RESUMO
Vicilins are seed proteins, and they constitute 70-80% of the total protein in leguminous seeds; with amolecular mass between 150 and 190 kDa, they are composed of subunits without disulfide bridges, with high affinity for chitin-binding. They are also associated with seed defense against insect pests. The chitin-binding vicilin from Anadenanthera colubrina seeds was purified by ammonium sulfate, followed by affinity chromatography on a chitin column, molecular exclusion on Superdex 75 Tricorn in FPLC system and Phenomenex C8 chromatography in HPLC system. The A. colubrina vicilin, named AcV, is a tetrameric glycoprotein composed of 1.55% carbohydrates and molecular weight determined by SDS-PAGE, consisting of 70, 73, 43 and 41 kDa. The AcV homogeneity was confirmed in native PAGE, where it was observed to be a unique band with slow mobility in this gel, with approximately 230 kDa. AcV added to the Callosobruchus maculatus diet in the bioassays resulted in a strong effect on adult emergence (ED50 of 0.096%), and in larvae caused a marked reduction in mass (WD50 of 0.32%) and lethality (LD50 of 0.33%) (w:w). The digestibility of AcV was evaluated in vitro with the digestive enzymes of larvae of C. maculatus of fourth instar, showing major fragments of 10 and 30 kDa. AcV showed reactivity against the anti-EvV antibody from Erythrina velutina vicilin. The deleterious effects of AcV are likely to be associated with the chitin-binding fragments generated by proteolysis in the bruchid gut, similarly to that found for vicilins from other leguminous plant species, Enterolobium contortisiliquum and Vigna unguiculata. AcV might be a candidate protein for a possible bioinsecticidal control of the bruchid weevil, C. maculatus.
RESUMO
Myrciaria floribunda (H. West ex Willd.) O. Berg, Myrtaceae, is popularly known as "camboim-amarelo" and was collected at Restinga de Jurubatiba (RJ, Brazil). Leaves from this species were submitted to hydrodistillation to extract its essential oil. Monoterpenes were the main compounds found (53.9%), and 1,8-cineole was the major constituent (38.4%). Studies were carried out to evaluate the effects of this essential oil on the development of two species of agricultural pests (Oncopeltus fasciatus and Dysdercus peruvianus). The essential oil was considered effective against D. peruvianus and O. fasciatus, causing mortality in both insects. The LD50 values (µg/insect) observed were 112.44 µg/insect (O. fasciatus) and 309.64 µg/insect (D. peruvianus) after one day of treatment, and 72.18 µg/insect (O. fasciatus) and 94.42 µg/insect (D. peruvianus) after 22 days of treatment. The present study reports for the first time the bioinsecticidal activity of essential oil of Myrciaria floribunda leaves, and provides important data regarding the use of essential oils in complementary programs for pest control.
RESUMO
The present study aims to provide new in vitro and in vivo biochemical information about a novel Kunitz trypsin inhibitor purified from Piptadenia moniliformis seeds. The purification process was performed using TCA precipitation, Trypsin-Sepharose and reversed-phase C18 HPLC chromatography. The inhibitor, named PmTKI, showed an apparent molecular mass of around 19 kDa, visualized by SDS-PAGE, which was confirmed by mass spectrometry MALDI-ToF demonstrating a monoisotopic mass of 19.296 Da. The inhibitor was in vitro active against trypsin, chymotrypsin and papain. Moreover, kinetic enzymatic studies were performed aiming to understand the inhibition mode of PmTKI, which competitively inhibits the target enzyme, presenting Ki values of 1.5 × 10(-8) and 3.0 × 10(-1) M against trypsin and chymotrypsin, respectively. Also, the inhibitory activity was assayed at different pH ranges, temperatures and reduction environments (DTT). The inhibitor was stable in all conditions maintaining an 80% residual activity. N-terminal sequence was obtained by Edman degradation and the primary sequence presented identity with members of Kunitz-type inhibitors from the same subfamily. Finally after biochemical characterization the inhibitory effect was evaluated in vitro on insect digestive enzymes from different orders, PmTKI demonstrated remarkable activity against enzymes from Anthonomus grandis (90%), Plodia interpuncptella (60%), and Ceratitis capitata (70%). Furthermore, in vivo bioinsecticidal assays of C. capitata larvae were also performed and the concentration of PmTKI (w/w) in an artificial diet required to LD50 and ED50 larvae were 0.37 and 0.3% respectively. In summary, data reported here shown the biotechnological potential of PmTKI for insect pest control.