Skip to main content
SpringerLink
Log in

Purification, characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (Vigna radiata L. Wilczek) seeds

  • Original Paper
  • Published:
Acta Physiologiae Plantarum Aims and scope Submit manuscript

Abstract

Protease inhibitors present in seeds of legumes possess strong inhibitory activity against trypsin and confer resistance against pests. In the present investigation, trypsin inhibitor activity was found in the seed flour extracts of all the eight selected varieties of mungbean under study which was further confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor was purified from mungbean seeds to near homogeneity with 58.1-fold and 22.8% recovery using heat denaturation, NH4(SO4)2 fractionation, ion-exchange chromatography on DEAE-Sephadex A-25 and gel filtration through Sephadex G-75. The molecular mass of the inhibitor was 47 kDa as determined by gel filtration and SDS-PAGE. The inhibitor retained 90% or more activity between pH 4 and 10, however, it was nearly inactive at extreme pH values. The inhibitor was stable up to 80°C but thereafter, the activity decreased gradually retaining nearly 30% of activity when heated at 100°C for 20 min. The inhibitor activity was undetectable at 121°C. Insect bioassay experiment using purified mungbean trypsin inhibitor showed a marked decline in survival (%) of larvae with increase in inhibitor concentration. The larval growth was also extended by the trypsin inhibitor. This study signifies the insecticidal potential of mungbean trypsin inhibitor which might be exploited for raising transgenic plants.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  • Annapurna SS, Ramadoss CS, Prasad DS (1991) Characterization of a trypsin/chymotrypsin inhibitor from jack fruit (Artocarpus integrifolia) seeds. J Sci Food Agric 54:605–618

    Article  CAS  Google Scholar 

  • Broadway RM (1995) Are insect resistant to plant proteinase inhibitors? J Insect Physiol 41:107–116

    Article  CAS  Google Scholar 

  • Chrispeels MJ, Baumgartner B (1978) Trypsin inhibitor in mungbean cotyledons. Plant Physiol 61:617–623

    Article  PubMed  CAS  Google Scholar 

  • Davis BJ (1964) Disc electrophoresis. II. Method and application to human serum proteins. Ann N Y Acad Sci 121:404–427

    Article  PubMed  CAS  Google Scholar 

  • deLeo F, Gallerani R (2002) The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littorallis larvae fed on transgenic plants. Insect Biochem Mol Biol 32:489–496

    Article  CAS  Google Scholar 

  • Dellaporta SL, Wood J, Hecks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19

    Article  CAS  Google Scholar 

  • Felicioli R, Garzelli B, Vaccari L, Melfi D, Balestreri E (1997) Activity staining of protein inhibitors of proteases on gelatin-containing polyacrylamide gel electrophoresis. Anal Biochem 244:176–179

    Article  PubMed  CAS  Google Scholar 

  • Ferrasson E, Quillien L, Gueguen J (1997) Proteinase inhibitors from pea seeds: purification and characterization. J Agric Food Chem 45:127–131

    Article  CAS  Google Scholar 

  • Gatehouse AMR, Gatehouse JA (1998) Identifying proteins with insecticidal activity: use of encoding genes to produce insect-resistant transgenic crops. Pest Sci 52:165–175

    Article  CAS  Google Scholar 

  • Gatehouse AMR, Gatehouse JA, Boulter D (1980) Isolation and characterization of trypsin inhibitor from cowpea (Vigna unguiculata). Phytochemistry 19:751–756

    Article  CAS  Google Scholar 

  • Gatehouse AMR, Norton E, Davis GM, Babbe SM, Newell CA, Gatehouse JA (1999) Digestive proteolytic activity in larvae of tomato moth, Lacanobia oleracea; effects of plant proteinase inhibitor in vitro and in vivo. J Insect Physiol 45:545–558

    Article  PubMed  CAS  Google Scholar 

  • Ghorpade VM, Kadam SS, Salunkhe DK (1986) Thermal stability and changes in trypsin inhibitor during germination and cooking of horse gram. J Food Sci Technol 23:164–165

    CAS  Google Scholar 

  • Giri AP, Harsulkar AM, Ku MSB, Gupta VS, Deshpande VV, Ranjekar PK, Franceschi VR (2003) Identification of potent inhibitors of Helicoverpa armigera gut proteinases from winged bean seeds. Phytochemistry 63:523–532

    Article  PubMed  CAS  Google Scholar 

  • Godbole SA, Krishna TG, Bhatia CR (1994) Purification and characterization of protease inhibitors from pigeon pea (Cajanus cajan) seeds. J Sci Food Agric 64:87–93

    Article  CAS  Google Scholar 

  • Gomes APG, Dias SC, Bloch C Jr, Melo FR, Furtado JR Jr, Monnerat RG, Grossi-de-Sá MF, Franco OL (2005) Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds. Comp Biochem Physiol B 140:313–319

    Article  CAS  Google Scholar 

  • Gupta P, Dhawan K, Malhotra SP, Singh R (2000) Purification and characterization of trypsin inhibitor from seeds of faba bean (Vicia faba L). Acta Physiol Plant 22:433–438

    Article  CAS  Google Scholar 

  • Hajela N, Sharma AH, Sharma DN, Rao S, Hajela K (1999) Studies on a doubleheaded protease inhibitor from Phaseolus mungo. J Plant Biochem Biotechnol 8:57–60

    CAS  Google Scholar 

  • Harsulkar AM, Giri AP, Patankar AG, Gupta VS, Sainani MN, Ranjekar PK, Despande VV (1999) Successive use of non-host plant proteinase inhibitors required for effective inhibition of Helicoverpa armigera gut proteinases and larval growth. Plant Physiol 121:497–506

    Article  PubMed  CAS  Google Scholar 

  • Johnston KA, Gatehouse JA, Anstee JH (1991) In vitro and In vivo studies of the effects of plant proteinases inhibitors on Helicoverpa armigera larvae. J Exp Bot 42:238

    Google Scholar 

  • Johnston KA, Gatehouse JA, Anstee JH (1993) Effect of soybean protease inhibitors on the growth and development of larval Helicoverpa armigera. Insect Physiol 39:657–664

    Article  CAS  Google Scholar 

  • Jouanin L, Bonade-Bottino M, Girard C, Morrot G, Giband M (1998) Transgenic plants for insect resistance. Plant Sci 131:1–11

    Article  CAS  Google Scholar 

  • Kakade ΜL, Simons Ν, Liener ΙΕ (1969) The evaluation of natural vs synthetic substrates for measuring the antitrypsin activities of soybean samples. Cereal Chem 46:518–526

    CAS  Google Scholar 

  • Kunitz M (1945) Crystallization of a trypsin inhibitor from soybean. Science 101:668

    Article  PubMed  CAS  Google Scholar 

  • Laemmli EK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Laskowski M Jr, Qasim MA (2000) What can the structures of enzyme–inhibitor complexes tell us about the structures of enzyme substrate complexes? Biochem Biophys Acta 1477:324–337

    PubMed  CAS  Google Scholar 

  • Lawn RJ, Ahn CS (1985) Mung bean (Vigna radiata (L) Wilczek/Vigna mungo (L) Hepper). In: Summerfield RJ, Roberts EH (eds) Grain legume crops. William Collins Sons & Co Ltd, London, pp 584–623

    Google Scholar 

  • Lawrence JC, Nielsen SS (2001) Partial characterization of a cysteine proteinase inhibitor from Lima bean (Phaseolus lunatus). J Agric Food Chem 49:1020–1025

    Article  PubMed  CAS  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:1265–1275

    Google Scholar 

  • Maggo S, Malhotra SP, Dhawan K, Singh R (1999) Purification and characterization of protease inhibitor from rice bean (Vigna umbellate). J Plant Biochem Biotechnol 8:61–64

    CAS  Google Scholar 

  • Murdock LL, Shade RE (2002) Lectins and protease inhibitors as plant defenses against insects. J Agric Food Chem 50:6605–6611

    Article  PubMed  CAS  Google Scholar 

  • Ryan CA (1991) Proteinase inhibitors in plants: genes for improving defenses against insects and pathogens. Annu Rev Plant Physiol 28:425–449

    Google Scholar 

  • Saini HS (1989) Thermal stability of protease inhibitors in some cereals and legumes. Food Chem 32:59–67

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, USA

    Google Scholar 

  • Singh A, Dudey A, Tyagi TK, Gaur AK, Mishra DP (2005) Screening of different Indian wheat varieties for alpha amylase inhibitor proteins/genes to develop insect-resistant transgenic plants. J Plant Biol 32:149–153

    CAS  Google Scholar 

  • Solomon M, Belenghi B, Delledone M, Menachem E, Levine A (1999) The involvement of cysteine proteinase and proteinase inhibitor genes in the regulation of programmed cell death in plants. Plant Cell 11:431–443

    Article  PubMed  CAS  Google Scholar 

  • Sudheendra K, Mulimani VH (2002) Effect of feeding legume proteinase inhibitors on Helicoverpa armigera gut proteinase activity. ICPN 9:51–53

    Google Scholar 

  • Telang M, Srinivasan A, Patankar A, Harsulkar A, Joshi V, Damle A, Deshapande V, Sainani M, Ranjekar P, Gupta G, Birah A, Rani S, Kachole M, Giri A, Gupta V (2003) Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura. Phytochemistry 63:643–652

    Article  PubMed  CAS  Google Scholar 

  • Veera Reddy GG, Bhattacharya AK (1990) Development of semi-synthetic diets for the rearing of Heliothis armigera (Hub). J Insect Sci 3:23–33

    Google Scholar 

  • Whitley EJ, Bowman DE (1975) Isolation and properties of navy beans proteinase inhibitor component I. Arch Biochem Biophys 169:42–50

    Article  PubMed  CAS  Google Scholar 

  • Xavier-Filho J (1992) The biological roles of serine and cysteine proteinase inhibitors in plants. Braz J Plant Physiol 4:1–6

    CAS  Google Scholar 

Download references

Acknowledgment

The authors want to thank National Agricultural Technology Project (Mission Mode), ICAR, New Delhi for the financial support.

Author information

Authors and Affiliations

  1. National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi, India

    Rekha Kansal, Ram Niwas Gupta & Kirpa Ram Koundal

  2. Department of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India

    Kalika Kuhar & Vijay Kumar Gupta

Authors
  1. Rekha Kansal
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ram Niwas Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kirpa Ram Koundal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kalika Kuhar
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vijay Kumar Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Vijay Kumar Gupta.

Additional information

Communicated by G. Klobus.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kansal, R., Gupta, R.N., Koundal, K.R. et al. Purification, characterization and evaluation of insecticidal potential of trypsin inhibitor from mungbean (Vigna radiata L. Wilczek) seeds. Acta Physiol Plant 30, 761–768 (2008). https://doi.org/10.1007/s11738-008-0178-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11738-008-0178-y

Keywords

Search

Navigation