Bioinsecticidal activity of a novel Kunitz trypsin inhibitor from Catanduva (Piptadenia moniliformis) seeds

Highlights

• Identification of a novel Kunitz trypsin inhibitor (PmTKI) purified from the Catanduva tree.

• In vitro and in vivo activity of PmTKI detected against different insect pests.

• Kinetics analyses of PmTKI were performed showing high specificity to trypsin-like proteinases.

Abstract

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.

Introduction

Proteinaceous inhibitors are proteins widely distributed in nature possessing the ability to interact with enzymes of different origins, suppressing partial or totally the catalytic activity of these enzymes. In plants, they have been found in storage tissues, such as leaves, fruits, tubers and seeds. A variety of physiological functions have been attributed for these proteins, especially its role in the plant-defense mechanisms against insect pest attack [1], [2]. Serine protease inhibitors of the Kunitz superfamily have gained particular attention due to the potent activity against insect larval midgut serine proteases. The use of proteinaceous inhibitors as candidates for control strategies of insects is hopeful, since insect digestive proteases are promising targets in the control of various insects. Kunitz inhibitors were capable of inhibiting the proteolytic activity of serine proteases from lepidopterans, such as the black cutworm (Agrotis ipsilon Hufnagel), the corn earworm (Heliothis zea), the tobacco budworm (Heliothis virenscens Fab.), the western spruce budworm (Choristeneura occidentalis) [3], and coleopterans such as the cotton boll weevil (Anthonomus grandis) [4], the cowpea weevil (Callosobruchus maculatus) [5], [6], [7], [8] and the bean weevil (Zabrotes subfasciatus) [8], [9] and dipterans such as Ceratitis capitata [6], [8], [9], [10]. Increased mortality, several deformations, decreased weight gain, and reduction of the fecundity and oviposition were common effects observed when Kunitz inhibitors were added to the artificial diets of insects [5], [6], [7], [11].

The fruit fly (Diptera: Tephritidae) is an insect pest that attacks a great variety of fruit-tree species that are economically important in Brazil such as mango, caja, caja-mirim, siriguela, guava, guabiroba, jabuticaba, rose apple, cherry, carambola, orange, tangerine, abiu and sapodilla trees. The species C. capitata (Wiedemann), commonly known as the Mediterranean fruit fly, is considered the main pest of fruticulture in the world, attacking an extensive host range including more than 350 plant species. Millions of dollars are annually lost and many programs were created to control and to eradicate this pest [12]. Great quantities of insecticides are often used, causing damage to the environment and consumer health. Solutions for this situation are based on the development of novel, target-specific compounds reducing the impact on the environment and increasing effectiveness in the combat against this insect [13], [14]. Compounds such as lectins, vicilins, proteases and amylases inhibitors can be considered possible alternatives to the use of pesticides to control insect pests [5], [6], [15].

In this view, the tropical Fabaceae tree of the Mimosoideae subfamily, Piptadenia moniliformis (Benth.), known popularly as Catanduva and angico-de-bezerro, typically found in the caatinga northeastern part of Brazil, had a Kunitz-type trypsin inhibitor purified and biochemically characterized. The inhibitory activity was tested: in vitro assays of insect pests from different orders and in vivo bioassays models during the larval development of C. capitata (fruit fly).