Soybean Trypsin Inhibitor Research Articles

ВИДІЛЕННЯ ІНГІБІТОРА ТРИПСИНУ НАСІННЯ ЛЮЦЕРНИ З ДОПОМОГОЮ АФФІННОЇ ХРОМАТОГРАФІЇ

A protein inhibitor of trypsin of the cultivar Eva alfalfa seeds was isolated and characterized. It has significant antiproteolytic activity and is promising for the creation of compositions that are designed to correct nutrition in various conditions, accompanied by increased activation of proteolytic enzymes. The stages of purification of the trypsin inhibitor from alfalfa seeds included: extraction of 0.05 M borate buffer (pH 7.6), fractionation of the protein component of the extract with ammonium sulfate, followed by dialysis of the fraction between 75% and 100% saturation and affinity chromatography on a biospecific sorbent trypsin - sepharose 4B. Calculations showed that from 100 g of alfalfa seeds of the cultivar Eva, 67.14 mg of trypsin inhibitor was obtained, the inhibitory activity of which is 27.6 IU / mg protein. Experimental data show that the obtained inhibitor reduces trypsin activity by 63.05% with a weight ratio of inhibitor: enzyme of 1: 1. It is known from literature that a soybean trypsin inhibitor reduces trypsin activity by 30%. The isolated trypsin inhibitor has significant inhibitory activity, the value of which is higher than the inhibitory activity of the plant trypsin inhibitor from soybeans. Affinity chromatography allowed us to obtain a pure, homogeneous trypsin inhibitor, which has significant antiproteolytic activity, which can be compared with the antiproteolytic activity of known plant trypsin inhibitors. From the obtained experimental data, it follows that both, the obtained inhibitor and the inhibitor, which is part of a commercial preparation, effectively reduce trypsin activity, slightly inhibit chymotrypsin activity, and do not affect α-amylase and protease activity. The above data indicate that the trypsin inhibitor of alfalfa seeds of the cultivar Eva is not a bifunctional inhibitor, since it does not affect the activity of amilolytic enzymes. The isolated inhibitor belongs to proteins with a high degree of hydrophobicity. The composition of amino acids, the side chains of which can take part in the formation of hydrogen bonds, is 25% of the total number of amino acid residues of the alfalfa seed trypsin inhibitor. For STI, this value is 23%.

Разработка иммуноферментных аналитических систем для контроля содержания основных немясных добавок в мясных продуктах

Abstract-Methods for control of the content of non-meat components (connective tissue of animals, eggs, soybeans) in meat products have been developed based on competitive enzyme immunoassay of biomarker proteins: collagen, ovalbumin, and soybean trypsin inhibitor. Polyclonal rabbit antibodies against the biomarkers were produced. Screening of the following analysis conditions was carried out using the most affine preparations: the duration of the stages, the concentrations of the reagents, the composition of the reaction medium to ensure the completeness and the minimum limits of detection of analytes. It was shown that the immunochemical stage can be transferred to the kinetic mode and reduced to 20 min, while the total analysis took only 1 h. The selected conditions synchronized detection stages and provided the same signal amplitudes from all three biomarkers. An additional advantage of the method is that the analysis can be carried out at room temperature. The detection limits for collagen, ovalbumin and soybean trypsin inhibitor in the final extracts were 0.025 μg/mL, 0.012 μg/mL, and 0.001 μg/mL, respectively. Approbation of the method showed the reliability of the conclusions about the composition of the tested meat products; the relative standard deviation in the analysis of the monitored biomarkers was 8-10%. Key words: enzyme-linked immunoassay, meat products, collagen, ovalbumin, soybean trypsin inhibitor The work was financially supported by the Russian Science Foundation (project no. 19-16-00108).

DEVELOPMENT OF IMMUNOENZYME ANALYTICAL SYSTEMS FOR CONTROL OF THE CONTENT OF MAIN NON-MEAT COMPOUNDS IN PROCESSED MEAT PRODUCTS

Methods have been developed to control the content of non-meat components (connective tissue of animals, eggs, soy) in processed meat products, based on enzyme immunoassay of biomarker proteins – collagen, ovalbumin, soybean trypsin inhibitor.

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.

Co-encapsulation by Flash NanoPrecipitation of Insulin, Trypsin Inhibitor and Caprate Permeabilization Enhancer for Oral Administration

<img src=” https://s3.amazonaws.com/production.scholastica/article/18519/large/1235741_graphical_abstract.jpg?1608410554”> We describe inverse-Flash NanoPrecipitation as a new scalable platform for co-encapsulation of insulin (a model therapeutic peptide) with soybean trypsin inhibitor (a peptidase inhibitor) and so-dium caprate (a permeation enhancer) forming 300 nm particles for oral delivery. The co-encapsulation of the protein therapeutic with a protease inhibitor and a permeation enhancer into nanoparticles addresses enzymatic attack on the protein drug and the transport across the gastroin-testinal tract barrier. Inverse-Flash NanoPrecipitation (iFNP) encompasses two sequential precipita-tion processes. First, insulin, soybean trypsin inhibitor (SBTI), and hydroxypropyl methylcellulose acetate-succinate polymer (HPMCAS, a stabilizing block copolymer)(at mass ratios of 1:4:5) are rapidly precipitated into an organic antisolvent forming the inverted nano-core (iNC) with 50 wt% insulin and SBTI. Encapsulation efficiencies are greater than 98%. The second step involves the precipitation of sodium caprate and a stabilizing polymer coating onto the iNCs surface. HPMCAS, chitosan, and PEG polymers are used to coat the iNCs to generate nanoparticles with anionic, cation-ic, and neutral surfaces, respectively. This demonstrates that the iFNP platform can encapsulate complex biologics mixtures at high loadings into particles with customizable surface properties.

Nickel Ferrite Nanoparticles as an Adsorbent for Immobilized Metal Affinity Chromatography of Proteins

A simple method of preparing amorphous nickel ferrite nanoparticles of about 5nm diameter is described. These particles were characterized by dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM) and selected area electron diffraction (SAED). The nanoparticles were evaluated for their use as a magnetic material for immobilized metal affinity chromatography (IMAC). The ferrite nanoparticles bound to bovine serum albumin (BSA) and the binding fitted Langmuir isotherm model. A high capacity of 916mg BSA/g dried nanoparticle was observed. Six proteins (Soybean trypsin inhibitor (STI), lactate dehydrogenase (LDH), papain, catalase, β-galactosidase and casein) were used and all were found to bind at >90% level (except papain which showed 84% binding). All the proteins except LDH and β-galactosidase could be eluted with 1M imidazole and with % activity recovery of >80%. Papain could be purified from its dried crude latex by 5-fold and purified papain showed a single band on SDS-PAGE. These nanoparticles constitute a high capacity and are magnetic material useful for IMAC and do not require any pre-functionalization.

Effect of feeding habits of fish on the characteristics of collagenolytic proteases isolated from the visceral waste.

In this study, influence of feeding habits of fish on the activity of collagenolytic proteases (CP) has been investigated. CP from the visceral waste of freshwater fish (Pangas, Rohu and Common carp) of different feeding habits was isolated and partially purified by 2-steps, (NH4)2SO4 fractionation and dialysis. Enzymatic activity and purification fold was determined in each step. The molecular mass of the enzymes were close to that of serine collagenases. Enzyme was assayed for temperature and pH optima, effect of sodium chloride and inhibitors. Optimum temperature and pH was 40°C and 7-8 respectively. Soybean trypsin inhibitor inhibited the enzyme activity, whereas, EDTA exerted no effect, led to confirmation of serine collagenases. CP of carnivore was more active over a wide range of temperature and pH compared to herbivore and omnivore. The study revealed that the feeding habit of fish play decides the optimal physiological conditions for maximum activity of CP.

Effect of interaction between tea polyphenols with soymilk protein on inactivation of soybean trypsin inhibitor

Soymilk is a popular health beverage worldwide, but its nutritional value is limited by soybean trypsin inhibitors (STIs). The interaction of tea polyphenols (TPs) with soymilk protein in the complex system was investigated in this work. TPs interacted with soymilk protein via static quenching process and hydrophobic interaction, with binding constant (Ka) of 5.22 × 103 L mol−1 at 298 K. Synchronous fluorescence suggested that the binding site of TPs to soymilk protein was mainly at Trp residues compared with Tyr residues. FTIR analysis revealed that hydrogen bonds were also observed in TPs-soymilk system. CD spectroscopy suggested that the protein conformation became more stable with addition of TPs by reducing β-sheet and random coil, and increasing α-helix and β-turn. The trypsin and chymotrypsin inhibitory activities (TIA and CIA) were reduced at 0.6 mg/g TPs in cooked soymilk from 788.3 ± 10.4 U/mL and 918.7 ± 18.0 U/mL to 388.3 ± 35.5 U/mL and 633.3 ± 52.8 U/mL, respectively.

Uremic solutes of indoxyl sulfate and p-cresol enhance protease-activated receptor-2 expression in vitro and in vivo in keratinocytes

Uremic pruritus is common in patients with chronic kidney disease (CKD). The retention of uremic solutes is thought to be associated with uremic pruritus. Meanwhile, activation of protease-activated receptor-2 (PAR-2) has been suggested to play an important role in pruritus. The present study was performed to investigate the effects of uremic solutes on the expression of PAR-2 in the skin. Indoxyl sulfate (IS), p-cresol (PC), and uremic sera from CKD patients were used to stimulate PAR-2 expression in normal human epidermal keratinocytes (NHEKs). Also, NHEKs were additionally pretreated with soybean trypsin inhibitor to evaluate its inhibitory effect on PAR-2 expression. Patterns of cutaneous PAR-2 expression were investigated in skin samples from five CKD patients and CKD mice. In NHEKs, IS, PC, and sera from CKD patients significantly induced PAR-2 mRNA and protein expression. Soybean trypsin inhibitor significantly decreased PAR-2 mRNA and protein expression in NHEKs treated with IS, PC, and CKD sera. NHEKs treated with IS and PC exhibited significant increases in protease activity. Skin from both CKD patients and mice exhibited marked upregulation of PAR-2 expression compared to control skin. Results from the present study suggest that uremic solutes either directly or indirectly affect PAR-2 expression in the skin of CKD subjects, potentially playing an important role in the pathogenesis of uremic pruritus.

Production of Extracellular Alkaline Serine Protease from Pediococcus acidilactici NCDC 252: Isolation, Purification, Physicochemical and Catalytic Characterization

Proteases are the most important industrial enzymes capable of hydrolyzing proteins into small peptides and amino acids and used commercially in food, textile and pharmaceutical industries. An alkaline serine protease from Pediococcus acidilactici NCDC 252 was purified using a simple two step purification procedure i.e. ammonium sulphate precipitation and gel filtration chromatography with purification fold of 5.9, a yield and specific activity of 11.8% and 23.3 U mg/ml respectively. Purified enzyme worked optimally at pH 8.5 and 37 °C. The enzyme was stable in the pH range of 6.5–9.5 and up to 50 °C. The Km for BAPNA was 38 µM and Vmax was 10.6 U/mg. Purified enzyme exhibited highest hydrolyzing activity for BAPNA. Inhibition of purified enzyme by soybean trypsin inhibitor, benzamide and β-ME suggested the enzyme to be alkaline serine protease with involvement of cysteine residues either in binding or regulation. In-silico characterization of alkaline serine protease revealed the presence of Ser, His and Asp residues at its catalytic site. This shows the resemblance of alkaline protease under study with other studied serine proteases.

Growth performance and amino acid digestibility responses of broiler chickens fed diets containing purified soybean trypsin inhibitor and supplemented with a monocomponent protease

Trypsin inhibitors (TI) resident in soybeans affects protein utilization. While heat treatment influences residual TI, it simultaneously affects the structure and solubility of the soybean proteins and confounds any response to exogenous proteases. Using purified TI, the effect of exogenous protease to TI can be dissociated from changes in the soybean protein. Thus, the current study was designed to evaluate the growth performance and protein utilization responses of broiler chickens to purified TI and exogenous protease. Soybean meal (SBM) was preanalyzed for basal TI (2,996 TIU/g SBM), formulated into nutritionally adequate experimental diets to contain 1,033 TIU/g diet, and purified TI was added at 9,000 TIU/g diet. A total of 320 Cobb-500 broiler chicks were allocated to 4 diets, each with 8 replicate cages and 10 birds per replicate. The experimental diets were arranged as a 2 × 2 factorial with factors being dietary TI (1,033 or 10,033 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). On day 7, 14, and 21 posthatching, protease supplementation improved the BW gain (P < 0.01) and gain to feed ratio (P < 0.05) of birds. On day 14 and 21 posthatching, the relative weight of pancreas increased (P < 0.05) with added TI but was reduced (P < 0.001) with protease supplementation. Apparent ileal digestibility of all amino acids, except methionine, decreased (P < 0.001) with added TI but increased (P < 0.05) with protease supplementation. Jejunal MUC-2 was downregulated (P < 0.01) and SCL7A-2 was upregulated (P < 0.05) by protease supplementation. Duodenal trypsin and chymotrypsin activities reduced (P < 0.05) with added TI but increased (P < 0.01) with protease supplementation. Exogenous protease produced longer villi (P < 0.05) and deeper crypts (P < 0.01) in the jejunal tissue. In conclusion, dietary addition of purified TI negatively affects nutrient utilization by broiler chickens. Furthermore, the study showed that the efficacy of the exogenous protease might be independent of dietary TI concentration.

Development of Immunoenzyme Assay for Detection of Soybean Raw Material in Food Products

An enzyme-linked immunosorbent assay (ELISA) has been developed to control the presence and estimate the content of soybean raw materials in meat food products. It is based on the binding of specific antibodies to the antigenic determinants of a soybean trypsin inhibitor. Due to the possible influence of enzymatic and high-temperature product treatments that can disrupt the native structure of the inhibitor and lead to its fragmentation, the analysis is based on the competitive interaction with antibodies of the antigen in the sample, which are immobilized in microplate wells. The conditions providing the minimum detection limit and high measurement accuracy were established to implement the ELISA. The developed assay was characterized by a duration of 60 min, a detection limit of 3.5 ng/mL, and a range of detectable concentrations of 7.7–110 ng/mL. It is shown that the proposed methodology can be used for the testing meat products.

Evaluation of silica nanoparticle mediated delivery of protease inhibitor in tomato plants and its effect on insect pest Helicoverpa armigera

The inert and surface tunable nature of silica nanoparticles (SiNPs) makes them suitable for different applications. We have evaluated the potential of SiNPs for delivering proteins in tomato (Lycopersicon esculentum) plants. SiNPs of 20 and 100 nm (Si20 and Si100) were functionalized with (3-aminopropyl) triethoxysilane (APTES) to obtain Si20APT and Si100APT, respectively, that were non-toxic toward plants. The functionalized nanoparticles were taken up by plants through roots as well as leaf surfaces. They were seen to be localized near the vasculature, particularly around the xylem. Si20APT and Si100APT nanoparticles were conjugated with soybean trypsin inhibitor (STI) to yield Si20APT-STI and Si100APT-STI, respectively. Based on the trypsin inhibitory activity of loaded nanoparticles, optimum loading was obtained for 0.4 mg of STI per 0.8 mg of NPs. Si20APT nanoparticles retained higher contents of STI than Si100APT. Exposure of STI-conjugated nanoparticles to 25°C or pH 8.0 aided release of the inhibitor. The particle bound STI inhibited bovine trypsin by 80% and Helicoverpa armigera gut proteinase (HGP) activity by 50%. Second instar H. armigera larvae ingesting STI-loaded particles (incorporated in artificial diet or leaves) showed significant retardation in growth. In choice assays, Si20APT-STI applied leaf discs were strikingly avoided by insect larvae. On the basis of the results obtained in this investigation, we recommend the use of Si20 nanoparticles for developing plant delivery vehicles in the future.

Identification of a novel alkaline serine protease from gazami crab (Portunus trituberculatus) hepatopancreas and its hydrolysis of myofibrillar protein

Serine proteases are thought to play a key role in the muscle softening of gazami crab (Portunus trituberculatus) during storage. A serine protease, Pt-sp2, was purified from the hepatopancreas of gazami crab using ammonium sulfate precipitation, anion-exchange and gel filtration chromatography, and was analyzed by mass spectrometry, transcriptome and bioinformatics. It revealed that Pt-sp2 was trypsin-like, with no 100% identical proteins in the NCBI database. The molecular weight of Pt-sp2 was approximately 37.2 kDa. Its optimum pH and temperature were 9.0 and 50 °C, respectively, using t-Butyloxy‑carbonyl-Phe-Ser-Arg-4-methyl-coumaryl-7-amide as a substrate. Pt-sp2 was activated in the presence of Ca2+. Both soybean trypsin inhibitor and Nα-Tosyl-l-lysine chloromethyl ketone hydrochloride completely suppressed Pt-sp2 activity, while it was only partially inhibited by phenylmethylsulfonyl fluoride and EDTA. However, PMSF, Pepstatin A and cystatin inhibitor E-64 showed no inhibition on Pt-sp2 protease activity. The Km value of Pt-sp2 was 0.82 μM, and Pt-sp2 effectively hydrolyzed myofibrillar protein at 37 °C.

Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways

AbstractStorage lesion–induced, red cell–derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV–induced coagulation activation, the ability of storage lesion–induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV–induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Inactivation of mesotrypsin by chymotrypsin C prevents trypsin inhibitor degradation

Mesotrypsin is an unusual human trypsin isoform with inhibitor resistance and the ability to degrade trypsin inhibitors. Degradation of the protective serine protease inhibitor Kazal type 1 (SPINK1) by mesotrypsin in the pancreas may contribute to the pathogenesis of pancreatitis. Here we tested the hypothesis that the regulatory digestive protease chymotrypsin C (CTRC) mitigates the harmful effects of mesotrypsin by cleaving the autolysis loop. As human trypsins are post-translationally sulfated in the autolysis loop, we also assessed the effect of this modification. We found that mesotrypsin cleaved in the autolysis loop by CTRC exhibited catalytic impairment on short peptides due to a 10-fold increase in Km , it digested β-casein poorly and bound soybean trypsin inhibitor with 10-fold decreased affinity. Importantly, CTRC-cleaved mesotrypsin degraded SPINK1 with markedly reduced efficiency. Sulfation increased mesotrypsin activity but accelerated CTRC-mediated cleavage of the autolysis loop and did not protect against the detrimental effect of CTRC cleavage. The observations indicate that CTRC-mediated cleavage of the autolysis loop in mesotrypsin decreases protease activity and thereby protects the pancreas against unwanted SPINK1 degradation. The findings expand the role of CTRC as a key defense mechanism against pancreatitis through regulation of intrapancreatic trypsin activity.

Comparison of physicochemical properties of recombinant buckwheat trypsin inhibitor (rBTI) and soybean trypsin inhibitor (SBTI)

The inhibitory activities of buckwheat trypsin inhibitor (rBTI) towards trypsin were compared with soybean trypsin inhibitor (SBTI) in terms of their sensitivities to temperature, pH, salt ions and organic solvents. Both rBTI and SBTI were stable over a broad pH range of 2.0–12.0. rBTI exhibited higher thermal stability than SBTI. The inhibitory activity of rBTI was decreased by Zinc ions (Zn2+), KSCN, vitamin C and urea, while that of SBTI remained unchanged. However, H2O2, Mg2+ and Cu2+ ions had no significant effects on the inhibitory activities of rBTI and SBTI. Acetonitrile enhanced the inhibitory activity of rBTI, but had no effect on SBTI, while dimethylacetamide (DMAC) increased the inhibitory effect of both rBTI and SBTI. On the contrary, the inhibitory activities of rBTI and SBTI were reduced by isopropyl alcohol and methanol. The inhibition constants Ki of rBTI and SBTI were calculated to be 7.41 × 10−9 M and 6.52 × 10−9 M, respectively.

Immunoreactivity of Lupine and Soybean Allergens in Foods as Affected by Thermal Processing.

Lupine and soybean are important technological aids for the food industry. However, they are also capable of inducing severe allergic reactions in food-sensitized/allergic individuals. In this context, this work intended to study the combined effects of thermal processing and food matrix on the immunoreactivity of lupine and soybean proteins used as ingredients in bakery and meat products, respectively. For this purpose, the effects of baking, mild oven cooking, and autoclaving on the protein profiles were evaluated, using model mixtures simulating the production of lupine-containing breads and soybean-containing cooked hams/sausages, by native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblotting using specific antibodies. The results showed that lupine gamma-conglutin immunoreactivity was slightly decreased in wheat flour mixtures compared to rice, but it was more pronounced in baked products. In meat mixtures, substantial protein fragmentation was noted after autoclaving, with decreased immunoreactivity of soybean trypsin inhibitor. The analysis of 22 commercial products enabled the identification of lupine gamma-conglutin in four bakery samples and soybean trypsin-inhibitor in five sausages, and further differentiated autoclaved from other milder thermally treated products. Generally, the immunoreactivity of target proteins was reduced by all the tested thermal treatments, though at a higher extent after autoclaving, being slightly altered by the food matrix.

IL-4 enhances interferon production by virus-infected human mast cells

IL-4 enhances interferon production by virus-infected human mast cells

Structural and functional relationship of Cassia obtusifolia trypsin inhibitor to understand its digestive resistance against Pieris rapae

Although digestive resistance of Kunitz protease inhibitors has been reported extensively, the molecular mechanism is not well established. In the present study, the first X-ray structure of Cassia obtusifolia trypsin inhibitor (COTI), a member of Kunitz protease inhibitors, was solved at a resolution of 1.9 Å. The structure adopted a classic β-trefoil fold with the inhibitory loop protruding from the hydrophobic core. The role of Phe139, a unique residue in Kunitz protease inhibitors, and Arg63 in the COTI structure was verified by F139A and R63E mutants. COTI was a specific inhibitor of bovine trypsin and the result was also verified by COTI-trypsin complex formation. Meanwhile, COTI showed equivalent inhibitory activity with that of soybean trypsin inhibitor against bovine trypsin and midgut trypsin from Pieris rapae. The F139 and R63E mutants further indicated that inhibitory specificity and efficiency of COTI were closely related to the global framework, the conformation and the amino acid composition of reactive loop. Finally, a midgut trypsin from P. rapae (PrSP40), which might be involve in the food digestion, was proposed to be a potential target of COTI and might be a promising target for future crop-protection strategy. The results supported the digestive resistance of COTI.