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

ABSTRACT: 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 K_m and a decreased V_max 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.

SUBMITTER: Xu Y

PROVIDER: S-EPMC7320042 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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

Xu Yanji Y Zhang Panpan P Liu Xiao X Wang Zhike Z Li Suxia S

Applied biochemistry and biotechnology 20200201 3

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 obtai ...[more]

PMID: 32006248

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Project description:A Kunitz-type trypsin inhibitor protein has been purified and characterized from seeds of Acacia nilotica L. LC-MS/MS analysis of Acacia nilotica trypsin inhibitor (AnTI) provided the N-terminal fragment of 11 amino acids which yielded 100% identity with already reported Kunitz-type trypsin inhibitor protein of Acacia confusa (AcTI) in UniProtKB database search. SDS-PAGE showed a single band of ~21 kDa under nonreduced condition and appearance of a daughter band (17 kDa) in the presence of β-mercaptoethanol indicating the presence of interchain disulfide linkage typical for Kunitz-type trypsin inhibitors. AnTI was purified from seed extract by using a combination of anion exchange and gel filtration chromatography. Since AnTI showed maximum homology with AcTI, a molecular structure of AcTI was predicted which showed highly β-sheeted molecular conformation similar to crystallographic structure of Enterolobium contortisiliquum trypsin inhibitor (EcTI). AnTI (20 µg) produces significant population inhibition against different human pathogenic bacteria along strong antifungal activity (50 µg). Entomotoxin potential of AnTI was evaluated against two stored grain insect pests Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera) and Sitophilus oryzae (Linnaeus) (Curculionidae: Coleoptera). Statistically significant mortality of T. castaneum adults was observed at 1.5 mg after 15 days in comparison to control. Additionally, number of total eggs, larvae, pupae, adults, and their male/female ratio were also severely reduced in comparison to control. Similarly, two generation progeny of S. oryzae was studied after mixing AnTI with rice kernels. Mean percent mortality of adult population was significantly higher after 9 days of exposure in comparison to control group. AnTI significantly reduced the F1 generation while little mortality was observed for F2 generation. Exploration of such potent molecules is the prerequisite of our time regarding the anticipation of postantibiotic era and the development of insect resistance against chemical pesticides.

Project description:Inhibitors of peptidases isolated from leguminous seeds have been studied for their pharmacological properties. The present study focused on purification, biochemical characterization and anti-inflammatory and anticoagulant evaluation of a novel Kunitz trypsin inhibitor from Erythrina velutina seeds (EvTI). Trypsin inhibitors were purified by ammonium sulfate (30-60%), fractionation followed by Trypsin-Sepharose affinity chromatography and reversed-phase high performance liquid chromatography. The purified inhibitor showed molecular mass of 19,210.48 Da. Furthermore, a second isoform with 19,228.16 Da was also observed. The inhibitor that showed highest trypsin specificity and enhanced recovery yield was named EvTI (P2) and was selected for further analysis. The EvTI peptide fragments, generated by trypsin and pepsin digestion, were further analyzed by MALDI-ToF-ToF mass spectrometry, allowing a partial primary structure elucidation. EvTI exhibited inhibitory activity against trypsin with IC50 of 2.2×10(-8) mol.L(-1) and constant inhibition (Ki) of 1.0×10(-8) mol.L(-1), by a non-competitive mechanism. In addition to inhibit the activity of trypsin, EvTI also inhibited factor Xa and neutrophil elastase, but do not inhibit thrombin, chymotrypsin or peptidase 3. EvTI was investigated for its anti-inflammatory and anti-coagulant properties. Firstly, EvTI showed no cytotoxic effect on human peripheral blood cells. Nevertheless, the inhibitor was able to prolong the clotting time in a dose-dependent manner by using in vitro and in vivo models. Due to anti-inflammatory and anticoagulant EvTI properties, two sepsis models were here challenged. EvTI inhibited leukocyte migration and specifically acted by inhibiting TNF-α release and stimulating IFN-α and IL-12 synthesis. The data presented clearly contribute to a better understanding of the use of Kunitz inhibitors in sepsis as a bioactive agent capable of interfering in blood coagulation and inflammation.

Project description:Trypsin inhibitors (TIs) are widely distributed in plants and are known to play a protective role against herbivores. TIs reduce the biological activity of trypsin, an enzyme involved in the breakdown of many different proteins, by inhibiting the activation and catalytic reactions of proteins. Soybean (Glycine max) contains two major TI classes: Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI). Both genes encoding TI inactivate trypsin and chymotrypsin enzymes, which are the main digestive enzymes in the gut fluids of Lepidopteran larvae feeding on soybean. In this study, the possible role of soybean TIs in plant defense against insects and nematodes was investigated. A total of six TIs were tested, including three known soybean trypsin inhibitors (KTI1, KTI2 and KTI3) and three genes encoding novel inhibitors identified in soybean (KTI5, KTI7, and BBI5). Their functional role was further examined by overexpression of the individual TI genes in soybean and Arabidopsis. The endogenous expression patterns of these TI genes varied among soybean tissues, including leaf, stem, seed, and root. In vitro enzyme inhibitory assays showed significant increase in trypsin and chymotrypsin inhibitory activities in both transgenic soybean and Arabidopsis. Detached leaf-punch feeding bioassays detected significant reduction in corn earworm (Helicoverpa zea) larval weight when larvae fed on transgenic soybean and Arabidopsis lines, with the greatest reduction observed in KTI7 and BBI5 overexpressing lines. Whole soybean plant greenhouse feeding bioassays with H. zea on KTI7 and BBI5 overexpressing lines resulted in significantly reduced leaf defoliation compared to non-transgenic plants. However, bioassays of KTI7 and BBI5 overexpressing lines with soybean cyst nematode (SCN, Heterodera glycines) showed no differences in SCN female index between transgenic and non-transgenic control plants. There were no significant differences in growth and productivity between transgenic and non-transgenic plants grown in the absence of herbivores to full maturity under greenhouse conditions. The present study provides further insight into the potential applications of TI genes for insect resistance improvement in plants.

Dataset Information

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

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

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