Indian Journal of Agricultural Research

  • Chief EditorV. Geethalakshmi

  • Print ISSN 0367-8245

  • Online ISSN 0976-058X

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Indian Journal of Agricultural Research, volume 53 issue 5 (october 2019) : 513-521

Purification and characterization of protease inhibitors from rice bean (Vigna umbellata) and their efficacy against Spodoptera litura (Fabricius) gut proteinases 

Kanika Sharma
1Department of Chemistry and Biochemistry, CSK Himachal Pradesh Krishi Vishwavidyalaya, Palampur-176 061, Himachal Pradesh, India.
Cite article:- Sharma Kanika (2019). Purification and characterization of protease inhibitors from rice bean (Vigna umbellata) and their efficacy against Spodoptera litura (Fabricius) gut proteinases. Indian Journal of Agricultural Research. 53(5): 513-521. doi: 10.18805/IJARe.A-5023.
A protease inhibitor from rice bean was isolated and purified to assess its potential against gut proteases of Spodoptera litura, a devastating pest of several economically important crops globally. The purified inhibitor showed a single band protein on SDS-PAGE corresponding to molecular weight of 24.0 kDa. The inhibitor was purified to near homogeneity with 181.55 fold purification and final yield of 29.18 per cent. The inhibitory potential of purified rice bean trypsin protease inhibitor on larvae of Spodoptera gut proteases was studied which showed maximum azocaseinolytic (1.93U), trypsin (0.66U) and chymotrypsin activity (0.37 U) in second larval instar which gradually decreased upto late fifth larval instar stage. Inhibitory assay revealed that rice bean trypsin inhibitors were strong inhibitor of Spodoptera litura and inhibited more than 80 per cent for trypsin and around 69 per cent for chymotrypsin activity. To further determine the efficacy of rice bean protease inhibitors, feeding assays were conducted by adding rice bean flour in larval artificial diet. High mortality rate was observed in Spodoptera litura larvae after 72 hrs when rice bean was given as sole diet (100% rice bean composition). The results obtained from present study provide important clues in designing strategies for sustainable use of rice bean protease inhibitors in developing insect-tolerant transgenic plants.
  1. Adedapo, A.A., Abatan, M.O., Olorunsogo, O.O. (2007). Efeects of some plants of the spurge family on the haematological and biochemical parameters of rats. Arhiv. 77: 29-38.
  2. Burkill, H.M. (1995). The Useful Plants of West Tropical African. Vol. 3, Families J-L., Royal Botanic Gardens, Kew.
  3. Clarke E.G.C. and Clarke, M.L. (1975). Veterinary toxicology. Balliere Tindall London.
  4. Fodstad O, Olsnes S, Pihl A (1976). Toxicity, distribution and elimination of the cancerostatic lectins abrin and ricin after parenteral injection into mice. Br J Cancer. 34: 418-25
  5. Galey F.D. (1996). Plants and other natural toxicants, In Large Animal Internal Medicine, 2nd ed., [edited by Bradford P Smith]. Mosby Publishers. Boston.
  6. Klaassen C.D. (2001). Casarett and Doull’s toxicology: The Basic Science of Poisoning, 6th ed. McGraw-Hill New York.
  7. Knight, A.P. and Walter, R.G. (2003). In: A Guide to Plant Poisoning of Animals in North America, (Eds.) Publisher: Teton NewMedia, Jackson WY
  8. Macfarlane, P.S., Reid, R., Callander, R. (2001). Pathology Illustrated, 5th ed. Churchill Livingstone. London:
  9. Olsen, C.T., Keller, W.C., Gerken, D.F. (1984). Suspected tremetol poisoning in horses. Journal of the American Veterinary Medical Association, 185:1001–1003.
  10. Reedman L., Shih R.D., Hung O. (2008). Survival after an intentional ingestion of crushed abrus seeds. Western Journal of Emergency Medicine. 9: 157–159. 
  11. Selvaraj P., Srinivasan S.R., Nambi A.P. (2007). Effect of Feed Additives on Rumen Microbial status in Ruminal Acidosis of Dairy Cattle. Intas Polivet. 8(2):443-446.
  12. Shih R. D.and Goldfrank L.R., (1998). Plants. In: Goldfrank’s Toxicologic Emergencies. [Goldfrank L.R., Flomenbaum N.E., Lewin N.A., Weisman R.S., Howland M.A., Hoffman R.S., editors]. 6. East Norwalk, Connecticut: Appleton and Lange.
  13. Sharma (2005). Biochemical studies on the effect of Abrus precatorious on the antifertility of male albino rats. Thesis for PhD (Zoology). Submitted to C.C.S.University, Meerut.
  14. Swenson, M.J. and Reece, O.W. (1993). Duke’s Physiology of Domestic Animals. Comstock Publishing Associates. Ithaca: Journal
  15. Tahirov, T.H, Lu, T.H, Liaw, Y.C, Chu, S.C, Lin, J.Y. (1994). A new crystal form of abrin-a from the seeds of Abrus precatorius. of Molecular Biology, 235:1152–1153.
  16. Windholz M. (1989). An Encyclopedia of Chemicals Drugs and Biologicals, in The Merck index, 3rd ed. Merck and Co. Inc. Rahway: S. Yogeshpriya, P. Selvaraj, S. Krishnakumar, M.Saravanan and P.K.Ramkumar. ECG Markers in Subclinical Hypocalcaemia Exacerbated Clinical Ketosis in Transition Cows. The Indian veterinary journal. 96(5):47-49.
  17. Ahmad, M., Arif, M.I. and Ahmad, M. (2007). Occurrence of insecticide resistance in field populations of Spodoptera litura (Lepidoptera: Noctuidae) in Pakistan. Crop Protec, 26: 809-817.
  18. Alarcon, F, J., Martýnez, T.F., Barranco, P., Cabello, T., Dýaz, M. and Moyano, F,J. (2002). Digestive proteases during development of larvae of red palm weevil, Rhynchophorus ferrugineus (Olivier, 1790) (Coleoptera: Curculionidae). Insect Biochem. and Mol. Biol. 32:265–274.
  19. Babu, S.R., Subrahmanyam, B., Srinivasan and Santha, I.M. (2012). In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae. J. of Biosci. 37(2): 269-276.
  20. Broadway, R.M. and Duffey, S.S. (1986a). Plant proteinase inhibitors: mechanisms of action and effect on the growth and physiology of larval Heliothis zea and Spodoptera exigua, J. of Insect Physiol. 32:827-834.
  21. Broadway, R.M. (1995). Are Insects resistant to plant proteinase inhibitors? J. of Insect Physiol. 41: 107-116.
  22. Chang, C.R. and Tsen, C.C. (1981). Isolation of trypsin inhibitors from rye, triticale and wheat samples Cereal Chem. 58:207-210.
  23. Charity, J.A., Anderson, M.A., Bittisnich, D.J., Whitecross, M. and Higgins, T.J.V. (1999). Transgenic tobacco and peas expressing a proteinase inhibitor from Nicotiana alata have increased insect resistance, Mol. Breeding, 5(4):357-365.
  24. Chitra, R. and Sadasivam, S. (1986). Studies on trypsin inhibitor of black gram [Vigna mungo (L.) Hepper] Food Chem. 21(4): 315-320.
  25. Elbadry, E.A., Abdel-Salam, F.A., Abo Elghar, M.R., Hassan, S.M. and Asal, M.A. (2009). The Effect of Four Different Host Plants on the Larval Development of the Cotton leafworm Spodoptera littoralis (Bois.) J of Applied Entomol. 68: 138-142.
  26. Erlanger, B., Kokowsky, N. and Cohen, W. (1961). The preparation and properties of two new chromogenic substrates of trypsin. Arch. of Biochem. and Biophys. 95: 271–278.
  27. Ferrasson, E., Quillien, L. and Gueguen, J. (1997). Proteinase inhibitors from pea seeds: purification and characterization. J. of Agric. and Food Chem. 45 (1): 127-131.
  28. Ferry, N., Edwards, M, G., Gatehouse, J.A. and Gatehouse, A.M.R. (2004). Plant-insect interactions: molecular approaches to insect resistance. Current Opinion in Biotech. 15:1–7
  29. Haq, S.K. and Khan, R.H. (2003). Characterization of a proteinase inhibitor from Cajanus cajan (L.) J. of Protein Chem. 22: 543-554.
  30. Klomklao, S., Benjakul, S., Kishimura, H. and Chaijan, M. (2011). Extraction, purification and properties of trypsin inhibitor from Thai mungbean (Vigna radiata (L.) R. Wilczek) Food Chem. 129: 1348–1354.
  31. Kowalska, J., Pszczoa, K., Wilimowska-Pelc, A., Lorenc-Kubis, I., Zuziak, E., Lugowski, M., Legowska, A., Kwiatkowska, A., Sleszynska, M., Lesner, A., Walewska, A., Zab³otna, E., Rolka, K. and Wilusz, T. (2007). Trypsin inhibitors from the garden four o’clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: Isolation, characterization and chemical synthesis. Phytochemistry. 68: 1487-1496.
  32. Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature, 277: 680–685.
  33. Lowry, O.H., Rosebrough, N.J., Fan, A.L. and Randall, R.J. (1951). Protein measurement with Folin phenol reagent J. of Biol. Chem. 193: 256–275.
  34. Macedo, M.L.R., Mello, G.C., Md, G.M., Freire, J.C., Novello, S., Marangon,i S. and de Matos, D.G.G. (2002). Effect of a trypsin inhibitor from Dimorphandra mollis seeds on the development of Callosobruchus maculates. Plant Physiol. and Biochem. 40: 891-898.
  35. Maggo, S., Malhotra, S.P., Dhawan, K. and Singh, R. (1999). Purification and characterization of protease inhibitor from rice bean (Vigna umbellata) J of Plant Biochem. Biotech. 8: 61-64
  36. Mendiola-Olaya, E., Valencia-Jimenez, A., Valdes-Rodriguez, S., Delano-Frier, J., and Blanco- Labra A. (2000). Digestive amylase from the larger grain borer, Prostephanus truncates Horn, Compar. Biochem. and Physiol. PartB, 126: 425-433.
  37. Nation, J.L. (2000). Insect Physiol. and Biochem. CRC Press p: 485.
  38. Paiva, P.M.G. and Coelho, L.C.B.B. (1992). Purification and partial characterization of two lectins isoforms from Cratylia mollis Mart. (Camaratu Bean), Applied Biochem. and Biotech. 36 (2): 113-118 
  39. Qin, H.G., Ye, Z.X., Huang, S.J., Ding, S.J. and Luo, J. (2004). The correlations of the different host plants with preference level, life duration and survival rate of Spodoptera litura Fabricius. Chinese J. of Eco-Agric. 12(2):40-42.
  40. Richardson, M.J. (1991). Seed storage proteins: The enzyme inhibitors. In: Richardson, M.J. ed. Methods in Plant Biochem., New York, Academic Press, 259.
  41. Saadati, F. and Bandani, A.R. (2011). Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn pest Eurygaster integriceps, J of Insect Sci, 11(72): 1-12.
  42. Satheesh, S.L. and Murugan, K. (2012) Protease inhibitors from Coccinia grandis (l.)Voigt. Leaves: purification, characterization and kinetic properties Int J of Pharmacy and Pharmac Sci. 4(1):565-573
  43. Shorey, H.H. and Hale, R.L. (1965). Mass-Rearing of the larvae of Nine Noctuid species on a simple artificial medium J of Econ. Entomol. 58:522–524.
  44. Srinivasan, A., Giri, A.P. and Gupta, V.S. (2006). Structural and functional diversities in lepidopteran serine proteases. Cellular & Mol. Biol Letters, 11(1): 132-154.
  45. Telang, M.A., Giri, A.P., Sainani, M.N. and Gupta, V.S. (2005). Elastase like proteinase of Helicoverpa armigera is responsible for inactivation of a proteinase inhibitor from chickpea J of Insect Physiol. 51:513–522. 

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