Characterization of a prolyl endoprotease from Eurygaster integriceps puton (Sunn pest) infested wheat

Abstract

Sunn pest, Eurygaster integriceps, Puton, infested and uninfested wheat seeds were obtained from the International Center for Agriculture Research in the Dry Areas (ICARDA), Aleppo, Syria, with the primary objective to identify the type of enzyme deposited by the Sunn pest on the wheat responsible for the gluten degradation. Enzyme levels were extremely low due to the enzyme being secreted by the insect in localized areas on the seed. Only extract from the infested wheat contained glutenase activity. Anion exchange, Cu(2+) sepharose, and gel filtration chromatography were used to partially purify and enrich protein samples from both infested wheat and uninfested wheat. An SDS-gluten assay was used to show gluten specificity while a commercially available chromogenic proline peptide, benzyloxycarbonyl-Gly-Pro-p-nitroanalide (ZGPpNA), was utilized to identify fractions containing the active proline specific enzyme activity and to determine Michaelis-Menten kinetics. Despite low levels of enzyme on the infested wheat, the enzyme was partially purified and enriched exhibiting a specific activity of 4.5 U/mg of total protein for gluten in a SDS gluten assay (1 U of enzyme activity was defined as the decrease in gel height in millimeters in 1 h) and exhibited a high-affinity Km of 65 microM for ZGPpNA, cleaving at the carboxy terminus of the proline residue. The enzyme exhibited optimal activity between pH 8 and 10.0 at temperatures between 20 degrees and 35 degrees C. The enzyme was identified to be a prolyl endoprotease.

Effects of natural protease inhibitors on high protease activity flours

“…This finding is consistent with previous study of Darkoh et al (2010) using phenylmethylsulphonyl floride (PMSF) as a commercial protease inhibitor for Eurygaster protease(s). The effect of EDTA-Na 2 confirmed that metal ions were essential to action or stability of the bug enzyme(s).…”

“…The major role of them is plant protection by blocking the digestive proteolytic enzymes of invading pests (Gatehouse, 1984). It was reported that a phenylmethylsulphonyl floride (PMSF), a commercial serine protease inhibitor, was inhibited Eurygaster and Nysius proteases (Darkoh et al, 2010;Every, 1993). The protease from Nysius huttoni was insensitive to most proteinaceous inhibitors except for potato inhibitor I and some divalent metal ions such as Co 2þ , Mn 2þ , Fe 2þ (Cressey, 1987;Every, 1993).…”

Prolyl-specific peptidases for applications in food protein hydrolysis

“…The PEP from E. integriceps (cf. above) was also tested for its ability to degrade gluten (Darkoh et al 2010). The enzyme activity was shown by use of an SDS-gluten assay, in which hydrolytic activity led to a degradation of the glutencontaining gel.…”

“…They detected 31 digestive enzymes, whereof four enzymes were identified as serine peptidases. Darkoh et al (2010) characterised a PEP from E. integriceps. In this study, the enzyme was not isolated from the beetle itself, but from infested wheat grains.…”

Wheat breeding for resistance to the Sunn pest (Eurygaster spp.): Does risk occur?

“…In recent years great progress has been made in the study of interaction mechanisms of bugs with plant seeds (Hosseininaveh et al, 2009;Darkoh et al, 2010;Mehrabadi et al, 2010;Zibaee et al, 2011).…”