Biochemical characterization and structural analysis of trypsin from Plodia interpunctella midgut: implication of determinants in extremely alkaline pH activity profile

Abstract

In the present study, trypsin from Plodia interpunctella (Hübner) is characterized to discover sequence, biochemical and structural features. This enzyme is purified by ion exchange chromatography using fast protein liquid chromatography on proteins from fifth‐instar larvae. The enzyme is optimally active at 50 °C and pH 11.0. The kinetic parameters (Km and Vmax) of the enzyme are 5.3 ± 0.6 µm and 31 ± 1.3 nmol min−1 mg−1, respectively (using Nα‐benzoyl‐l‐arginine ρ‐nitroanilide hydrochloride as substrate). The enzyme is inhibited by the addition of Cu2+ and Mn2+, whereas it is activated by Li+ at high concentrations. Moreover, the enzyme is almost completely inhibited in the presence of Nα‐tosyl‐l‐lysine chloromethyl ketone hydrochloride and phenylmethanesulphonyl fluoride. To understand some characteristics of P. interpunctella trypsin, including active site structure and alkaline pH profile, a reliable structural model of P. interpunctella trypsin is built based on the Fusarium oxisporum (Schlecht) trypsin cystal structure (Protein Data Bank code: 1GDU). The secondary structure content of the purified trypsin from near‐ultraviolet circular dichroism data shows considerable similarities with that of P. interpunctella trypsin predicted structure. Analysis of pKa values of active site residues, a type of amino acid residue in the active site cleft and the surface charges of the model and Tribolium castaneum (Herbst) trypsin structure as an insect species from different orders reveals some differences between them. These differences might effect on the microenvironment of the active site cleft and consequently shift its pH profile. The application of multiple theoretical and experimental techniques is well adapted to predict the enzyme structure with high accuracy and this could help in the design of a powerful inhibitor for trypsin with ideal properties.

Biochemical characteristics of sorghum cultivars affect life table parameters, feeding performance, and digestive enzyme activities of Helicoverpa armigera

“…The hemolymph was eluted with pre‐chilled water, excess tissues were removed, and the midguts of all 20 larvae per cultivar were pooled and homogenized in 3 mL distilled water on ice. After that, the homogenates were centrifuged for 10 min (16000 g at 4 °C), and the clean supernatant fractions were aliquoted into microtubes and stored at −20 °C until the tests were carried out (Hemmati et al., 2017).…”

Feeding responses and digestive function of Spodoptera littoralis (Boisd) on various leafy vegetables exhibit possible tolerance traits

“…The midguts of 50 larvae of S. littoralis were homogenized on ice and prepared as described by Hemmati et al . (2017). Homogenates were centrifuged at 15,000 × g at 4°C for 10 min, and then supernatants were collected and stored at −20°C for enzymatic assays.…”

“…Preparation of larval midgut extract Sixth instar S. littoralis larvae were anesthetized on ice and dissected under a stereomicroscope. The midguts of 50 larvae of S. littoralis were homogenized on ice and prepared as described by Hemmati et al (2017). Homogenates were centrifuged at 15,000 × g at 4°C for 10 min, and then supernatants were collected and stored at −20°C for enzymatic assays.…”

Interaction between feeding efficiency and digestive physiology of Plodia interpunctella Hubner (Lepidoptera: Pyralidae) on different date fruit varieties

“…The weight of pupal stages was also measured 24 h after their appearance on each date variety. In order to determine the dry weight of larvae, the samples of food and larval frass were weighed, then dried at 60°C for 48 h, and weighed again (Hemmati et al 2017).…”