility of the protein. To this aim, a commercial air‐classified pea protein‐enriched flour (PPEF) was hydrolyzed by different proteases (trypsin, Savinase, papain, and pepsin) to different degrees of hydrolysis (DH 2%–4% and 10%–12%) and levels of phytochemicals and in vitro protein digestibility corrected amino acid score were examined.FindingsProtease treatments reduced the contents of condensed tannins and total phenolics, as well as trypsin inhibitor and chymotrypsin inhibitor activities. Hydrolysis at 10%–12% DH (degrees of hydrolysis) improved the amino acid score of the PPEF, but it was still limiting sulfur amino acids, whereas low levels of hydrolysis (DH 2%–4%) decreased the amino acid score. The in vitro protein digestibility score increased slightly following hydrolysis, with the greatest improvement in digestibility occurring with the papain treatment (DH 10%–12%). The in vitro protein digestibility corrected amino acid score with trypsin hydrolysis at DH 2%–4% was the lowest at 59.17%, whereas pepsin hydrolysis (10%–12% DH) gave the highest score (72.89%).ConclusionsSpecific hydrolysis treatments could be used to improve the overall nutrition of PPEF, enhancing the potential utilization of modified pea protein ingredients.Significance and noveltyThis study utilized four enzymes with different specificities resulting in PPEF hydrolysates with different protein quality even when the DH was the same. Increasing the nutritional quality of a PPEF may give it a competitive advantage over other pea ingredients.

Occurrence of serine proteinase inhibitors in seeds of some plants

Serine proteinase inhibitors, produced by plants, inhibiting protein digestion in herbivorous insects, can be used as one of the best ecofriendly IPM tactics. In this context, laboratory assay was conducted to identify the potential source of serine proteinase inhibitors from the seeds of 38 plant species corresponding to nine families. Among them, extracts from 20 species exhibited both trypsin and chymotrypsin inhibitory activity (TIA and CIA respectively) and 15 plant species possess TIA. The maximum TIA [9878 TIU (Trypsin Inhibitory Unit) g−1 of seed] was observed in Momordica balsamina seed extract. Erythrina variegata was found to possess both TIA (2737 TIU g−1 of seed) and CIA (2800 CIU Chymotrypsin Inhibitory Unit g−1 of seed). Seeds of two species may be utilized as a potential source of proteinase inhibitor in insect control programme. These presence of trypsin inhibitory activity in M. balsamina is reported for the first time.

Discovery and Rational Design of a Novel Bowman-Birk Related Protease Inhibitor.

Anuran amphibian skin secretions are a rich source of peptides, many of which represent novel protease inhibitors and can potentially act as a source for protease inhibitor drug discovery. In this study, a novel bioactive Bowman-Birk type inhibitory hexadecapeptide of the Ranacyclin family from the defensive skin secretion of the Fukien gold-striped pond frog, Pelophlax plancyi fukienesis, was successfully isolated and identified, named PPF-BBI. The primary structure of the biosynthetic precursor was deduced from a cDNA sequence cloned from a skin-derived cDNA library, which contains a consensus motif representative of the Bowman-Birk type inhibitor. The peptide was chemically synthesized and displayed a potent inhibitory activity against trypsin (Ki of 0.17 µM), as well as an inhibitory activity against tryptase (Ki of 30.73 µM). A number of analogues of this peptide were produced by rational design. An analogue, which substituted the lysine (K) at the predicted P1 position with phenylalanine (F), exhibited a potent chymotrypsin inhibitory activity (Ki of 0.851 µM). Alternatively, a more potent protease inhibitory activity, as well as antimicrobial activity, was observed when P16 was replaced by lysine, forming K16-PPF-BBI. The addition of the cell-penetrating peptide Tat with a trypsin inhibitory loop resulted in a peptide with a selective inhibitory activity toward trypsin, as well as a strong antifungal activity. This peptide also inhibited the growth of two lung cancer cells, H460 and H157, demonstrating that the targeted modifications of this peptide could effectively and efficiently alter its bioactivity.

Lathyrus sativus Originating from Different Geographical Regions Reveals Striking Differences in Kunitz and Bowman-Birk Inhibitor Activities.

Grass pea (Lathyrus sativus L.) is an important legume commonly grown in arid and semi-arid regions. This protein-rich legume performs well even under harsh environmental conditions and is considered to be a strategic famine food in developing countries. Unfortunately, its potential usage is greatly limited as a result of the presence of antinutritional factors, including the neuroexcitatory amino acid β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP) and protease inhibitors. β-ODAP is responsible for a neurodegenerative syndrome that results in the paralysis of lower limbs, while protease inhibitors affect protein digestibility, resulting in reduced growth. Concerted research efforts have led to development of grass pea cultivars with reduced β-ODAP content. In contrast, very little information is available on the protease inhibitors of L. sativus. In this study, we have conducted biochemical characterization of 51 L. sativus accessions originating from different geographical regions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of seed globulins and prolamins revealed striking similarity in their protein profile, although geographic-specific variations in profiles was also evident. Measurement of Bowman-Birk chymotrypsin inhibitor (BBi) and Kunitz trypsin inhibitor (KTi) activities in accessions revealed striking differences among them. Amino acid sequence alignment of grass pea BBi and KTi revealed significant homology to protease inhibitors from several legumes. Real-time polymerase chain reaction analysis demonstrated high-level expression of BBi and KTi in dry seeds and weak expression in other organs. Our study demonstrates substantial variation in BBi and KTi among grass pea accessions that could be exploited in breeding programs for the development of grass pea lines that are devoid of these antinutritional factors.

A Novel Kunitzin-Like Trypsin Inhibitor Isolated from Defensive Skin Secretion of Odorrana versabilis.

Protease inhibitors that were identified from amphibian skin secretions with low molecular weights and potent inhibitory activity were thought to be potential candidates for novel peptide drugs. Here, a novel peptide with trypsin inhibitory activity was found in the skin secretion of the Chinese bamboo leaf odorous frog, Odorrana versabilis. Based on the sequence alignments of sequencing results, the novel peptide (ALKYPFRCKAAFC) was named as Kunitzin-OV. The synthetic replicate of Kunitzin-OV was subjected to a series of functional assays, and it exhibited a trypsin inhibitory activity with a Ki value of 3.042 µM, whereas, when Lys-9 at P1 position was substituted by Phe, trypsin inhibitory activity was undetected and the chymotrypsin inhibitory activity was optimized with a Ki value of 2.874 µM. However, its protease-binding loop was catabolized by trypsin during the trypsin cleavage test. In conclusion, Kunizin-OV is a novel peptide with trypsin inhibitory activity as a member of kunitzins, which is a non-typical Kunitz-like trypsin inhibitor with a highly conserved reactive site (K-A) and quite a short sequence.

Purification and characterization of Bowman-Birk and Kunitz isoinhibitors from the seeds of Rhynchosia sublobata (Schumach.) Meikle, a wild relative of pigeonpea

Rhynchosia sublobata, a wild relative of pigeonpea, possesses defensive proteinase/protease inhibitors (PIs). Characterization of trypsin specific PIs (RsPI) separated from seeds by column chromatography using 2-D gel electrophoresis and Edman degradation method identified R. sublobata possessed both Bowman-Birk isoinhibitors (RsBBI) and Kunitz isoinhibitors (RsKI). A quick method was developed to separate RsBBI and RsKI from RsPI based on their differential solubility in TCA and acetate buffer. N-terminus sequencing of RsBBI and RsKI by MALDI-ISD ascertained the presence of Bowman Birk and Kunitz type isoinhibitors in R. sublobata. RsBBI (9216 Da) and RsKI (19,412 Da) exhibited self-association pattern as revealed by western blotting with anti-BBI antibody and MALDI-TOF peptide mass fingerprint analysis, respectively. RsBBI and RsKI varied significantly in their biochemical, biophysical and insecticidal properties. RsBBI inhibited the activity of trypsin (Ki = 128.5 ± 4.5 nM) and chymotrypsin (Ki = 807.8 ± 23.7 nM) while RsKI (Ki = 172.0 ± 9.2 nM) inhibited the activity of trypsin alone, by non-competitive mode. The trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities of RsBBI were stable up to 100 °C. But, RsBBI completely lost its TI and CI activities on reduction with 3 mM DTT. Conversely, RsKI lost its TI activity on heating at 100 °C and retained >60% of its TI activity in presence of 3 mM DTT. CD spectroscopic studies on RsBBI and RsKI showed their secondary structural elements in the following order: random coils > β-sheets/β-turns > α-helix. However, RsKI showed reversible denaturation midpoint (Tm) of 75 °C. Further, the significant inhibitory activity of RsBBI (IC50 = 24 ng) and RsKI (IC50 = 59 ng) against trypsin-like gut proteases of Achaea janata (AjGPs) and Helicoverpa armigera (HaGPs) suggest them as potential biomolecules in the management of A. janata and H. armigera, respectively.

Glycation affects differently the main soybean Bowman-Birk isoinhibitors, IBB1 and IBBD2, altering their antiproliferative properties against HT29 colon cancer cells.

Naturally-occurring serine protease inhibitors of the Bowman-Birk family, particularly abundant in legume seeds, exert their potential chemopreventive and/or therapeutic properties via protease inhibition. Processing of legume seeds, including soybeans, has been proposed as a major cause for their loss of bioactivity due to glycation. In order to assess how glycation affected the protease inhibitory activities of major soybean Bowman-Birk isoinhibitors (BBI) and their antiproliferative properties, IBB1 and IBBD2 were purified and subjected to glycation under controlled conditions using glucose at high temperature. Both soybean isoinhibitors showed remarkable heat stability. In the presence of glucose, IBBD2 lost most of its trypsin inhibitory activity while IBB1 maintains similar trypsin and chymotrypsin inhibitory activities as in the absence of sugar. Glycation patterns of both BBI proteins were assessed by MALDI-TOF spectrometry. Our results show that the glycation process affects IBBD2, losing partially its antiproliferative activity against HT29 colon cancer cells, while glycated-IBB1 was unaffected.

Influence of Different Cowpea (<i>Vigna unguiculata</i> (L.) Walp.) Genotypes from Burkina Faso on Proteases Inhibition

Cowpea is an important protein crop widespread in Africa. The purpose of this research was to determine the content of trypsin and chymotrypsin inhibitors in different genotypes of cowpea seeds. Trypsin percentage inhibition showed about 13.5-fold variation (5.12% ± 1.47 to 70.52% ± 6.58) between the cowpea genotypes respectively for KVx 421-2J and Kondesyoungo local. The chymotrypsin inhibitory activity varies among cowpea genotypes from 21.19 to 76.94%. The highest percent inhibitory activity was exhibited by KVx 396-4-5-2D on chymotrypsin. This study also showed significant correlations between type of cowpea genotype and trypsin inhibition potential and also between colour and chymotrypsin inhibition potential of cowpea seeds. Kondesyoungo local, a landraceae genotype exhibited the high potential to inhibit the trypsin enzyme. Coloured seeds of cowpea genotypes possess higher percentage inhibition of chymotrypsin than the colourless ones (p < 0.05). The calculated mean of trypsin and chymotrypsin inhibition activities showed that Labagela local genotype possess the highest inhibition activity of both protease enzymes. The PCA components analysis and the dendrogram performed basis on the protease inhibitory activities divided the thirty-one genotypes of cowpea used in this study into three classes. The results presented in this work can contribute greatly to the planning of a cowpea breeding program aimed at reducing the content of proteases inhibitors in order to improve the nutritional value of seeds or to increase PI content for tolerance to stored grain pests.

Bldesin, the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities

Fungus defensin is a kind of important natural peptide resource, such as plectasin from the soil fungus Pseudoplectania nigrella with potential application in the antimicrobial peptide lead drug discovery. Here, a fungus defensin named Bldesin with Kv1.3 channel and serine protease inhibitory activities was first explored. By GST-Bldesin fusion expression and enterokinase cleaving strategy, recombinant Bldesin was obtained successfully. Antimicrobial assays showed that Bldesin had potent activity against Gram-positive Staphylococcus aureus, but had no effect on Gram-negative Escherichia coli. Electrophysiological experiments showed that Bldesin had Kv1.3 channel inhibitory activity. Serine protease inhibitory associated experiments showed that Bldesin had unique chymotrypsin protease inhibitory, elastase protease inhibitory, and serine protease-associated coagulation inhibitory activities. To the best of our knowledge, Bldesin is the first functionally characterized pathogenic fungus defensin with Kv1.3 channel and chymotrypsin inhibitory activities and highlighted novel pharmacological effects of fungus-derived defensin peptides.

Inducible defense responses in Populus alba berolinensis to Pb stress

To better understand the effects of heavy metal stress on inducible defenses of woody plants, contents of nutritional substances and secondary metabolites, plus the activities of defense enzymes and protease inhibitors in the leaves of Populus alba berolinensis seedlings that were planted in either non-contaminated soil (control) or Pb-contaminated soil (at 300, 500 and 700mg/kg) were studied. Our results showed that protein and soluble sugar contents in leaves 30–50 days after the Pb stress were significantly lower than those in the control group in a dose-dependent manner, whereas the lignin and flavonoid contents were significantly higher. Polyphenol oxidase (PPO) and phenylalnine ammonialyase (PAL) activities in leaves were significantly enhanced by the low Pb concentration stress (300 mg/kg) for all three sampling dates (30, 40 and 50 days after the Pb treatments), while at the two higher Pb concentrations (500 and 700 mg/kg), PPO activities were always inhibited and PAL activities presented a tendency of “promoting first and then restraining” with the increase of exposure duration. The response patterns of trypsin inhibitor (TI) and chymotrypsin inhibitor (CI) activities to Pb stress were similar to that of PPO, and presented a “low-promotion, high-inhibition” effect of Pb concentrations in leaves. These results suggest that the inducible defense responses in P. alba berolinensis leaves to Pb stress depend on both the Pb concentrations and the exposure duration, and the low Pb concentration at 300 mg/kg seemed to induce the strongest defense responses in the poplar seedlings, much stronger than those at the higher Pb concentrations.

Protein Separation Coacervation with Carboxymethyl Cellulose of Different Substitution Degree: Noninteracting Behavior of Bowman-Birk Chymotrypsin Inhibitor.

We first observed that protein/polysaccharide interaction exhibited noninteracting behavior which makes Bowman-Birk chymotrypsin inhibitor (BBI) always free of complexation, being separated from another protein with similar isoelectric points, Kunitz trypsin inhibitor (KTI). Turbidity titrations showed that the electrostatic attractions were much stronger between KTI/BBI (KBi) and carboxymethyl cellulose of higher substitution degree. Unchanged chymotrypsin inhibitory activity (CIA) indicated that BBI had negligible contribution to protein recovery and trypsin inhibitory activity (TIA). Tricine-SDS-PAGE revealed that, at r = 20:1-2:1, unbound BBI was left in the supernatant when bound KTI transferred into precipitates, even if there was excess negative charge. Thus, purified KTI or BBI was achieved easily at the given conditions. The noninteracting behavior of BBI was further confirmed by ITC, where the binding enthalpy of BBI to CMC was negligible compared with the high binding affinity ( Kb) of KTI. This work will be beneficial to protein purification based on protein-polysaccharide coacervation.

Effect of Fermentation on the Protein Digestibility and Levels of Non-Nutritive Compounds of Pea Protein Concentrate.

In order to determine the impact of fermentation on protein quality, pea protein concentrate (PPC) was fermented with Lactobacillus plantarum for 11 h and total phenol and tannin contents, protease inhibitor activity, amino acid composition and in vitro protein digestibility were analyzed. Phenol levels, expressed as catechin equivalents (CE), increased on dry mass basis from 2.5 at 0 h to 4.9 mg CE per 1 g of PPC at 11 h. Tannin content rose from 0.14 at 0 h to a maximum of 0.96 mg CE per 1 g of PPC after 5 h, and thereafter declined to 0.79 mg/g after 11 h. After 9 h of fermentation trypsin inhibitor activity decreased, however, at all other fermentation times similar levels to the PPC at time 0 h were produced. Chymotrypsin inhibitor activity decreased from 3.7 to 1.1 chymotrypsin inhibitory units (CIU) per mg following 11 h of fermentation. Protein digestibility reached a maximum (87.4%) after 5 h of fermentation, however, the sulfur amino acid score was reduced from 0.84 at 0 h to 0.66 at 11 h. This reduction in sulfur content altered the in vitro protein digestibility-corrected amino acid score from 67.0% at 0 h to 54.6% at 11 h. These data suggest that while fermentation is a viable method of reducing certain non-nutritive compounds in pea protein concentrate, selection of an alternative bacterium which metabolises sulfur amino acids to a lesser extent than L. plantarum should be considered.

Nutritional values and metabolic profile with and without boiled treatment of 'Gallo Matese' beans (Phaseolus vulgaris L.), a landrace from Southern Italy.

'Gallo Matese' beans are known as a typical legume of Southern Italy and continue to be consumed as a traditional food preserving the diversity of this region. Nonetheless, no information about the nutritional values of this legume is available. The objective of the present investigation was to determine the nutritional value and metabolic profile of 'Gallo Matese' beans. Results. 'Gallo Matese' beans contain high levels of proteins (22.64 g/100 g) and essential amino acids (8.3 g/100 g). Furthermore, differentunsaturated fatty acidscontributetothetotalamountoflipids (0.97 g/100 g); among them, the essential PUFA α-linolenic (0.48 g/100 g) and linoleic (0.39 g/100 g) acids are the most abundant. The total phenol content was revealed and ABTS and ORAC-fluorescein methods were applied to determine the radical scavenging capabilities of the extract with and without boiled treatment. Finally, a de- crease in trypsin and chymotrypsin inhibitory activities was estimated before and after the boiling procedure. Conclusion. The data obtained show that 'Gallo Matese' beans are a functional food with healthy qualities. Overall, these results are useful to promote their cultivation and consumption, thus preserving Italian beans biodiversity due to consumer interest in choosing a healthy diet, such as the Mediterran. nd. 'Gallo Matese' beans are known as a typical legume of Southern Italy and continue to be consumed as a traditional food preserving the diversity of this region. Nonetheless, no information about the nutritional values of this legume is available. The objective of the present investigation was to determine the nutritional value and metabolic profile of 'Gallo Matese' beans. 'Gallo Matese' beans contain high levels of proteins (22.64 g/100 g) and essential amino acids (8.3 g/100 g). Furthermore, differentunsaturated fatty acidscontributetothetotalamountoflipids (0.97 g/100 g); among them, the essential PUFA α-linolenic (0.48 g/100 g) and linoleic (0.39 g/100 g) acids are the most abundant. The total phenol content was revealed and ABTS and ORAC-fluorescein methods were applied to determine the radical scavenging capabilities of the extract with and without boiled treatment. Finally, a de- crease in trypsin and chymotrypsin inhibitory activities was estimated before and after the boiling procedure. The data obtained show that 'Gallo Matese' beans are a functional food with healthy qualities. Overall, these results are useful to promote their cultivation and consumption, thus preserving Italian beans biodiversity due to consumer interest in choosing a healthy diet, such as the Mediterranean diet.

Recovery of Protease Inhibitors from Potato Fruit Water by Expanded Bed Adsorption Chromatography in Pilot Scale

The current study was conducted to investigate the recovery of native potato protein from potato fruit water (PFW) by expanded bed adsorption (EBA) chromatography. The eluted proteins were concentrated by ultrafiltration and spray-dried into powder. The SDS-PAGE showed that the recovered proteins were potato protease inhibitors (PPIs). The trypsin and chymotrypsin inhibitor activities of the recovered PPIs were 377.93 ± 8.22 and 12.90 ± 0.03 mg g−1 protein, respectively. The recovery yield of protease inhibitors was 74.88%. The glycoalkaloid assay showed that the recovered PPIs contained 30.31 ± 0.15 μg g−1 of α-chaconine and 92.77 ± 0.52 μg g−1 of α-solanine, and these values were much lower than those in potato protein concentrate (PPC) obtained by traditional thermal coagulation. The most abundant amino acid in the PPIs was serine. The results indicated that the EBA can be used to effectively recover native potato protein from PFW.

Effects of Disulfide Bond Reduction on the Conformation and Trypsin/Chymotrypsin Inhibitor Activity of Soybean Bowman-Birk Inhibitor.

Soybean seeds contain three groups (A, C, and D) of Bowman-Birk inhibitors (BBIs). In this study, highly purified BBI-A (approximately 96%) was obtained from soybean whey at the 0.1 g level by the complex coacervation method. BBI-A has seven disulfide bonds (SS) and no sulfhydryl group and exhibits trypsin inhibitor activity (TIA) and chymotrypsin inhibitor activity (CIA). The X-ray structure has shown that BBI-A has five exposed SS and two buried SS. Because of steric hindrance, it was reasonable to consider that dithiothreitol first attacks the five exposed SS and then the two buried SS, which was supported by the results that SS reduction with dithiothreitol could be divided into quick and slow stages, and the critical point was close to 5/7. The effects of SS reduction on TIA and CIA could be divided into three stages: when one exposed SS was reduced, both TIA and CIA decreased to approximately 60%; with further reduction of exposed SS, CIA gradually decreased to 8% and TIA gradually decreased to 26%; with further reduction of buried SS, CIA gradually decreased to 2% and TIA slightly decreased to 24%. Far-ultraviolet (far-UV) circular dichroism (CD) spectra showed that the secondary structure of BBI-A was slightly changed, whereas near-ultraviolet (near-UV) CD spectra showed that the conformation of BBI-A was substantially changed after the five exposed SS were reduced; further reduction of buried SS affected the conformation to some extent. The results of Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from a C8 column showed the same trend as near-UV CD spectra. BBI-A has a structural peculiarity in that two hydrophobic patches are exposed to the exterior (in contrast to typical soluble proteins), which was attributed to the seven SS by some researchers. These results support the hypothesis that hydrophobic collapse of the exposed hydrophobic patches into a regular hydrophobic core occurred after the reduction of SS in BBI-A.

Novel Kazal-type proteinase inhibitors from the skin secretion of the Splendid leaf frog, Cruziohyla calcarifer

Peptidase inhibitors have an important role controlling a variety of biological processes. Here, we employed a peptidomic approach including molecular cloning, tandem mass spectrometry and enzymatic assays to reveal 7 Kazal-type proteinase inhibitors (CCKPs) (18 variants) in the skin secretion of the unexplored frog, Cruziohyla calcarifer. All 18 proteins shared the Kazal pattern C-X(7)-C-X(6,7)-C-X(6,7)-Y-X(3)-C-X(2)-C-X(15-21)-C and 3 disulphide bridges. Based on structural comparative analysis, we deemed trypsin and chymotrypsin inhibitory activity in CCKP-1, 4 and CCKP 2, 5, 7, respectively. These peptidase inhibitors presumably play a role to control the balance between other functional peptides produced in the amphibian skin secretions.

Changes in levels of enzyme inhibitors during soaking and cooking for pulses available in Canada.

The effects of processing (soaking and cooking) on enzyme inhibitors (α-amylase, trypsin and chymotrypsin inhibitors) in a range of pulses (4 peas, 9 lentils, 3 chickpeas, 2 faba beans and 4 beans) were investigated, using soybean as a control. Analysis of variance indicated that pulse type, treatment and their interaction had significant effects on levels of all enzyme inhibitors. Soybean contained the highest levels of trypsin inhibitory activity (TIA) and chymotrypsin inhibitory activity (CIA) among all seeds. α-Amylase inhibitory activity was absent from peas, lentils, chickpeas and faba beans, but was present in beans and soybean. TIA was found to be low in peas but high in beans. Beans contained relatively high CIA levels followed by chickpeas, lentils, peas and faba beans. Soaking markedly decreased the activity of enzyme inhibitors. Cooking of presoaked seeds was even more effective as greater reductions (78.7-100%) were observed for all pulses. The content of enzyme inhibitors in pulses varied widely, but levels of protease inhibitors were generally lower that those found in soybean. Processing, in particular heat treatments, drastically reduced these levels.

An advance for removing antinutritional protease inhibitors: Soybean whey purification of Bowman-Birk chymotrypsin inhibitor by combination of two oppositely charged polysaccharides

Two successive and selective coacervations induced by chitosan (Ch) and carrageenan (CG) were applied to remove antinutritional protease inhibitors and purify Bowman–Birk protease inhibitor (BBI) from soybean whey. At the first coacervation induced by Ch (66.7, 200, and 510kDa), only Kunitz trypsin inhibitor (KTI) and BBI complexed with Ch were extracted, while β-amylase and soybean agglutinin remained in supernatant. The binding constants for the interaction increased on the order Ch-66.7<Ch-200<Ch-510. At the second selective complexation, we observed a competitive binding behavior between KTI/BBI and CG. At a mixing weight ratio of 3:1 (pH 3.0 for ι-CG, and pH 3.11 for λ-CG), the preferential binding of KTI to CG led to the single enrichment of BBI in the supernatant. Our results indicated that the purified BBI was a good source for further study of its anti-carcinogenic properties, due to its high bioactivity (669.5U/mg chymotrypsin-inhibitory activity and 2260U/mg trypsin-inhibitory activity).

Protease inhibitors in various flours and breads: Effect of fermentation, baking and in vitro digestion on trypsin and chymotrypsin inhibitory activities.

In this study trypsin (TIA) and chymotrypsin inhibitory (CIA) activities were determined in the extracts of wheat, rye mix, mixed cereals and, whole wheat flours and, breads made with these flours. In addition, effects of fermentation, baking and in vitro digestion on TIA and CIA were studied. Whole wheat flour, dough, and bread did not show any TIA. Other flours displayed TIA. Contrary to, all flours, doughs, and breads exhibited CIA. Although TIA was not detected in the protein extracts obtained from wheat and rye mix breads, protein extract of rye mix flour exhibited TIA. Following in vitro digestion process, TIA of wheat bread was found as 5.91units/mL gastric dialysate and 9.17units/mL intestine dialysate. CIA was determined in dialysates obtained from wheat (2.12CI/mL and 3.78CI/mL for gastric and intestinal dialysate respectively) and rye breads (4.16CI/mL and 2.46CI/mL for gastric and intestinal dialysate respectively).