Purification and properties of the chymotrypsin inhibitor from wild emmer wheat (Triticum dicoccoides) of Israel and its toxic effect on beet armyworm, Spodoptera exigua

A novel chymotrypsin inhibitor, which detected in the seed of wild emmer wheat (Triticum dicoccoides), was purified by ion-exchange chromatography, affinity chromatography and Ultracentrifugation. On the basis of its specificity, this inhibitor was named WeCI (wild emmer chymotrypsin inhibitor). SDS-PAGE analysis displayed that the purified WeCI is a single chain polypeptide with a molecular weight of approximately 13 kDa. The inhibition constants (Ki) for amylase and bovine pancreatic chymotrypsin were 1.12 × 10− 9 M and 2.41 × 10− 9 M, respectively. Automated sequencing and mass spectrometry analyses revealed that WeCI is a neutral monomeric protein consisting of 119 residues. In vitro, WeCI strongly suppressed bovine pancreatic chymotrypsin as well as chymotrypsin-like activities separated from the midgut of the beet armyworm Spodoptera exigua. No inhibitory activities were found against bovine pancreatic trypsin, bacterial subtilisin, or porcine pancreatic elastase. The primary structure of WeCI was markedly similar (46-95%) to those of several proteins belonging to the wheat crop chymotrypsin/α-amylase inhibitor superfamily and displayed the typical sequence motif of the α-amylase inhibitor-seed storage protein group. WeCI significantly inhibited the growth and development of Spodoptera exigua, dependent on inhibitor concentration. WeCI significantly increased the mortality rate of Spodoptera exigua and caused a significant decrease in its fertility.