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      Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization

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          Abstract

          Background

          The defensive capacities of plant protease Inhibitors (PI) rely on inhibition of proteases in insect guts or those secreted by microorganisms; and also prevent uncontrolled proteolysis and offer protection against proteolytic enzymes of pathogens.

          Methods

          An array of chromatographic techniques were employed for purification, homogeneity was assessed by electrophoresis. Specificity, Ki value, nature of inhibition, complex formation was carried out by standard protocols. Action of SNTI on insect gut proteases was computationally evaluated by modeling the proteins by threading and docking studies by piper using Schrodinger tools.

          Results

          We have isolated and purified Soap Nut Trypsin Inhibitor (SNTI) by acetone fractionation, ammonium sulphate precipitation, ion exchange and gel permeation chromatography. The purified inhibitor was homogeneous by both gel filtration and polyacrylamide gel electrophoresis (PAGE). SNTI exhibited a molecular weight of 29 kDa on SDS-PAGE, gel filtration and was negative to Periodic Acid Schiff’s stain. SNTI inhibited trypsin and pronase of serine class. SNTI demonstrated non-competitive inhibition with a Ki value of 0.75 ± 0.05×10-10 M. The monoheaded inhibitor formed a stable complex in 1:1 molar ratio. Action of SNTI was computationally evaluated on larval gut proteases from Helicoverpa armigera and Spodoptera frugiperda. SNTI and larval gut proteases were modeled and docked using Schrodinger software. Docking studies revealed strong hydrogen bond interactions between Lys10 and Pro71, Lys299 and Met80 and Van Der Waals interactions between Leu11 and Cys76amino acid residues of SNTI and protease from H. Armigera. Strong hydrogen bonds were observed between SNTI and protease of S. frugiperda at positions Thr79 and Arg80, Asp90 and Gly73, Asp2 and Gly160 respectively.

          Conclusion

          We conclude that SNTI potentially inhibits larval gut proteases of insects and the kinetics exhibited by the protease inhibitor further substantiates its efficacy against serine proteases.

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          Author and article information

          Contributors
          gvd.divya@gmail.com
          0891-2840464 (0) , rachelr68@gmail.com
          kunal.zaveri22@gmail.com
          kiranmayi.patnala@gmail.com
          Journal
          BMC Biochem
          BMC Biochem
          BMC Biochemistry
          BioMed Central (London )
          1471-2091
          22 October 2015
          22 October 2015
          2015
          : 16
          : 23
          Affiliations
          [ ]Assistant professor, Department of Biochemistry/Bioinformatics, Institute of Science, GITAM University, Rushikonda, Visakhapatnam, 530045 Andhra Pradesh India
          [ ]Department of Biotechnology, Institute of Science, GITAM University, Rushikonda, Visakhapatnam, 530045 Andhra Pradesh India
          Article
          52
          10.1186/s12858-015-0052-7
          4618930
          26489418
          6ff05059-07e7-4482-9b15-c502e195bdd4
          © Gandreddi et al. 2015

          Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

          History
          : 10 August 2015
          : 30 September 2015
          Categories
          Research Article
          Custom metadata
          © The Author(s) 2015

          Biochemistry
          ki,monoheaded inhibitor,page,snti,specificity,gut proteases,protein-protein docking
          Biochemistry
          ki, monoheaded inhibitor, page, snti, specificity, gut proteases, protein-protein docking

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