Question

Nephilium protease inhibitors characterisation and purification

Answer 1

Rekha KansalI; Mukesh KumarI; Kalika KuharII; Ram N. GuptaI; Bhattiprolu SubrahmanyamIII; Kirpa R. KoundalI; Vijay K. GuptaII,*

INational Research Centre on Plant Biotechnology, Indian Agricultural Research Institute (PUSA), New Delhi, India 
IIDepartment of Biochemistry, Kurukshetra University, Kurukshetra, Haryana, India
IIIDivision of Entomology, Indian Agricultural Research Institute (PUSA), New Delhi, India

 

 

ABSTRACT

Protease inhibitors in legumes are one of the most promising weapons that confer resistance against insects by inhibiting proteases present in the gut of insect larvae. In the present study, trypsin inhibitor activity was detected in the seed flour extracts of 10 selected varieties of chickpea. The presence of inhibitor was confirmed by dot blot analysis. All the varieties showed inhibitory activity in vitro against the gut protease of Helicoverpa armigera (HGP). Trypsin inhibitor has been purified to near homogeneity to 60.46 fold and 29.20% recovery from chickpea seeds using heat denaturation, ammonium sulphate fractionation, DEAE-Sephadex A-25 and Sephadex G-75. The purified inhibitor showed a single band on SDS-PAGE corresponding to molecular mass of 30,000 Da. The purified inhibitor was active over a wide pH range although it retained maximum activity between pH 6 and 10. The inhibitor protein was stable up to 80°C but retained only 40% of activity when heated at 100°C for 20 min. The inhibitor lost its activity completely at 121°C. The chickpea trypsin inhibitor exhibited inhibitory activity against Helicoverpa armigera both in vitro and in vivo. In insect bioassay, a progressive decline in larval weight, growth and survival as well as temporal extension of larval growth was observed after feeding H. armigera larvae on diet supplemented with increasing concentrations of chickpea trypsin inhibitor. The adult emergence was also adversely affected by the inhibitor. It may be concluded that chickpea trypsin inhibitor has insecticidal potential against H. armigera.

Answer 2

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