Soybean Protease Inhibitor Research Articles

Lipoxygenases regulate digestive enzyme inhibitor activities in developing seeds of field-grown soybean against the southern green stink bug (Nezara viridula).

Soybean (Glycine max ) is the world's most widely grown seed legume. One of the most important pests that decrease seed quality and reduce yield of soybean crops is the southern green stink bug (Nezara viridula ). Insect damage triggers accumulation of defensive compounds such as protease inhibitors (PIs), isoflavonoids and reactive oxygen species, which are regulated by the lipoxygenase (LOX)-regulated jasmonic acid (JA) to stop insect feeding. This study identified and characterised the role of LOX isoforms in the modulation of chemical defences in seeds of field-grown soybean that decreased digestive enzyme activities of N. viridula after insect attack. Stink bugs attack increased LOX 1 and LOX 2 expression, and activities of LOX 1 and LOX 3 isoenzymes in developing soybean seeds. In addition, stink bug damage and methyl jasmonate application induced expression and activity of both cysteine PIs and trypsin PIs in developing soybean seeds, suggesting that herbivory induced JA in soybean seeds. High PI activity levels in attacked seeds decreased cysteine proteases and α-amylases activities in the gut of stink bugs that fed on field-grown soybean. We demonstrated that LOX isoforms of seeds are concomitantly induced with JA-regulated PIs by stink bugs attack, and these PIs inhibit the activity of insect digestive enzymes. To our knowledge, this is the first study to investigate the participation of LOX in modulating JA-regulated defences against stink bugs in seeds of field-grown soybean, and our results suggest that soybean PIs may inhibit α-amylase activity in the gut of N. viridula .

Intestinal proteases profiling from Anticarsia gemmatalis and their binding to inhibitors.

Although the importance of intestinal hydrolases is recognized, there is little information on the intestinal proteome of lepidopterans such as Anticarsia gemmatalis. Thus, we carried out the proteomic analysis of the A. gemmatalis intestine to characterize the proteases by LC/MS. We examined the interactions of proteins identified with protease inhibitors (PI) using molecular docking. We found 54 expressed antigens for intestinal protease, suggesting multiple important isoforms. The hydrolytic arsenal featured allows for a more comprehensive understanding of insect feeding. The docking analysis showed that the soybean PI (SKTI) could bind efficiently with the trypsin sequences and, therefore, insect resistance does not seem to involve changing the sequences of the PI binding site. In addition, a SERPIN was identified and the interaction analysis showed the inhibitor binding site is in contact with the catalytic site of trypsin, possibly acting as a regulator. In addition, this SERPIN and the identified PI sequences can be targets for the control of proteolytic activity in the caterpillar intestine and serve as a support for the rational design of a molecule with greater stability, less prone to cleavage by proteases and viable for the control of insect pests such as A. gemmatalis.

Targeted inactivation of soybean proteinase inhibitors using zinc

In this study the potential targeted use of zinc to inactivate proteinase inhibitors (PI) has been investigated as an alternative to the widely applied heat treatment used industrially for inactivation of PI. Zinc was utilized for the reduction of disulfide bonds leading to the structural changes in proteins, thus affecting the decreased affinity between PI and proteinases. The protein disulfide bond reduction mechanism was studied using a newly developed micellar electrokinetic capillary chromatography (MECC) with the glutathione redox reaction with dithiothreitol (DTT) as model system. This model proved efficient in monitoring the reduction of disulfide bonds in the Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). The use of zinc as a reductant resulted in a significant reduction of trypsin inhibitor activity (TIA) of 72% for KTI and 85% for BBI, highlighting zinc as a promising potential agent to reduce the activity of PI as an alternative to heat treatment.

Efficacy of Partially Purified Soybean Protease Inhibitor on The Development of Cotton Leafworm, Spodoptera littoralis (Bosid.)

Plant protease inhibitors) PIs) are one of the plant strategies to maintain favorable growth and survival. Soybean trypsin inhibitor is the most well-known of the plant serine proteinase. The present study aims to detect the inhibitory activity of two concentrations of partially purified trypsin inhibitors from seeds of soybean(Glycine max). Comparing the results of feeding larvae on partially purified soybean inhibitors with control, revealed that the larval weight reduction caused by soybean PI was noticed after seven days of treatment. The highest larval weight reduction was obtained due to treatment with SPI at levels SPI 30% and SPI 60%, and there was no significant difference between the two levels. Feeding larvae on SPI 60% caused higher larval mortality about 20% than the control followed by SPI 30% which caused 16% larval mortality. The pupation in treated larvae was delayed two days by comparing to control. The highest weight reduction in pupal weight was 3.5± 0.3 gm at SPI 60% compared with control. Besides, the pupation rate decreased from 83% at SPI 60% to 80% at SPI 30%. Also, the reduction in adult emergency % was recorded at SPI level 30% by 41% and at SPI level 60% by 16% compared with the control. Results reveal that soybean protease inhibitors inhibit the growth and development of S. littoralis, and may be used in integrated pest control strategies.

Soybean protease inhibitors increase Bacillus thuringiensis subs. israelensis toxicity against Hypothenemus hampei

Introduction. The coffee berry borer (Hypothenemus hampei Ferrari, CBB) is one of the most devastating pests on coffee plantations around the world. Although CBB is susceptible to the effect of δ–endotoxins of Bacillus thuringiensis subs. israelensis (Bti) at laboratory level, the efficacy of this control method is poor in the field, presumably due to the inactivation by digestive proteases different to those required for protoxin activation. Objective. To study whether the addition of a soybean flour extract enriched with protease inhibitors (PI), mixed with Bti crystals and spores (Bti-sc) in an artificial diet, could improve the toxicity of Bti against CBB. Materials and methods. This study was performed in San José, Costa Rica, between 2012 and 2013. A set of adult female CBB insects was exposed to a mixture containing different concentrations of a partially purified soybean meal extract with active PI and lyophilized Bti-sc, and were tested through a bioassay in artificial diet to estimate the sub-lethal concentration (LC50). The mortality results were validated by observing the dissected midgut, whose ultrastructure was analyzed by transmission electron microscopy. Results. The soybean extracts partially degraded the Bti-sc complex, it reduced its LC50 by almost four times (from 1.135 to 0.315 µg µl-1) and enhanced CBB mortality in a concentration-dependent manner. Histological analyses of the midgut confirmed this synergistic effect, since severe epithelial damage to the intestinal epithelium of CBB exposed to Bti-sc + PI was visualized compared to Bti-sc alone. Conclusions. The combination of a soybean extract enriched in PI and Bti-sc enhanced the mortality effect over CBB, which was confirmed by the midgut collapse. Soybean flour is a cost-effective supplement that could increase Bti effectiveness against CBB and delay the appearance of biological resistance.

Larval development and proteolytic activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) exposed to different soybean protease inhibitors.

Anticarsia gemmatalis represents a relevant factor for lowering soybean and other legumecropproductivities. Protease inhibitors affectprotein degradation and reducethe availability of amino acids, impairing the development and survival of insect pests. To evaluate the possible use of proteinaceous protease inhibitors in the management of this pest, the activities of midgut proteases andthe growth and development of A. gemmatalis larvae exposed to soybean Bowman-Birk trypsin-chymotrypsin inhibitor (SBBI) and soybean Kunitz trypsin inhibitor (SKTI) were determined. The survival curves obtained using Kaplan-Meier estimators indicated that SKTI and SBBI stimulated larval survival. However, the development of A. gemmataliswas delayed, and prepupal weight decreased in the presence of both inhibitors. The results showed that SKTI and SBBI inhibited the trypsin-like and total proteolytic activities of larvae on the 12th day after eclosion. On the 15th day after eclosion, larvae exposed to SKTI increased the activities of trypsin and total proteases. Although SKTI and SBBI did not affect the survival of the insect, they had effects on midgut proteases in a stage whereinA. gemmatalis fedvoraciously, increased the larval cycle, and decreased prepupal weight. These findings provide baseline informationabout the potential of proteinaceous protease inhibitors to manage the velvetbean caterpillar, avoiding chemical pesticides.

Characterized non-transient microbiota from stinkbug (Nezara viridula) midgut deactivates soybean chemical defenses.

The Southern green stinkbug (N. viridula) feeds on developing soybean seeds in spite of their strong defenses against herbivory, making this pest one of the most harmful to soybean crops. To test the hypothesis that midgut bacterial community allows stinkbugs to tolerate chemical defenses of soybean developing seeds, we identified and characterized midgut microbiota of stinkbugs collected from soybean crops, different secondary plant hosts or insects at diapause on Eucalyptus trees. Our study demonstrated that while more than 54% of N. viridula adults collected in the field had no detectable bacteria in the V1-V3 midgut ventricles, the guts of the rest of stinkbugs were colonized by non-transient microbiota (NTM) and transient microbiota not present in stinkbugs at diapause. While transient microbiota Bacillus sp., Micrococcus sp., Streptomyces sp., Staphylococcus sp. and others had low abundance, NTM microbiota was represented by Yokenella sp., Pantoea sp. and Enterococcus sp. isolates. We found some isolates that showed in vitro β-glucosidase and raffinase activities plus the ability to degrade isoflavonoids and deactivate soybean protease inhibitors. Our results suggest that the stinkbugs´ NTM microbiota may impact on nutrition, detoxification and deactivation of chemical defenses, and Enterococcus sp., Yokenella sp. and Pantoea sp. strains might help stinkbugs to feed on soybean developing seeds in spite of its chemical defenses.

Partially purified Glycine max proteinase inhibitors: potential bioactive compounds against tobacco cutworm, Spodoptera litura (Fabricius, 1775) (Lepidoptera: Noctuidae)

Spodoptera litura (Fabricius, 1775), a polyphagous defoliator with broad host spectrum, causes significant damage to agriculturally important crops. Serine protease is primarily responsible for most of the proteolytic activity in the larval gut of lepidopteran insects. Second-instar larvae were reared on an artificial diet containing serine trypsin inhibitor partially purified from Glycine max seeds. Different concentrations were amended in the diet, i.e. 25, 50, 100, 200, and 400 μg/mL. The larval development period, total developmental period, longevity, fecundity, larval weight, and mean relative growth rate were decreased at the 100 μg/mL concentration. However, with further increase in concentration, the above parameters were all found to increase. Nutritional indices revealed reduction in efficiency of conversion of digested food and approximate digestibility, whereas increase in efficiency of conversion of ingested food had significant influence on food assimilation. Trypsin activity was suppressed at 100 μg/mL with increase in exposure interval. Results reveal that soybean protease inhibitor inhibited S. Litura gut proteinase and it was more effective at lower concentrations. Thus, low levels of inhibitor would be enough to affect the growth and development of the target pest for the development of transgenic plants.

Protease purification and characterization of a serine protease inhibitor from Egyptian varieties of soybean seeds and its efficacy against Spodoptera littoralis

Abstract Serine inhibitors have been described in many plant species and are universal throughout the plant kingdom. Trypsin inhibitors are the most common type. In the present study, trypsin and chymotrypsin inhibitory activity was detected in the seed flour extracts of four Egyptian varieties of soybean (Glycine max). The soybean variety, Giza 22, was found to have higher trypsin and chymotrypsin inhibitory potential compared to other tested soybean varieties. For this reason, Giza 22 was selected for further purification studies which used ammonium sulphate fractionation and DEAE-Sephadex A-25 column. Soybean purified proteins showed a single band on SDS-PAGE corresponding to a molecular mass of 17.9 kDa. The purified inhibitor was stable at temperatures below 60°C and was active at a wide range of pH, from 2 to 12 pH. The kinetic analysis revealed a non-competitive type of inhibition against trypsin and chymotrypsin enzymes. The inhibitor constant (Ki) values suggested that the inhibitor has higher affinity toward a trypsin enzyme than to a chymotrypsin enzyme. Purified inhibitor was found to have deep and negative effects on the mean larval weight, larval mortality, pupation, and mean pupal weight of Spodoptera littoralis. It may be concluded, that soybean protease inhibitor gene(s) could be potential targets for those future studies which are concerned with developing insect resistant transgenic plants

The effect of legume protease inhibitors on native milk and bacterial proteases

Protease inhibitors from legume seed extracts (soybean, cowpea and marama beans) and purified soybean protease inhibitor were evaluated with regards to their abilities to inhibit proteases produced by important milk contaminating bacteria, i.e. Bacillus spp. and Pseudomonas spp., and native milk protease, plasmin. Although heat treatment is the most common mean of inactivating enzymes, some heat-stable enzymes can survive the ultra-high temperature (UHT) processing of milk and cause sensory and consistency defects during storage at room temperature. The legume protease inhibitors reduced the activity of plasmin and proteases produced by Bacillus spp. by up to 94% and 97%, respectively, while it showed low inhibitory activity towards Pseudomonas fluorescens proteases (19%) in a buffer system. The protease inhibitors reduced the activity of plasmin (41%) and Bacillus proteases (50%) in UHT milk, however to a lesser extent as compared to inhibition in the buffer system; while it had little or no effect on proteases form Pseudomonas spp. Legume protease inhibitors show great potential in preventing or reducing proteolytic activity of Bacillus proteases and plasmin and may be exploited in various applications where these proteases cause sensory or consistency defects in the product.

Advances of Research on Glycinin and β-Conglycinin: A Review of Two Major Soybean Allergenic Proteins

Being an important crop, soybean is widely used in the world and plays a vital role in human and animal nutrition. However, it contains several antinutritional factors (ANFs) including soybean agglutinin, soybean protease inhibitors, soybean allergenic proteins, etc., that may result in poor food utilization, decreased growth performance, and even disease. Among these ANFs, soybean allergenic proteins can lead to allergic reactions in human and animals, which has become a public problem all over the world, but our knowledge on it is still inadequate. This paper aims to provide an update on the characteristics, detection or exploration methods, and in vivo research models of soybean allergenic proteins; especially glycinin and β-conglycinin are deeply discussed. Through this review, we may have a better understanding on the advances of research on these two soybean allergenic proteins. Besides, the ingredient processing used to reduce the allergenicity of soybean is also reviewed.

Isolation and in vitro identification of proteinase inhibitors from soybean seeds inhibiting helicoverpa gut proteases

Helicoverpa armigera is a polyphagous pest damaging vast numbers of different crops leading to decrease in total production. Use of Bt transgenic to control H. armigera has worked well but has increased resistance against Bt in H. armigera and controversies about the Bt transgenic making it imperative to find another strategy to control attack. Soybean is a nonhost plant for H. armigera; reason could be laid in the defense system of the soybean. Proteinase Inhibitor (PIs) have been extensively studied for development of resistance against insect pest. Two cultivars developed by our university were investigated for the presence of proteinase inhibitors namely, MAUS-158 and MAUS-61. Partially purified inhibitors were showed inhibition of total protease activity of gut extract by 91.34±1.49 and 89.95±0.96% by MAUS-158 and MAUS-61, respectively. While inhibition of trypsin like proteases were found between 65 and 71% and inhibition of chymotrypsin like proteases ranges between 40 and 42%. The partial purification study shows stability of PIs up to 60°C. Soybean PIs are also showing more prominent inhibition pattern against trypsin than chymotrypsin.

Prevention of cancer and inflammation by protease inhibitors

Several plant-based nutrients and non-nutrients that can inhibit mutagenesis and cell proliferation have been identified. Some of the most promising compounds identified as chemopreventive and anti-metastatic agents include soybean-derived protease inhibitors (PIs), Bowman-Birk Inhibitor (BBI) and Kunitz-Trypsin Inhibitor (KTI). A growing body of evidence suggests that BBI could act as anti-carcinogenic agent and KTI is considered to prevent cancer invasion and metastasis. These inhibitors are non-toxic, are of low cost and can be taken orally or as a part of the daily diet. PIs are undergoing investigation in the clinical setting as potential agents for chemoprevention and anti-metastasis. A complex scenario about the interaction between PIs and cell signaling has been emerging. Soybean PIs are not just anti-proteolytic proteins, but can also be modulators of cell signal transduction. Cancer and inflammatory treatment strategies modulating signal transduction need further investigation.

Growth and development of melon fruit fly larvae under the influence of soybean trypsin inhibitors

Protease inhibitors (PIs) retard growth and development and cause mortality in a range of insect pests belonging to various orders. Some transgenic plants expressing PI genes have also been effective against insect pest attack. The effects of two purified soybean PIs, i.e. trypsin inhibitor (Kunitz type; SBTI) and trypsin-chymotrypsin inhibitor (Bowman-Birk type; SBBI), were investigated on first and third instar larvae of the melon fruit fly Bactrocera Cucurbitae (Coquillett) in laboratory feeding bioassays. The larval period of both the first and third instar larvae was prolonged when the larvae were treated with various concentrations of SBTI and SBBI. The total development period prolonged significantly in both the instars of larvae treated with SBTI, but in SBBI-treated larvae, the development period was delayed significantly only in the third instar larvae. A decrease in percentage pupation and adult emergence was observed in the first and third instar larvae treated with different concentrations of both SBTI and SBBI. The present studies showed that SBTI and SBBI have a potential in inhibiting the growth and development of melon fruit fly, but further detailed and molecular level studies are required before their use in transgenics.

Characteristics of binding of zwitterionic liposomes to water-soluble proteins

The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to (31)P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).

Direct allowance for the effects of thermodynamic nonideality in the quantitative characterization of protein self-association by osmometry

A procedure is described for the direct analysis of osmotic pressure data for reversibly dimerizing proteins that makes allowance for effects of thermodynamic nonideality on the statistical–mechanical basis of the potential-of-mean-force between molecules. Detailed consideration is also given to calculation of the magnitudes of the required virial coefficients. After illustration of the approach with analysis of simulated osmotic pressure data, the method is used to obtain dimerization constants from published osmotic pressure data for soybean proteinase inhibitor, hemoglobin and α-chymotrypsin.

Liposomal formulations of protein proteinase inhibitors: Preparation and specific activity

Here we investigated encapsulation of water-soluble proteins into multilayer liposomes of soybean zwitterionic phospholipid mixtures (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)). The influence of the PC/PE w/w ratio on the incorporation efficiency of the Bowman-Birk soybean proteinase inhibitor (BBI) and aprotinin (BPTI) into liposomes was studied. Protein encapsulation did not affect liposome sizes. Confocal laser scanning microscopy demonstrated that proteins were located in the central part of the spherical particle and between the bilayers. The biological activity (antitrypsin and antichymotrypsin) assay of the protein entrapped in liposomes showed its active sites were spatially shielded. The effect of an ionic detergent on the activity of the encapsulated BBI and BPTI confirmed this hypothesis and suggested that this shielding is reversible. The liposomes stability in three various media-artificial gastric juice and intestinal fluids was also examined. The liposomes prepared seem to be promising formulations for BBI and BPTI delivery.

Novel alleles among soybean Bowman-Birk proteinase inhibitor gene families

Trypsin inhibitors have been found in various animals, plants and microorganisms. There were two types of trypsin inhibitors in soybean including Bowman-Birk protease inhibitors (BBI) and Kunitz inhibitors (KTI). The different BBI genes from wild soybean (G. soja) and cultivated soybean (G. max) formed a multigene family. We constructed a cDNA library of cultivar 'SuiNong 14' seed at the R7 growth stage using the SMART Kit. Seventeen contigs or singletons were highly homologous to soybean protease inhibitors. Contigs of 5, 35, 8 and 9 were highly homologous to BBI family members BBI-A1, BBI-A2, BBI-C and BBI-D, respectively. Sequence analyses showed there were novel allelic variations among the 4 BBI members in SuiNong 14. Based on the comparison of soybean seed cDNA libraries from different developmental stages, it was apparent that the expression of trypsin inhibitors increased during seed development in soybean. Phylogenetic analysis of BBI gene sequences among dicotyledonous and monocotyledonous plants demonstrated that these genes shared a common progenitor.

Effects of soybean proteinase inhibitors on development of the soil mite Scheloribates praeincisus (Acari: Oribatida)

Proteinase inhibitors (PI) are present in plant tissues, especially in seeds, and act as a defense mechanism against herbivores and pathogens. Serine PI from soybean such as Bowman-Birk (BBPI) and Kunitz have been used to enhance resistance of sugarcane varieties to the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), the major pest of this crop. The use of these genetically-modified plants (GM) expressing PI requires knowledge of its sustainability and environmental safety, determining the stability of the introduced characteristic and its effects on non-target organisms. The objective of this study was to evaluate direct effects of ingestion of semi-purified and purified soybean PI and GM sugarcane plants on the soil-dwelling mite Scheloribates praeincisus (Berlese) (Acari: Oribatida). This mite is abundant in agricultural soils and participates in the process of organic matter decomposition; for this reason it will be exposed to PI by feeding on GM plant debris. Eggs of S. praeincisus were isolated and after larvae emerged, immatures were fed milled sugarcane leaves added to semi-purified or purified PI (Kunitz and BBPI) or immatures were fed GM sugarcane varieties expressing Kunitz and BBPI type PI or the untransformed near isogenic parental line variety as a control. Developmental time (larva-adult) and survival of S. praeincisus was evaluated. Neither Kunitz nor BBPI affected S. praeincisus survival. On the other hand, ingestion of semi-purified and purified Kunitz inhibitor diminished duration of S. praeincisus immature stages. Ingestion of GM senescent leaves did not have an effect on S. praeincisus immature developmental time and survival, compared to ingestion of leaves from the isogenic parental plants. These results indicate that cultivation of these transgenic sugarcane plants is safe for the non-target species S. praeincisus.

General up regulation of Spodoptera frugiperda trypsins and chymotrypsins allows its adaptation to soybean proteinase inhibitor

The existence of a diverse serine proteinase gene family in lepidopteran insects suggests they play a significant role in the insect adaptation to plant proteinase inhibitors. These proteinases have been shown to be involved in the process of proteolytic digestion in insect larvae. We carried out a selective transcriptome study of midguts from Spodoptera frugiperda larvae fed on a diet supplemented with soybean proteinase inhibitor (SPI). Using subtracted cDNA libraries made of gut-expressed transcripts, a total of 2100 partial sequences were obtained, of those 38% were related to digestive process. Two large and diverse groups of chymotrypsins and trypsins were obtained, and some of these proteinase-encoding genes were further characterized by quantitative RT-PCR. The transcription analyses revealed two groups: one group of genes constitutively expressed in the control larvae that is up regulated by introducing SPI to the diet, and a second group that is absent in the control but is induced by the SPI-rich diet. This observation suggests that adaptation of S. frugiperda to SPI involves de novo synthesis and also up regulation of existing enzymes. Proteases from intestines of larvae reared on a diet with SPI showed insensitivity to the inhibitor. The proteases were also insensitive to a broad-spectrum potato proteinase inhibitor preparation. We propose that adaptation of S. frugiperda to SPI follows a “shotgun” approach, based on a general up regulation of a large set of endoproteinases.