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Characterization of Trypsin Like Protease from Helicoverpa armigera (Hubner) and Its Potential Inhibitors

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Proceedings of the National Academy of Sciences, India Section B: Biological Sciences Aims and scope Submit manuscript

Abstract

Helicoverpa armigera is the most devastating pest of important crops causing heavy yield losses. The larval midgut harbours proteases and their inhibition starves insects to death or may disrupt their normal metabolism. So there is need to characterize gut protease and its potential inhibitors. A serine protease (trypsin like) from H. armigera gut was purified ~37 folds with 22 % yield and its molecular weight was found to be ~18.8 kilo Dalton. The Michaelis constant using N-α-benzoyl-DL-arginine-p-nitroanilide as a substrate was 0.31 mM. Maximum reaction rate was determined to be 3.47 nmol p-nitroaniline/min. Free energy of binding was found to be −20.8 kJ/mol. The optimum temperature of enzyme was found to be 50 °C and around 17 % activity was maintained on heating at 80 °C for 30 min. The optimum pH for trypsin like activity was 11 with wide range of activity between 9 and 12 indicating that midgut digestive trypsin of H. armigera is active over wide range of alkaline pH. Metal ions Cu2+, Zn2+, Cd2+ and Hg2+ were found to have the potential to inhibit the enzyme. Maximum inhibition was observed with Cu2+ (73 %) and minimum inhibition with Cd2+ (47 %). Copper inhibited trypsin non-competitively and zinc inhibited uncompetitively. Ascorbic acid, EDTA, leupeptin and benzamidine were found to be competitive inhibitors of H. armigera gut protease. The Ki values for Cu2+, ascorbic acid, EDTA, leupeptin and benzamidine were found to be 1.32, 38.23, 2.51 mM, 37.13 µM and 5.66 mM respectively.

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Acknowledgments

The authors are thankful to Pulses section of Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana for providing the research material.

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Authors and Affiliations

  1. Department of Biochemistry, Punjab Agricultural University, Ludhiana, India

    Sheetanshu Grover, Satvir Kaur & Anil Kumar Gupta

  2. Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India

    Gaurav Kumar Taggar & Jagmeet Kaur

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  1. Sheetanshu Grover
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  2. Satvir Kaur
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Correspondence to Satvir Kaur.

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Grover, S., Kaur, S., Gupta, A.K. et al. Characterization of Trypsin Like Protease from Helicoverpa armigera (Hubner) and Its Potential Inhibitors. Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci. 88, 49–56 (2018). https://doi.org/10.1007/s40011-016-0732-0

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  • DOI: https://doi.org/10.1007/s40011-016-0732-0

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