Purification and characterization of trypsin inhibitor from Cicer arietinum L. and its efficacy against Helicoverpa armigera

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 whereas 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 complete activity 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.

Effect of thermal processing on anti nutritional factors and in vitro bioavailability of minerals in desi and kabuli cultivars of chick pea grown in North India.

“…It is very difficult to compare enzyme inhibitory potential of legumes, as reported by different authors, primarily, because of the difference in the methods and units used, Smith et al,(1980) reported trypsin inhibitor values (using the same method as used in this work) 16.6 mg/g to 30 mg/g of raw soybeans meal. Results also found in agreement with Kansal et al, (2008) All data are the mean ± SEm of three replicates. All data are expressed on dry weight basis.…”

Exploring the potential of seed flour from pigeon pea genotypes to retard the growth and development of Helicoverpa armigera

“…This result was supporting the in vitro results of our earlier study, where AL 1495 was shown to contain least amount of trypsin inhibitor units (114 TIU/g), lower phenolics (0.28 mg/g) and phytic acid (0.32 mg/g) content than AL 1677 (158 TIU/g, 0.43 mg/g and 0.44 mg/g, respectively). A significant reduction (α=0.05) in larval weight and increase in mortality rate was observed when protease inhibitors from Capsicum annuum, chickpea and kidney bean was incorporated in artificial diet for bioassay during feeding trials of H. armigera (Tamhane et al, 2005;Kansal et al, 2008;Mittal et al, 2014). In a study to test, the efficacy of phenolics on growth and survival of H. armigera and Spodoptera litura, rutin was found to cause inhibition of H. armigera larvae which might be due to its interference with the prothoracicotrophic hormone and ecdysteroid action, thus, affecting molting process (Curtis et al, 1984;Jadhav et al, 2012a).…”

Serine protease inhibitors from squid ovary: extraction and its effect on proteolysis and gel properties of surimi

“…MW of Sephadex G-75 fraction showing the highest inhibitor activity was estimated to be in the range of 9.05-11.47 kDa. MW of serine protease inhibitor from SOSPI was lower than those of trypsin inhibitor from chickpeas (28 kDa), soybean (19 kDa) and mustard seeds (20 kDa) (Kansal et al, 2008). Trypsin inhibitor from yellow tuna fish roe was reported to have MW of 70 kDa based on size exclusion chromatography (Klomklao et al 2015).…”