Identification and characterisation of gut proteases in the fig tree skeletoniser moth, Choreutis nemorana Hübner (Lepidoptera: Choreutidae)

Abstract

The biochemical properties of proteases from the digestive system of the fig tree skeletonizer moth, Choreutis nemorana, were determined. Gut extracts of C. nemorana larvae were analysed using different specific peptide substrates and proteinase inhibitors. The optimal pH and temperature for proteolytic activities using azocasein as substrate were obtained as pH 11 and 45°C, respectively. In the case of N-benzoyl-l-arg-p-nitroanilide as substrate, the enzyme showed the maximum tryptic activity at pH 11. The kinetic parameters of trypsin-like proteases indicated that the K m and V max values of trypsin in the gut of C. nemorana were 0.157 ± 0.006mM and 0.188 ± 0.005 µmol/min/mg protein. Using specific proteolytic inhibitors, the inhibitors including phenyl methane sulfonyl fluoride, N-p-tosyl-l-lys chloromethyl ketone and ethylene diamine tetraacetic acid showed the greatest inhibitory effect on total proteolytic activity. These results indicated that serine proteinases accounted for the major proteases in the gut of C. nemorana. Inhibition assays and zymogram analysis showed that only small amounts of cysteine proteases are present in the digestive system of C. nemorana.

Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization

“…The mid gut region of insect digestive system comprises of digestive proteases which catalyses the breakdown of proteins into small molecules [20]. Protease inhibitors are proficient in interfering with digestive enzymes of insect gut and hence are able in controlling them [21]. JSTI (Jack fruit Seed Trypsin Inhibitor) has effectively shown insecticidal activity against proteases of larval mid gut [18].…”

Effect of Soybean Leaf Protease Inhibitor on the Mean Leaf Area Consumed by Spodopetra litura and Spilosoma obliqua larvae

“…Franco et al, (2004) reported that Enzymatic assays using gut extracts from larval and adult boll weevil have demonstrated the presence of digestive serine proteinase-like activities and in vitro assays showed that soybean Kunitz trypsin inhibitor (SKTI) was able to inhibit these enzymes. Gholamzadeh et al, (2013) investigated that, the Proteolytic activity of azocasein as a protein substrate in the gut of C. nemorana was 7.267 ± 0.37 μmol/min/mg protein. In addition, the trypsin and chymotrypsin activity was 1.53 ± 0.03 and 1.42 ± 0.1 μmol/min/mg protein, respectively.…”

Functional Characterization of Outer Membrane Proteins (OMPs) in Xenorhabdus nematophila and Photorhabdus luminescens through Insect Immune Defense Reactions

“…The samples were incubated at 4 °C for 30 min, the centrifuged at 10,000× g for 10 min and 100 µL of the supernatant mixed with NaOH 1 M (100 µL). The activity of protease was expressed as µmol dye/min/mg protein using the extinction coefficient of the chromogenic azo group produced by the cleavage of casein [18].…”