Search Results (24)

Drug-resistant bacteria cause millions of global infections each year, and the development of alternative antimicrobial drugs has become a serious undertaking. Currently, peptides with antimicrobial activity represent potential candidates for new antibiotic discovery as they are less likely to cause drug resistance in bacteria. In this study, bifunctional peptides with potent trypsin-inhibitory activity and antimicrobial activity were obtained by rational computation-based structural modifications to a novel Bowman–Birk-type inhibitor (BBI) peptide. The analogues not only displayed potent bacterial killing ability against two drug-resistant bacteria strains of E. coli but also an excellent safety profile, as assessed by low haemolytic activity and low anti-proliferation activity on HaCaT cells. Throughout the molecular dynamics simulations, the peptides exhibited stable adsorption onto the mixed POPE/POPG membrane; most amino acid residues of the AMPs remained bound to the membrane surface, with a few amino acid residues partially penetrating the membrane interior. This showed that the electrostatic interactions were the dominant driving force mediating the peptide–membrane associations. In addition, the tested peptides displayed a degree of stability in the presence of salt ions, serum, and trypsin. These modified peptides thus possess potential as clinical antibacterial agents, and the strategies used in structural modification may also provide a different path to developing new antimicrobial peptides. Full article

Soybean meal (SBM) is extensively used as a predominant protein source in animal feed. However, raw soybean cannot be directly utilized in animal feed, due to the presence of the Kunitz trypsin inhibitor (KTi) and the Bowman–Birk protease inhibitor (BBi). These antinutritional factors inhibit the digestive enzymes in animals, trypsin and chymotrypsin, resulting in poor animal performance. To inactivate the activity of protease inhibitors, SBM is subjected to heat processing, a procedure that can negatively impact the soybean protein quality. Thus, it would be beneficial to develop soybean varieties with little or no trypsin inhibitors. In this study, we report on the creation of experimental soybean lines with significantly reduced levels of Bowman–Birk protease inhibitors. RNA interference (RNAi) technology was employed to generate several transgenic soybean lines. Some of these BBi knockdown soybean lines showed significantly lower amounts of both trypsin and chymotrypsin inhibitor activities. Western blot analysis revealed the complete absence of BBi in selected RNAi-derived lines. RNA sequencing (RNAseq) analysis demonstrated a drastic reduction in the seed-specific expression of BBi genes in the transgenic soybean lines during seed development. Confocal fluorescence immunolabeling studies showed that the accumulation of BBi was drastically diminished in BBi knockdown lines compared to wild-type soybeans. The absence of BBi in the transgenic soybean did not alter the overall protein, oil, and sulfur amino acid content of the seeds compared to wild-type soybeans. The seed protein from the BBi knockdown lines were more rapidly hydrolyzed by trypsin and chymotrypsin compared to the wild type, indicating that the absence of BBi enhances protein digestibility. Our study suggests that these BBi knockdown lines could be a valuable resource in order for plant breeders to incorporate this trait into commercial soybean cultivars, potentially enabling the use of raw soybeans in animal feed. Full article

Soybean Trypsin Inhibitors (STIs) in soy-based foods have negative effects on soybean protein digestion and pancreatic health of humans. The inactivation of STIs is a critical unit operation aimed at enhancing the nutritional properties of soy-based foods during processing. This paper reviews the structure of STIs and soybean proteins, as well as the mechanisms of digestion. Various technologies (physical, chemical, biological) have been used to inactivate STIs. Their parameter settings, operating procedures, advantages, and disadvantages are also described. Mechanisms of inactivation of STIs (Kunitz trypsin inhibitor (KTI) and Bowman–Birk inhibitor (BBI)) conformations under different treatments are clarified. In addition, emerging technologies, e.g., Ohmic Heating, Electron Beam Irradiation, Dielectric-Barrier Discharge, and probiotics, have demonstrated great potential to inactivate STIs. We advise that multiple emerging technologies should combine with other unit operating systems to maximize inactivation efficiency. Full article

Wheat is one of the most extensively grown crops in the world; however, its productivity is reduced due to salinity. This study focused on millimeter wave (MMW) irradiation to clarify the salt-stress tolerance mechanism in wheat. In the present study, wheat-root growth, which was suppressed to 77.6% of the control level under salt stress, was recovered to the control level by MMW irradiation. To reveal the salt-stress tolerance mechanism of MMW irradiation on wheat, a proteomic analysis was conducted. Proteins related to cell cycle, proliferation, and transport in biological processes, as well as proteins related to the nucleus, cytoskeleton, and cytoplasm within cellular components, were inversely correlated with the number of proteins. The results of the proteomic analysis were verified by immunoblot and other analyses. Among the proteins related to the scavenging reactive-oxygen species, superoxide dismutase and glutathione reductase accumulated under salt stress and further increased in the MMW-irradiated wheat. Among pathogen-related proteins, pathogenesis-related protein 1 and the Bowman–Birk proteinase inhibitor decreased under salt stress and recovered to the control level in the MMW-irradiated wheat. The present results indicate that MMW irradiation of wheat seeds improves plant-growth recovery from salt stress through regulating the reactive oxygen species-scavenging system and the pathogen-related proteins. These genes may contribute to the development of salt-stress-tolerant wheat through marker-assisted breeding and genome editing. Full article

Plant defense polypeptides play a crucial role in providing plants with constitutive immunity against various biotic and abiotic stressors. In this study, we explored a complex of proteins from wheatgrass (Elytrigia elongata) spikelets to estimate their role in the plant’s tolerance to various environmental factors. The current research shows that in vitro protein extracts from E. elongata spikelets possess antifungal activity against certain Fusarium species, which are specific cereal pathogens, at concentrations of 1–2 mg/mL. In this study, we reproduced these antifungal activities using a 4 mg/mL extract in artificial fungal infection experiments on wheat grain (Triticum aestivum) under controlled laboratory conditions. Furthermore, the tested extract demonstrated a protective effect on Saccharomyces cerevisiae exposed to hyper-salinity stress at a concentration of 2 mg/mL. A combined scheme of fractionation and structural identification was applied for the estimation of the diversity of defense polypeptides. Defensins, lipid-transfer proteins, hydrolase inhibitors (cereal bifunctional trypsin/alpha-amylase inhibitors from a Bowman–Birk trypsin inhibitor), and high-molecular-weight disease resistance proteins were isolated from the extract. Thus, wheatgrass spikelets appear to be a reservoir of defense polypeptides. Our findings contribute to a deeper understanding of plant defense proteins and peptides and their involvement in the adaptation to various stress factors, and they reveal the regulatory effect at the ecosystem level. Full article

Despite the high quality of soybean protein, raw soybeans and soybean meal cannot be directly included in animal feed mixtures due to the presence of Kunitz (KTi) and Bowman–Birk protease inhibitors (BBis), which reduces animal productivity. Heat treatment can substantially inactivate trypsin and chymotrypsin inhibitors (BBis), but such treatment is energy-intensive, adds expense, and negatively impacts the quality of seed proteins. As an alternative approach, we have employed CRISPR/Cas9 gene editing to create mutations in BBi genes to drastically lower the protease inhibitor content in soybean seed. Agrobacterium-mediated transformation was used to generate several stable transgenic soybean events. These independent CRISPR/Cas9 events were examined in comparison to wild-type plants using Sanger sequencing, proteomic analysis, trypsin/chymotrypsin inhibitor activity assays, and qRT-PCR. Collectively, our results demonstrate the creation of an allelic series of loss-of-function mutations affecting the major BBi gene in soybean. Mutations in two of the highly expressed seed-specific BBi genes lead to substantial reductions in both trypsin and chymotrypsin inhibitor activities. Full article

This study explores the impact of RNAi in terms of selectively inhibiting the expression of the OsBBTI5 gene, with the primary objective of uncovering its involvement in the molecular mechanisms associated with salt tolerance in rice. OsBBTI5, belonging to the Bowman–Birk inhibitor (BBI) family gene, is known for its involvement in plant stress responses. The gene was successfully cloned from rice, exhibiting transcriptional self-activation in yeast. A yeast two-hybrid assay confirmed its specific binding to OsAPX2 (an ascorbate peroxidase gene). Transgenic OsBBTI5-RNAi plants displayed insensitivity to varying concentrations of 24-epibrassinolide in the brassinosteroid sensitivity assay. However, they showed reduced root and plant height at high concentrations (10 and 100 µM) of GA₃ immersion. Enzyme activity assays revealed increased peroxidase (POD) and superoxide dismutase (SOD) activities and decreased malondialdehyde (MDA) content under 40-60 mM NaCl. Transcriptomic analysis indicated a significant upregulation of photosynthesis-related genes in transgenic plants under salt stress compared to the wild type. Notably, this study provides novel insights, suggesting that the BBI gene is part of the BR signaling pathway, and that OsBBTI5 potentially enhances stress tolerance in transgenic plants through interaction with the salt stress-related gene OsAPX2. Full article

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-Phe^P(OPh)₂]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-Phe^P(OPh)₂ (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman–Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin. Full article

Inflammation and cancer are diseases caused by genetic and environmental factors as well as altered microbiota. Diet plays a role, with leguminous such as beans (Phaseolus vulgaris, Vicia faba), chickpeas (Cicer arietinum), lentils (Lens culinaris), peas (Pisum sativum) and soybeans (Glycine max), known to prevent such diseases. Processing of food leguminous yields aqueous side streams. These products are nothing short of water extracts of leguminous, containing albumin, globulin, saponins, and oligosaccharides. This review analysed the most recent findings on the anticancer activities of legume-soluble nutrients. Albumin from chickpeas and peas inhibits the pro-inflammatory mediator interleukins, while soy Bowman–Birk Inhibitor inhibits serine proteases. The peptide vicilin activates peroxisome proliferator-activated receptor, mediating triglyceride metabolism. Soyasaponins promote apoptosis of cancer cells by activating caspases and by enhancing the concentration of intracellular calcium. Soyasapogenol regulates specific protein pathways, leading to apoptosis. Oligosaccharides such as raffinose and stachyose promote the synthesis of short chain fatty acids, balancing the intestinal microbiota, as result of their prebiotic activity. Verbascoside also modulate signalling pathways, leading to apoptosis. In closing, water extracts of leguminous have the potential to be efficient anticancer ingredients, by means of numerous mechanisms based on the raw material and the process. Full article

The objective of the investigation was to understand the biochemical activities of hydrolysate of soybean milk protein (SMP). Hydrolysis was carried out by different concentrations of papain (0.008 g·L⁻¹, 0.016 g·L⁻¹, 0.032 g·L⁻¹ and 0.064 g·L⁻¹). The antioxidant capacity was measured by the ferric-reducing ability of plasma (FRAP) and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assays. The anti-angiotensin activity of hydrolysate was measured by the recombinant angiotensin converting enzyme and substrate Abz-FRK(Dnp)-P. The contributions of the Kunitz trypsin inhibitor (KTI) and Bowman–Birk inhibitor (BBI) on antigenicity, and the in vitro digestion of papain-hydrolyzed SMP were studied. Rabbit polyclonal anti-KTI and anti-BBI antibodies together with peroxidase-labelled goat anti-Rb IgG secondary antibody were used to identify the antigenicity of KTI and BBI in unhydrolyzed and papain-hydrolyzed SMP. The antioxidant capacity and anti-angiotensin activity of SMP were increased after the papain hydrolysis of SMP. The KTI- and BBI-specific antigenicity were reduced in SMP by increasing the concentration of papain. However, there was interaction between papain-hydrolyzed SMP and trypsin in native gel, while interaction with chymotrypsin was absent. The interaction between trypsin and SMP was reduced due to the hydrolysis of papain in a concentration-dependent manner. According to the in vitro gastrointestinal digestion simulation protocol (Infogest), the digestibility of SMP was not statistically increased. Full article

The Bowman–Birk protease inhibitor (BBI) family is a prototype group found mainly in plants, particularly grasses and legumes, which have been subjected to decades of study. Recently, the discovery of attenuated peptides containing the canonical Bowman–Birk protease inhibitory motif has been detected in the skin secretions of amphibians, mainly from Ranidae family members. The roles of these peptides in amphibian defense have been proposed to work cooperatively with antimicrobial peptides and reduce peptide degradation. A novel trypsin inhibitory peptide, named livisin, was found in the skin secretion of the green cascade frog, Odorrana livida. The cDNA encoding the precursor of livisin was cloned, and the predicted mature peptide was characterized. The mature peptide was found to act as a potent inhibitor against several serine proteases. A comparative activity study among the native peptide and its engineered analogs was performed, and the influence of the P₁ and P_2′ positions, as well as the C-terminal amidation on the structure–activity relationship for livisin, was illustrated. The findings demonstrated that livisin might serve as a potential drug discovery/development tool. Full article

A new approach to on-resin detection of three model proteases (trypsin, chymotrypsin, and thrombin) has been developed, while at the same time already described methodology for simultaneous detection of two enzymes (trypsin and chymotrypsin) has been additionally generalized. Appropriate immobilized substrates, comprising specifically cleavable peptide sequences capped with fluorescent dyes, have been synthesized on Rink Amide PEGA resin or Amino PEGA resin modified with backbone amide linker (BAL). Resulting solid support-bound probes were then dispersed into Tris-HCl buffer solution (pH = 8.0) and subjected to enzymatic cleavage. Liberated fluorophores have been tracked by fluorescence measuring. The competitive activities of studied proteases towards the thrombin probe have been efficiently limited and controlled by employing a Bowman-Birk inhibitor into a system. Full article

Plant-derived protease inhibitors (PI), such as Bowman-Birk inhibitors and Kunitz-type inhibitors, have been suggested to negatively affect dietary protein digestion by blocking the activity of trypsin and chymotrypsin in the human gastrointestinal system. In addition, some PIs may possess proinflammatory activities. However, there is also scientific evidence on some beneficial effects of PIs, for example, gut-related anti-inflammatory and chemopreventive activities in vitro and in vivo. Some PIs are sensitive to processing and digestion; thus, their survival is an important aspect when considering their positive and negative bioactivities. The aim of this review was to evaluate the relevance of PIs in protein digestion in humans and to discuss the potential of PIs from whole foods and as purified compounds in decreasing symptoms of bowel-related conditions. Based on the reviewed literature, we concluded that while the complex interactions affecting plant protein digestibility and bioavailability remain unclear, PI supplements could be considered for targeted purposes to mitigate inflammation and gastric pain. Full article

Environmental factors, particularly diet, are considered central to the pathogenesis of the inflammatory bowel diseases (IBD), Crohn’s disease and ulcerative colitis. In particular, the Westernization of diet, characterized by high intake of animal protein, saturated fat, and refined carbohydrates, has been shown to contribute to the development and progression of IBD. During the last decade, soybean, as well as soy-derived bioactive compounds (e.g., isoflavones, phytosterols, Bowman-Birk inhibitors) have been increasingly investigated because of their anti-inflammatory properties in animal models of IBD. Herein we provide a scoping review of the most studied disease mechanisms associated with disease induction and progression in IBD rodent models after feeding of either the whole food or a bioactive present in soybean. Full article

Bowman-Birk inhibitors (BBIs) are found primarily in seeds of legumes and in cereal grains. These canonical inhibitors share a highly conserved nine-amino acids binding loop motif CTP1SXPPXC (where P1 is the inhibitory active site, while X stands for various amino acids). They are natural controllers of plants’ endogenous proteases, but they are also inhibitors of exogenous proteases present in microbials and insects. They are considered as plants’ protective agents, as their elevated levels are observed during injury, presence of pathogens, or abiotic stress, i.a. Similar properties are observed for peptides isolated from amphibians’ skin containing 11-amino acids disulfide-bridged loop CWTP1SXPPXPC. They are classified as Bowman-Birk like trypsin inhibitors (BBLTIs). These inhibitors are resistant to proteolysis and not toxic, and they are reported to be beneficial in the treatment of various pathological states. In this review, we summarize up-to-date research results regarding BBIs’ and BBLTIs’ inhibitory activity, immunomodulatory and anti-inflammatory activity, antimicrobial and insecticidal strength, as well as chemopreventive properties. Full article