Purification and characterization of a trypsin–papain inhibitor from Pithecelobium dumosum seeds and its in vitro effects towards digestive enzymes from insect pests

A novel trypsin–papain inhibitor, named PdKI-2, was purified from the seeds of Pithecelobium dumosum seeds by TCA precipitation, Trypsin–Sepharose chromatography and reversed-phase HPLC. PdKI-2 had an Mr of 18.1 kDa as determined by SDS–PAGE and was composed of a single polypeptide chain. The inhibition on trypsin was stable at pH range 2–10, temperature of 50 °C and had a Ki value of 1.65 × 10−8 M, with a competitive inhibition mechanism. PdKI-2 was also active to papain, a cysteine proteinase, and showed a noncompetitive inhibition mechanism and Ki value of 5.1 × 10−7 M. PdKI-2 was effective against digestive proteinase from bruchids Zabrotes subfasciatus and Callosobruchus maculatus; Dipteran Ceratitis capitata; Lepidopterans Plodia interpunctella and Alabama argillacea, with 74.5%, 70.0%, 70.3%, 48.7%, and 13.6% inhibition, respectively. Results support that PdKI-2 is a member of Kunitz-inhibitor family and its effect on digestive enzyme larvae from diverse orders indicated this protein as a potent insect antifeedant.