Kunitz Soybean Trypsin Inhibitor Research Articles

Purification of soybean Kunitz trypsin inhibitor and the mechanism of its passivation by lysine and disulfide bond modifications

Soybean trypsin inhibitor（STI）was selectively recovered from soybean whey, a by-product of soybean protein production, by ammonium sulfate salting and the STI was purified up to 5.46-fold (Compared to STI in soybean whey). After purification with a Superdex 75 increase gel filtration preloader column, a 91.5% electrophoretically pure soybean Kunitz trypsin inhibitor（SKTI）was obtained by optical density analysis with a purification ploidy of 33.32-fold and a specific activity of 1072.9 U/mg, and a single band protein with a molecular weight of 20 kDa on SDS-PAGE. SKTI's 85.65% inhibitory activity was maintained after heating at 80 °C for 30 min, and there was no significant change in inhibitory activity in the range of pH 3–10. The secondary structure of SKTI did not change with temperature and pH showing good stability. By chemical bond modification of SKTI, the activity of SKTI was found to have an important connection with lysine and disulfide bond. This study set up a process for the rapid preparation of STI by salinization and the related properties of SKTI was explored to provide more potential for the treatment of soy protein production waste and the application of STI in the food field in the future.

Canonical or noncanonical? Structural plasticity of serine protease-binding loops in Kunitz-STI protease inhibitors.

The Kunitz-Soybean Trypsin Inhibitor (Kunitz-STI) family is a large family of proteins with most of its members being protease inhibitors. The versatility of the inhibitory profile and the structural plasticity of these proteins, make this family a promising scaffold for designing new multifunctional proteins. Historically, Kunitz-STI inhibitors have been classified as canonical serine protease inhibitors, but new inhibitors with novel inhibition mechanisms have been described in recent years. Different inhibition mechanisms could be the result of different evolutionary pathways. In the present work, we performed a structural analysis of all the crystallographic structures available for Kunitz-STI inhibitors to characterize serine protease-binding loop structural features and locations. Our study suggests a relationship between the conformation of serine protease-binding loops and the inhibition mechanism, their location in the β-trefoil fold, and the plant source of the inhibitors. The classical canonical inhibitors of this family are restricted to plants from the Fabales order and bind their targets via the β4-β5 loop, whereas serine protease-binding loops in inhibitors from other plants lie mainly in the β5-β6 and β9-β10 loops. In addition, we found that the β5-β6 loop is used to inhibit two different families of serine proteases through a steric blockade inhibition mechanism. This work will help to change the general perception that all Kunitz-STI inhibitors are canonical inhibitors and proteins with protease-binding loops adopting noncanonical conformations are exceptions. Additionally, our results will help in the identification of protease-binding loops in uncharacterized or newly discovered inhibitors, and in the design of multifunctional proteins.

Trypsin Inhibitor from Soybean Whey Wastewater: Isolation, Purification and Stability

Soybean trypsin inhibitor (STI) was obtained from simulated soybean whey wastewater through a sustainable method consisting of isoelectric precipitation, ammonium sulfate salting out, and gel filtration chromatography, and the effect of temperature, pH, and pepsin on the stability of STI was also discussed. The results showed that the recovery rate of the trypsin inhibitory activity was 89.47%, the purity and the specific activity of STI were 71.11%, and 1442.5 TIU/mg in the conditions of pH 4.0 and 40% ammonium sulfate saturation. The soybean Kunitz trypsin inhibitor (KTI) and soybean Bowman–Brik trypsin inhibitor (BBI) were obtained via gel filtration chromatography, and their specific activity levels were 1733.5 TIU/mg and 2588.3 TIU/mg, respectively. The STI displayed good stability over a wide temperature and pH range. The STI, KTI, and BBI were all resistant to pepsin hydrolysis, and their ability was ranked as BBI > STI > KTI. These findings will provide a theoretical basis for recycling STI from soybean whey wastewater and promoting better active compound utilization.

Bovine pancreatic trypsin inhibitor and soybean Kunitz trypsin inhibitor: Differential effects on proteases and larval development of the soybean pest Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Pest management is challenged with resistant herbivores and problems regarding human health and environmental issues. Indeed, the greatest challenge to modern agriculture is to protect crops from pests and still maintain environmental quality. This study aimed to analyze by in silico, in vitro, and in vivo approaches to the feasibility of using the inhibitory protein extracted from mammals – Bovine Pancreatic Trypsin Inhibitor (BPTI) as a potential inhibitor of digestive trypsins from the pest Anticarsia gemmatalis and comparing the results with the host-plant inhibitor – Soybean Kunitz Trypsin Inhibitor (SKTI). BPTI and SKTI interacts with A. gemmatalis trypsin-like enzyme competitively, through hydrogen and hydrophobic bonds. A. gemmatalis larvae exposed to BPTI did not show two common adaptative mechanisms i.e., proteolytic degradation and overproduction of proteases, presenting highly reduced trypsin-like activity. On the other hand, SKTI-fed larvae did not show reduced trypsin-like activity, presenting overproduction of proteases and SKTI digestion. In addition, the larval survival was reduced by BPTI similarly to SKTI, and additionally caused a decrease in pupal weight. The non-plant protease inhibitor BPTI presents intriguing element to compose biopesticide formulations to help decrease the use of conventional refractory pesticides into integrated pest management programs.

Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid

Protein-based targeting reagents, such as antibodies and non-antibody scaffold proteins, are rapidly inactivated in the upper gastrointestinal (GI) tract. Hydrochloric acid in gastric juice denatures proteins and activates pepsin, concentrations of which reach 1 mg/mL in the mammalian stomach. Two stable scaffold proteins (nanobody and nanofitin), previously developed to be protease-resistant, were completely digested in less than 10 min at 100-fold lower concentration of pepsin than found in the stomach. Here we present gastrobodies, a protein scaffold derived from Kunitz soybean trypsin inhibitor (SBTI). SBTI is highly resistant to the challenges of the upper GI tract, including digestive proteases, pH 2 and bile acids. Computational prediction of SBTI’s evolvability identified two nearby loops for randomization, to create a potential recognition surface which was experimentally validated by alanine scanning. We established display of SBTI on full-length pIII of M13 phage. Phage selection of gastrobody libraries against the glucosyltransferase domain of Clostridium difficile toxin B (GTD) identified hits with nanomolar affinity and enzyme inhibitory activity. Anti-GTD binders retained high stability to acid, digestive proteases and heat. Gastrobodies show resilience to exceptionally harsh conditions, which should provide a foundation for targeting and modulating function within the GI tract.

Proteolytic enzymes in the salivary glands of the Neotropical brown stink bugEuschistus heros: Reduced activities in imidacloprid‐resistant strains

AbstractProteases present in the salivary glands of phytosuccivorous insects are responsible for the initial breakdown of dietary protein into the amino acids necessary for insect growth and development. Here, we characterised the total proteases and trypsin‐like activities in the salivary glands of the Neotropical brown stink bug,Euschistus heros(F.) and used in silico and biochemical approaches to determine the interactions between trypsin‐like enzymes and the soybean Kunitz trypsin inhibitor (SKTI). Additionally, we evaluated whether these enzyme activities were affected in individuals resistant to the actions of the insecticide imidacloprid. We used an originally field‐collected strain to select individuals resistant to imidacloprid alone (ImiGoiasRes) or synergised with piperonyl butoxide (ImiPBORes). A laboratory‐susceptible (ImiSusc) strain was also used to select piperonyl butoxide synergised imidacloprid‐resistant (ImiPBOSel) individuals. Our results revealed that pH and temperature significantly affected the specific trypsin‐like activities in imidacloprid‐susceptible individuals. Significant alterations in the total protease activities were recorded for field‐ (ImiGoiasRes) and laboratory‐selected imidacloprid‐resistant individuals (ImiPBOSelandImiPBORes). Our computational docking analysis demonstrated that the molecular interactions and the presence of the SKTI inhibitor (0.5 mg mL−1) could reduce up to 40% of the trypsin‐like activities of imidacloprid‐susceptible individuals. However, all imidacloprid‐resistant strains exhibited significantly less trypsin‐like activities and exhibited lower hydrolysing velocity (VMAX) and affinity (KM) rates, and the prior exposure to SKTI only synergised the imidacloprid toxicity for individuals ofImiFieldResstrain. Collectively, our findings demonstrate, for the first time, insecticide resistance detrimentally affected the activities of proteolytic enzymes in phytosuccivorous stink bug pests.

Preparation and Irreversible Inhibition Mechanism Insight into a Recombinant Kunitz Trypsin Inhibitor from Glycine max L. Seeds

Soybean Kunitz trypsin inhibitor (SKTI), extracted from soybean (Glycine max L.) seeds, possesses insect resistance and anti-tumor properties. But its specific mechanisms of action are not yet known. This article reports an efficient method to produce recombinant SKTI (rSKTI) in Escherichia coli, reveals some biochemical properties of rSKTI, and discusses the inhibition mechanism of SKTI. The rSKTI was expressed as inclusion body in E. coli BL21 (DE3). After refolding, the active rSKTI was obtained and was further purified with anion-exchange chromatography (DEAE-FF) efficiently. There were similar biochemical properties between SKTI and rSKTI. The optimum pH and the optimum temperature were pH 8.0 and 35 °C, respectively, being stable during pH 7.0–11.0 and below 37 °C. The activity against trypsin was inhibited by Co2+, Mn2+, Fe3+, Al3+, and epoxy chloropropane. Inhibition kinetic assay of SKTI against trypsin as Lineweaver-Burk plots analysis both showed an unchanged Km and a decreased Vmax with N-benzoyl-l-arginine ethyl ester (BAEE) as substrate. Molecular modeling showed Arg63 of SKTI (active residue of SKTI) that interacts with four residues of trypsin, including three catalytic site (His57, Asp102, and Ser195) and one binding site (Asp189), forming five interactions. These provide reference for understanding the inhibition mechanism of such kind of Kunitz trypsin inhibitors.

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.

Soybean Trypsin Inhibitors: Selective Inactivation at Hydrolysis of Soybean Proteins by Some Enzymatic Complexes

Inactivation of the Kunitz and Bowman-Birk soybean trypsin inhibitors at hydrolysis of soybean proteins by enzymatic complexes was studied. These complexes are derived from king crab hepatopancreas, cod fish pyloric caeca, and a commercial enzymatic complex of protosubtilin. Analysis of the soybean protein hydrolysates showed that these enzymatic complexes completely digested the Kunitz trypsin inhibitor (60–70% of the total trypsin inhibitors) and had almost no effect on the Bowman-Birk trypsin and chymotrypsin inhibitor. All of the enzymatic complexes contain different sets of enzymes with different proteolytic specificity. This allow to make the conclusion that Bowman-Birk inhibitor is highly resistant to proteolysis and is not inactivated at enzymatic hydrolysis.

Macromolecular properties and partial amino acid sequence of a Kunitz-type protease inhibitor from okra (Abelmoschus esculentus) seeds

A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seeds by a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performance liquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circular dichroism spectrum showed that the protein contains approx. 39% beta-sheets but only approx. 5% alpha-helices. The protein is thermally quite stable, and exhibits a cooperative thermal unfolding transition at approx. 70 degree C, as determined by circular dichroism spectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS) allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identity with a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 and Cys156 was also detected. The protein showed approx 24 and approx 25% sequence identity with alpha-amylase/subtilisin inhibitor from barley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structural fold similar to other Kunitz-type TIs. The presence of Cys149-Cys156 disulfide bond as detected by MS and a second disulfide bond connecting Cys44-Cys91, conserved in all Kunitz-type TIs, is also identified in the model.

Worm wounding increases levels of pollen-related food allergens in soybean (Glycine max)

The levels of food allergens in worm-wounded or non-wounded green soybeans (edamame) and mature soybeans were investigated by immunoblotting and enzyme-linked immunosorbent assay (ELISA), using allergen-specific antibodies. Non-wounded and worm-wounded soybeans showed similar total protein profiles after Coomassie brilliant blue staining, but some protein bands were observed to have been changed by worm wounding. Immunoblotting with specific antibodies for major soybean allergens (Gly m 5, Gly m 6, Gly m Bd 30K, and Kunitz soybean trypsin inhibitor) revealed that protein band profiles and intensities were not significantly changed by worm wounding. In contrast, levels of the pollen-related soybean allergens Gly m 4 and Gly m 3 were strongly increased by worm wounding in both green and mature soybeans, as detected by immunoblotting and ELISA. These results suggested that the pollen-related food allergen risk (i.e., oral allergy syndrome; OAS) from soybeans might be enhanced by worm wounding of soybeans.

Combining 2-DE immunoblots and mass spectrometry to identify putative soybean (Glycine max) allergens

Soybean is recognized as a commonly allergenic food, but the identity of important allergens is not well studied. Recently, some global regulatory agencies started requiring quantitative analysis of individual allergens, including unproven allergens, as part of the risk assessment for genetically engineered (GE) soybeans. We sought to identify soybean proteins that bind IgE from any of 10 individual soybean-sensitized subjects. Soybean IgE binding proteins were identified by 2-DE immunoblots using sera from four soy-allergic and plasma from six soy-sensitized human subjects. Corresponding spots were excised from stained gels, digested, and analyzed using a quadrupole TOF Synapt G2-S tandem mass spectrometer. Results showed the major IgE binding proteins were subunits of either β-conglycinin (Gly m 5) or glycinin (Gly m 6). Soybean Kunitz trypsin inhibitor (SKTI) was a significant IgE binding protein for four subjects. Soybean agglutinin, seed biotinylated protein (SBP) of 65 kDa, late embryogenesis protein (LEP), and sucrose-binding protein were identified as IgE binding only for soy-sensitized subjects. We conclude that the major soybean allergens are isoforms of Gly m 5, Gly m 6, and possibly SKTI and that requirements for quantitative measurement of proteins that are not clear allergens is not relevant to safety.

Inga laurina trypsin inhibitor (ILTI) obstructs Spodoptera frugiperda trypsins expressed during adaptive mechanisms against plant protease inhibitors

Plant protease inhibitors (PIs) are elements of a common plant defense mechanism induced in response to herbivores. The fall armyworm, Spodoptera frugiperda, a highly polyphagous lepidopteran pest, responds to various PIs in its diet by expressing genes encoding trypsins. This raises the question of whether the PI-induced trypsins are also inhibited by other PIs, which we posed as the hypothesis that Inga laurina trypsin inhibitor (ILTI) inhibits PI-induced trypsins in S. frugiperda. In the process of testing our hypothesis, we compared its properties with those of selected PIs, soybean Kunitz trypsin inhibitor (SKTI), Inga vera trypsin inhibitor (IVTI), Adenanthera pavonina trypsin inhibitor (ApTI), and Entada acaciifolia trypsin inhibitor (EATI). We report that ILTI is more effective in inhibiting the induced S. frugiperda trypsins than SKTI and the other PIs, which supports our hypothesis. ILTI may be more appropriate than SKTI for studies regarding adaptive mechanisms to dietary PIs.

IMGN632: A CD123-Targeting Antibody-Drug Conjugate (ADC) with a Novel DNA-Alkylating Payload, Is Highly Active and Prolongs Survival in Acute Myeloid Leukemia (AML) Xenograft Models

ADCs targeting cytotoxic payloads to cancer cells using antibodies that recognize tumor-associated antigens are an expanding therapeutic area. CD123, the IL-3 receptor alpha-subunit, is an attractive cancer target implicated in AML cell survival and proliferation. While CD123 is expressed on AML blasts and is associated with aggressive disease, it is also differentially expressed on AML stem cells relative to normal hematopoietic cells. We report the pre-clinical evaluation of IMGN632, an ADC comprising a CD123-binding antibody and a DNA-alkylating, indolino-benzodiazepine payload (termed an IGN), in disseminated and subcutaneous (SC) AML xenograft models which possess poor prognosis markers or demonstrate drug resistance.Novel anti-CD123 antibodies were generated in mice by immunization with a human CD123-expressing cell line. Following antibody selection and humanization in the IgG1 format, IMGN632 was produced by conjugating a novel DNA-alkylating payload (DGN549), via a cleavable peptide linker, to the anti-CD123 antibody at engineered cysteine residues, resulting in an ADC with ~2 DGN549 molecules per antibody. The in vitro cytotoxicity of IMGN632 and of a non-targeted control ADC (the DGN549 payload conjugated to an antibody against Kunitz soybean trypsin inhibitor) on human cancer cell lines were evaluated by determining cell viability using the WST-8 reagent after continuous ADC exposure for up to 7 days. The IC50 was determined for each ADC and the specificity ratio (IC50 of control ADC: IC50 of IMGN632) was calculated for each cell line. The antitumor activity of IMGN632 and the control ADC were assessed in vivo in immuno-deficient mice bearing Molm-13, Kasumi-3-Luc or MV4-11 disseminated or EOL-1 sc human AML xenografts. Subcutaneous tumor volumes were measured twice weekly. Mice bearing Kasumi-3-Luc were live-animal imaged approximately twice weekly to quantify bioluminescent tumor burden. The maximum tolerated dose (MTD) of IMGN632 was determined by administering single intravenous (IV) injections of IMGN632 to non-tumor bearing female CD-1 mice. In all studies, assessment was terminated for weight loss > 20% or for clinical signs or, in the EOL-1 model, when SC tumor volume reached approximately 1000 mm3.IMGN632 was highly potent in vitro towards CD123-expressing human AML cell lines with poor prognostic markers (EOL-1, Molm-13 (FLT3-ITD), MV4-11 (FLT3-ITD) and Kasumi-3 (p53, MDR1+)), with IC50 values of < 3 pMol and high specificity ratios ranging from 100 to 2000. In contrast, IMGN632 had IC50 values of > 8000 pMol and a specificity ratio of 1 in CD123-negative human cell lines (Namalwa, K562). These data confirm the CD123-directed activity of IMGN632 in vitro.IMGN632 displayed antigen-specific antitumor activity in immuno-deficient mice bearing disseminated xenografts, resulting in prolonged survival, with an increase in life span from 50% to > 262%. In these models, the control ADC was inactive, with no increase in life span. In the Kasumi-3-Luc disseminated model, treatment with highly active doses of 240 or 800 mcg/kg IMGN632 (by antibody) resulted in decreased bioluminescent tumor burden. In the other disseminated models, IMGN632 was highly active at doses of 80 or 240 mcg/kg in MV4-11 and at doses as low as 8 mcg/kg in Molm-13, which, when taken together with the MTD of 8000 mcg/kg, generated a therapeutic index of 1000. In the azacitidine- and cytarabine-resistant EOL-1 SC model, IMGN632 was highly active at a dose of 240 mcg/kg, resulting in 8/8 long-term complete responses, while unconjugated DGN549, anti-CD123 antibody and control ADC were inactive at IMGN632-matched doses.In conclusion, IMGN632 exhibits potent, CD123-specific in vitro activity against AML cell lines, including those with markers of poor prognosis such as MDR1, p53 and FLT3-ITD. IMGN632 is highly active in vivo against Molm-13, Kasumi-3 and MV4-11 disseminated xenografts, resulting in prolonged survival and reduced tumor burden. In addition, IMGN632 is highly active against EOL-1 xenografts, which appear to be resistant to azacitidine and cytarabine in vivo, but highly sensitive to the ADC, resulting in tumor regression and prolonged tumor-free survival. These findings support advancing IMGN632 into clinical trials. DisclosuresAdams:ImmunoGen, Inc.: Employment. Wilhelm:ImmunoGen, Inc.: Employment. Harvey:ImmunoGen, Inc.: Employment. Bai:ImmunoGen, Inc.: Employment. Yoder:ImmunoGen, Inc.: Employment. Kovtun:ImmunoGen, Inc.: Employment. Chittenden:ImmunoGen, Inc.: Employment. Pinkas:ImmunoGen, Inc.: Employment.

Adaptive regulation of digestive serine proteases in the larval midgut of Helicoverpa armigera in response to a plant protease inhibitor

Protease inhibitors (PIs) are direct defenses induced by plants in response to herbivory. PIs reduce herbivore digestive efficiency by inhibiting insects' digestive proteases; in turn insects can adapt to PIs by generally increasing protease levels and/or by inducing the expression of PI-insensitive proteases. Helicoverpa armigera, a highly polyphagous lepidopteran insect pest, is known for its ability to adapt to PIs. To advance our molecular and functional understanding of the regulation of digestive proteases, we performed a comprehensive gene expression experiment of H. armigera exposed to soybean Kunitz trypsin inhibitor (SKTI) using a custom-designed microarray. We observed poor larval growth on the SKTI diet until 24 h, however after 48 h larvae attained comparable weight to that of control diet. Although initially the expression of several trypsins and chymotrypsins increased, eventually the expression of some trypsins decreased, while the number of chymotrypsins and their expression increased in response to SKTI. Some of the diverged serine proteases were also differentially expressed. The expression of serine proteases observed using microarrays were further validated by qRT-PCR at different time points (12, 24, 48, 72 and 96 h) after the start of SKTI ingestion. There were also large changes in transcriptional patterns over time in the control diet. Carbohydrate metabolism and immune defense genes were affected in response to SKTI ingestion. Enzyme assays revealed reduced trypsin-specific activity and increased chymotrypsin-specific activity in response to SKTI. The differential regulation of trypsins and chymotrypsins at the transcript and protein levels accompanying a rebound in growth rate indicates that induction of SKTI-insensitive proteases is an effective strategy of H. armigera in coping with this protease inhibitor in its diet.

Identification of a new soybean kunitz trypsin inhibitor mutation and its effect on bowman-birk protease inhibitor content in soybean seed.

Soybean seed contains antinutritional compounds that inactivate digestive proteases, principally corresponding to two families: Kunitz trypsin inhibitors (KTi) and Bowman-Birk inhibitors (BBI). High levels of raw soybean/soybean meal in feed mixtures can cause poor weight gain and pancreatic abnormalities via inactivation of trypsin/chymotrypsin enzymes. Soybean protein meal is routinely heat-treated to inactivate inhibitors, a practice that is energy-intensive and costly and can degrade certain essential amino acids. In this work, we screened seed from 520 soybean accessions, using a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots with anti-Kunitz trypsin inhibitor antibodies. A soybean germplasm accession was identified with a mutation affecting an isoform annotated as nonfunctional (KTi1), which was determined to be synergistic with a previously identified mutation (KTi3-). We observed significant proteome rebalancing in all KTi mutant lines, resulting in dramatically increased BBI protein levels.

Detection and quantification of soymilk in cow–buffalo milk using Attenuated Total Reflectance Fourier Transform Infrared spectroscopy (ATR–FTIR)

Milk consumption is steadily increasing, especially in India and China, due to rising income. To bridge the gap between supply and demand, unscrupulous milk vendors add milk-like products from vegetable sources (soymilk) to milk without declaration. A rapid detection technique is required to enforce the safety norms of food regulatory authorities. Fourier Transform Infrared (FTIR) spectroscopy has demonstrated potential as a rapid quality monitoring method and was therefore explored for detection of soymilk in milk. In the present work, spectra of milk, soymilk (SM), and milk adulterated with known quantity of SM were acquired in the wave number range of 4000–500cm−1 using Attenuated Total Reflectance (ATR)–FTIR. The acquired spectra revealed differences amongst milk, SM and adulterated milk (AM) samples in the wave number range of 1680–1058cm−1. This region encompasses the absorption frequency of amide-I, amide-II, amide-III, beta-sheet protein, α-tocopherol and Soybean Kunitz Trypsin Inhibitor. Principal component analysis (PCA) showed clustering of samples based on SM concentration at 5% level of significance and thus SM could be detected in milk using ATR–FTIR. The SM was best predicted in the range of 1472–1241cm−1 using multiple linear regression with coefficient of determination (R2) of 0.99 and 0.92 for calibration and validation, respectively.

Allergenicity of an enzymatic hydrolysate of soybean2Sprotein

This study was performed to examine how the characteristics of soybean 2S protein influence allergenicity after enzymatic hydrolysis. Soybean 2S protein was extracted and enzymatic hydrolysis was performed using pepsin and chymotrypsin. Allergenicity was observed using soybean-sensitive patients' sera. Only 13.3% (6/45) of soybean-sensitive patients reacted to soybean Kunitz trypsin inhibitor (SKTI), known as the major allergen of soybean 2S protein. After peptic hydrolysis for 90 min at pH 1.2, the intensity of SKTI decreased to 25% but was still visible on SDS-PAGE. Chymotryptic hydrolysis following peptic hydrolysis at pH 8 for 60 min showed a limited hydrolytic effect on soybean 2S protein. Peptic hydrolysis of soybean 2S protein partially reduced the allergenicity of soybean 2S protein, while chymotryptic hydrolysis following peptic hydrolysis increased slightly the allergenicity. Food allergy caused by soybean 2S protein occurred in part of the soybean-sensitive patients. SKTI was partially digested after peptic hydrolysis for 90 min. The allergenicity was decreased with peptic hydrolysis, while subsequent treatment of chymotrypsin increased slightly the allergenicity.

Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema

Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 μM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy.

Use of Protease Inhibitors in Composite Polyelectrolyte Microparticles in Order to Increase the Bioavailability of Perorally Administered Encapsulated Proteins

Protein protease inhibitors (aprotinin, soybean Bowman–Birk inhibitor, and Kunitz soybean trypsin inhibitor) possessing different specificity with respect to trypsin, chymotrypsin, and elastase were encapsulated together with a cargo protein in polyelectrolyte microparticles using layer-by-layer (LbL) deposition techniques. The most efficient inclusion of the inhibitors occurred at the formation stage of the insoluble protein complex with the polyanion. Simultaneous immobilization of the inhibitor and protein did not influence the physicochemical properties of the microparticles, specifically their pH-sensitive behavior under conditions modeling the passage through various parts of the human gastrointestinal tract after peroral administration. The most effective protection against the action of proteolytic enzymes of pancreatic juice and the small intestine was achieved for simultaneous release of cargo protein and inhibitor from the microparticles. Soybean Bowman–Birk inhibitor, which is most similar to insulin with respect to physicochemical properties, in addition to the soybean extract enriched with protease inhibitors were the most suitable agents for protection of human insulin or its rapidly acting analogs (lispro and aspart). These findings suggested that simultaneous microencapsulation of both protein and protein protease inhibitor was a promising way to increase the protein bioavailability upon peroral administration of polyelectrolyte microparticles.