Purification and characterization of a Kunitz inhibitor from Poincianella pyramidalis with insecticide activity against the Mediterranean flour moth (original) (raw)
2009
Two trypsin inhibitors (called PdKI-3.1 and PdKI-3.2) were purified from the seeds of the Pithecellobium dumosum tree. Inhibitors were obtained by TCA precipitation, affinity chromatography on Trypsin-Sepharose and reversed-phase-HPLC. SDS-PAGE analysis with or without reducing agent showed that they are a single polypeptide chain, and MALDI-TOF analysis determined molecular masses of 19696.96 and 19696.36 Da, respectively. The N-terminal sequence of both inhibitors showed strong identity to the Kunitz family trypsin inhibitors. They were stable over a wide pH (2-9) and temperature (37 to 100 o C) range. These inhibitors reduced over 84% of trypsin activity with inhibition constant (Ki) of 4.20 x 10 -8 and 2.88 x 10 -8 M, and also moderately inhibited papain activity, a cysteine proteinase. PdKI-3.1 and PdKI-3.2 mainly inhibited digestive enzymes from Plodia interpunctella, Zabrotes subfasciatus and Ceratitis capitata guts. Results show that both inhibitors are members of the Kunitzinhibitor family and that they affect the digestive enzyme larvae of diverse orders, indicating a potential insect antifeedant.
… and Physiology Part B: …, 2010
Digestive endoprotease activities of the coconut palm weevil, Homalinotus coriaceus (Coleoptera: Curculionidae), were characterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitivity to protease inhibitors. Trypsin-like proteinases were major enzymes for H. coriaceus, with minor activity by chymotrypsin proteinases. More importantly, gut proteinases of H. coriaceus were inhibited by trypsin inhibitor from Inga laurina seeds. In addition, a serine proteinase inhibitor from I. laurina seeds demonstrated significant reduction of growth of H. coriaceus larvae after feeding on inhibitor incorporated artificial diets. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. Dietary utilization experiments show that 0.05% I. laurina trypsin inhibitor, incorporated into an artificial diet, decreases the consumption rate and fecal production of H. coriaceus larvae. We have constructed a three-dimensional model of the trypsin inhibitor complexed with trypsin. The model was built based on its comparative homology with soybean trypsin inhibitor. Trypsin inhibitor of I. laurina shows structural features characteristic of the Kunitz type trypsin inhibitor. In summary, these findings contribute to the development of biotechnological tools such as transgenic plants with enhanced resistance to insect pests.
Mechanism of Resistance to Deltamethrin inCydia pomonella(L.) (Lepidoptera: Tortricidae)
Pesticide Biochemistry and Physiology, 1997
The pathways for deltamethrin detoxification were studied in fifth instar larvae of two resistant strains of Cydia pomonella obtained by mass selection (with deltamethrin (Rv) or diflubenzuron (Rt)) of resistant field populations from southeastern France. Compared to a laboratory susceptible strain (S), these strains exhibited respectively a 3170-and 1600-fold resistance ratio to topically applied deltamethrin. The esterase inhibitor S,S,S-tributyl phosphorotrithioate synergized deltamethrin in both resistant and susceptible strains, whereas the mixed-function oxidase (mfo) inhibitor piperonyl butoxide (PBO) synergized deltamethrin only in R strains (Rv and Rt). The two inhibitors together totally overcame the deltamethrin resistance of Rv and Rt strains. While ethoxycoumarin deethylase activities, measured at individual level in microtitration plates, were higher in R strains than with S, those of glutathione S-transferases showed little difference and those of esterase showed no difference. Moreover, the involvement of mfo in the resistance to deltamethrin was confirmed by in vivo metabolization of [ 14 C] deltamethrin by guts of fifth instar larvae. Metabolites resulting from the ester bond hydrolysis, such as phenocyanol and PBald, were common to S and R strains. The mfo-dependent hydroxylated derivatives (t or c-CHO-t-deltamethrin, 4Ј or 5-HO-deltamethrin and 4Ј or 5-HO-PB-acid) were generated only in Rv and Rt strains and were inhibited by PBO. These results may provide the basis for further studies in genetic of resistance in C. pomonella. ᭧1997 Academic Press
Journal of Agricultural and Food Chemistry, 2013
Anagasta kuehniella is a polyphagous pest that causes economic losses worldwide. This species produces serine proteases as its major enzymes for protein digestion. In this study, a new serineprotease inhibitor was isolated from Acacia polyphylla seeds (AcKI). Further analysis revealed that AcKI is formed by two polypeptide chains with a relative molecular mass of ~20kDa. The effects of AcKI on the development, survival, and enzymatic activity of Anagasta kuehniella larvae were evaluated, by incorporating AcKI in an artificial diet. Bioassays revealed a reduction in larval weight of ~50% with the lower concentration of AcKI used in the study (0.5%). Although additional assays showed an increase in endogenous trypsin and chymotrypsin activities, with a degree of AcKI-insensivity, AcKI produces an anti nutritional effect on A. kuehniella, indicating AcKI as a promising bioinsecticide protein for engineering plants that are resistant to insect pests.
Journal of Agricultural and Food Chemistry, 2009
A new pheromone antagonist of the codling moth Cydia pomonella is reported. Presaturation of the antennae of the insects with vapors of the antagonist (E,E)-8,10-dodecadienyl trifluoromethyl ketone, analogue of the main component of the pheromone (codlemone), resulted in lower electrophysiological responses to the pheromone relative to untreated insects. In the wind tunnel, the compound elicited a remarkable disruptive effect on all types of behavior of males flying toward a source baited with a pheromone/antagonist blend in 1:1, 1:5, and 1:10 ratios. The insects displayed erratic flights in the presence of the antagonist, as shown by their flight parameters in comparison to insects attracted to the pheromone alone. In the field, traps baited with mixtures of 1:10 codlemone/antagonist attracted considerably lower numbers of males than the natural attractant. The antagonist, however, did not inhibit the pheromone-degrading enzymes present in male antennae, suggesting that trifluoromethyl ketones are not sufficiently electrophilic to produce a stable intermediate adduct with a cysteine residue of the enzyme, a mechanism previously proposed for oxidase inhibition in insects. Overall and taking into account our previous reports and, particularly, the reduction in damage induced in maize fields by a trifluoromethyl ketone analogue of the pheromone of Sesamia nonagrioides (Solé, J.; Sans, A.; Riba, M.; Rosa, E.; Bosch, M. P.; Barrot, M.; Palencia, J.; Castell a, J.; Guerrero, A. Reduction of damage by the Mediterranean corn borer, Sesamia nonagrioides, and the European corn borer, Ostrinia nubilalis, in maize fields by a trifluoromethyl ketone pheromone analog. Entomol. Exp. Appl. 2008, 126, 28-39), the antagonist might be a new candidate to consider in future strategies to control the codling moth.
Pesticide Biochemistry and Physiology, 2009
Plant defense a b s t r a c t Crop improvement generally focuses on yield, seed quality and nutritional characteristics, rather than resistance to biotic and abiotic stresses. A clear consequence of this approach is the absence of natural anti-feedant toxins in some improved seed materials, allowing predation of commercial crops by insect herbivores. Cowpea (Vigna unguiculata), commonly cultivated by small farmers, is particularly affected by insect-pests that reproduce and develop inside stored seeds. One alternative to conventional pesticides for pest control is the use of biotechnological tools, such as the digestive enzyme inhibitors, that could be introduced in transgenic crops to enhance resistance. In this study, it was verified that the in vivo bioassays using artificial seeds containing 0.5%, 1.0% and 1.5% (w/w) of Delonix regia rich fraction, containing a-amylase inhibitors with effectiveness toward insect a-amylases and other sources, caused remarkable reduction in development and increased mortality of Callosobruchus maculatus cowpea weevil and to cotton boll weevil Anthonomus grandis. Therefore, attempts were made to isolated those inhibitors by SP-Sepharose ion exchange chromatography followed by high performance liquid chromatography on a Vydac C18-TP analytical column. Four inhibitor peaks were obtained with molecular masses of 6.0, 20 and 24 kDa. Their N-termini showed high sequence similarities with Kunitz-like inhibitor family members. These results provide evidence that D. regia synthesizes a multiple family of Kunitz-like a-amylase inhibitors, with different molecular masses and a wide biotechnological potential to control insect-pests.
Journal of the Korean Society for Applied Biological Chemistry, 2014
Diamondback moth, Plutella xylostella, is one of the most destructive insect pests of several crops worldwide. Effects of Paenibacillus elgii HOA73 and its combined application with organic sulfur pesticide on Plutella xylostella were evaluated. Results showed that M3 medium composition indicated the best medium for optimization of bacterial colony growth, hydrolytic enzyme production, and insecticidal activity and was selected for culturing P. elgii HOA73 in further assays. The highest colony growth of P. elgii HOA73 was identified at 5 days after inoculation. Extracted crude enzyme and crude insecticidal compound from P. elgii HOA73 cultured in the optimized medium kill the second instar larvae of Diamondback moth in 40 and 50% at 220 mg mL −1 of crude enzyme and 2% crude extract, respectively. Combined application of organic sulfur pesticide with bacterial suspension significantly killed 85% second instar larvae of Diamondback moth, when compared to use of single application: bacterial suspension (65%) and organic sulfur pesticide (38%), suggesting that P. elgii HOA73 combined with organic sulfur pesticide could be used to control P. xylostella.
Insect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor
European Journal of Biochemistry, 2001
Insect silk is made up of structural fibrous (fibroins) and sticky (sericins) proteins, and contains a few small peptides of hitherto unknown functions. We demonstrate that two of these peptides inhibit bacterial and fungal proteinases (subtilisin, proteinase K and pronase). These`silk proteinase inhibitors' 1 and 2 (SPI 1 and 2) are produced in the middle section of the silk-secreting glands prior to cocoon spinning and their production is controlled at transcription level. The full length cDNA of pre-SPI 1 contains 443 nucleotides and encodes a peptide of 76 amino-acid residues, of which 20 make up a signal sequence. The mature SPI 1 (6056.7 Da, 56 residues) is a typical thermostable Kunitztype proteinase inhibitor with Arg in P1 position. The cDNA of pre-SPI 2 consists of 260 nucleotides and yields a putative secretory peptide of 58 amino-acid residues. The functional SPI 2 (3993 Da, 36 residues) is a single-domain Kazal-type proteinase inhibitor with unique structural features: free segment of the N-terminus is reduced to a single amino-acid residue, lack of CysI and CysV precludes formation of the A-ring and provides increased flexibility to the C-ring, and absence of several residues around the normal position of CysV shortens and changes the a helix segment of the protein. The structure reveals that the length and arrangement of the B-ring, including exposure of the P1 residue, and the position of the C-terminus relative to the B-loop, are essential for the activity of the Kazal-type inhibitors.