Molecular Identification of Four Different α-amylase Inhibitors from Baru (Dipteryx alata) Seeds with Activity Toward Insect Enzymes

  • Bonavides, Krishna B. (Centro de Analises Proteomicas e Bioquimicas, Universidade Catolica de Brasilia) ;
  • Pelegrini, Patricia B. (Centro de Analises Proteomicas e Bioquimicas, Universidade Catolica de Brasilia) ;
  • Laumann, Raul A. (Embrapa-Cenargen) ;
  • Grossi-De-Sa, Maria F. (Embrapa-Cenargen) ;
  • Bloch, Carlos Jr. (Embrapa-Cenargen) ;
  • Melo, Jorge A.T. (Embrapa-Cenargen) ;
  • Quirino, Betania F. (Centro de Analises Proteomicas e Bioquimicas, Universidade Catolica de Brasilia) ;
  • Noronha, Eliane F. (Centro de Analises Proteomicas e Bioquimicas, Universidade Catolica de Brasilia) ;
  • Franco, Octavio L. (Centro de Analises Proteomicas e Bioquimicas, Universidade Catolica de Brasilia)
  • Published : 2007.07.31


The endophytic bruchid pest Callosobruchus maculatus causes severe damage to storage cowpea seeds, leading to economical losses. For this reason the use of $\alpha$-amylase inhibitors to interfere with the pest digestion process has been an interesting alternative to control bruchids. With this aim, $\alpha$-amylase inhibitors from baru seeds (Dipteryx alata) were isolated by affinity chromatographic procedures, causing enhanced inhibition of C. maculatus and Anthonomus grandis $\alpha$-amylases. To attempt further purification, this fraction was applied onto a reversed-phase HPLC column, generating four peaks with remarkable inhibition toward C. maculatus $\alpha$-amylases. SDS-PAGE and MALDI-ToF analysis identified major proteins of approximately 5.0, 11.0, 20.0 and 55 kDa that showed $\alpha$-amylase inhibition. Results of in vivo bioassays using artificial seeds containing 1.0% (w/w) of baru crude extract revealed 40% cowpea weevil larvae mortality. These results provide evidence that several $\alpha$-amylase inhibitors classes, with biotechnological potential, can be isolated from a single plant species.


$\alpha$-amylase inhibitors;Bean weevils;Callosobruchus maculates;Dipteryx alata


  1. Batalia, M. A., Monzingo, A. F., Ernst, S., Roberts, W. and Robertus, J. D. (1996) The crystal structure of the antifungal protein zeamatin, a member of the thaumatin-like, Pr-5 protein family. Nat. Struct. Biol. 3, 19-23.
  2. Bernfeld, P. (1955) Amylases a and b. Meth. Enzymol. 1, 149-154.
  3. Bloch Jr., C. and Richardson, M. (1991) A new family of small (5kDa) protein inhibitors of insect ${\alpha}-amylases$ from seeds pf sorghum (Sorghum bicolor (L) Moench) have sequence homologies with wheat g-purothionins. FEBS Lett. 79, 101-104.
  4. Chrispeels, M. J. and Raikhel, N. V. (1991) Lectins, lectin genes, and their role in plant defense. Plant Cell 3, 1-9.
  5. Colilla, F. J., Rocher, A. and Mendez, E. (1990) g-Purothionins: amino acid sequence of two polypeptides of a new family of thionins from wheat endosperm. FEBS Lett. 270, 191-194.
  6. Da Silva, M. C. M., Grossi-de-Sa, M. F., Chrispeels, M. J., Togawa, R. C. and Neshich, G. (2000) Analysis of structural and physico-chemical parameters involved in the specificity of binding between ${\alpha}-amylases$ and their inhibitors. Protein Eng. 13, 167-177.
  7. Dias, S. C., Franco, O. L., Magalhaes, C. P, de Oliveira-Neto, O. B., Laumann, R. A., Figueira, E. L. Z., Melo, F. R. and Grossi-de-Sá, M. F. (2005) Molecular cloning and expression of an ${\alpha}-amylase$ inhibitor from rye with potential for controlling insect pests. Protein J. 24, 113-123.
  8. Feng, G. H., Richardson, M., Chen, M. S., Kramer, K. J., Morgan, T. D. and Reeck, G. R. (1996) ${\alpha}-Amylase$ inhibitors from wheat: amino acid sequences and patterns of inhibition of insect and human ${\alpha}-amylases$. Insect Biochem. Mol. Biol. 26, 419-426.
  9. Franco, O. L., Rigden, D. J., Melo, F. R., Bloch Jr., C., Silva, C. P. and Grossi-de-Sa, M. F. (2000) Activity of wheat ${\alpha}-amylase$ inhibitors towards bruchid ${\alpha}-amylases$ and structural explanation of observed specificities. Eur. J. Biochem. 267, 2166-2173.
  10. Franco, O. L., Rigden, D. J., Melo, F. R. and Grossi-de-Sa, M. F. (2002) Plant ${\alpha}-amylase$ inhibitors and their interaction with insect ${\alpha}-amylases$. Structure, function and potential crop protection. Eur. J. Biochem. 269, 397-412.
  11. Franco, O. L., Melo, F. R., Mendes, P. A., Paes, N. S., Yokoyama, M., Coutinho, M. V., Bloch Jr, C. and Grossi-de-Sa, M. F. (2005) Characterization of two Acanthoscelides obtectus ${\alpha}-amylases$ and their inactivation by wheat inhibitors. J. Agric. Food Chem. 53, 1585-1590.
  12. Gomes, A. P. G., Dias, S. C., Bloch, C. Jr., Melo, F. R., Furtado, J. R. Jr., Monnerat, R. G., Grossi-de-Sa, M. F. and Franco, O. L. (2005) Toxicity to cotton boll weevil Anthonomus grandis of a trypsin inhibitor from chickpea seeds. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 140, 313-319.
  13. Grossi-de-Sa, M. F. and Chrispeels, M. J. (1997) Molecular cloning of bruchid (Zabrotes subfasciatus) ${\alpha}-amylase$ cDNA and interaction of the expressed enzyme with bean ${\alpha}-amylase$ inhibitors. Insect Biochem. Mol. Biol. 27, 271-281.
  14. Ishimoto, M. and Kitamura, K. (1989) Growth inhibitory effects of an ${\alpha}-amylase$ inhibitor from kidney bean, Phaseolus vulgaris (L.) on three species of bruchids (Coleoptera: Bruchidae), Appl. Ent. Zool. 24, 281-286.
  15. Ishimoto, M., Yamada, T. and Kaga, A. (1999) Insecticidal activity of an ${\alpha}-amylase$ inhibitor-like protein resembling a putative precursor of ${\alpha}-amylase$ inhibitor in the common bean, Phaseolus vulgaris L. Biochim. Biophys. Acta 1432, 104-112.
  16. Kasahara, K., Hayashi, K., Arakawa, T., Philo, J. S., Wen, J., Hara, S. and Yamaguchi, H. (1996) Complete sequence, subunit structure, and complexes with pancreatic ${\alpha}-amylase$ of an ${\alpha}-amylase$ inhibitor from Phaseolus vulgaris white kidney beans. J. Biochem. 120, 177-183.
  17. Kluh, I., Horn, M., Hyblova, J., Hubert, J., Doleckova-Maresova, L., Voburka, Z., Kudlíkova, I., Kocourek, F. and Mares, M. (2005) Inhibitory specificity and insecticidal selectivity of ${\alpha}-amylase$ inhibitor from Phaseolus vulgaris. Phytochem. 66, 31-39.
  18. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227, 680-685.
  19. Macedo, M. L. R., de Sa, C. M., Freire, M.das G.M. and Parra, J. R. P. (2004) A Kunitz-type inhibitor of Coleopteran Proteases, isolated from Adenanthera pavonina L. Seeds and its effect on Callosobruchus maculatus. J. Agric. Food Chem. 52, 2533-2540.
  20. Malehorn, D. E., Borgmeyer, J. R., Smith, C. E. and Shah, D. M. (1994) Characterization and expression of an antifungal zeamatin-like protein (Zlp) gene from Zea mays. Plant Physiol. 106, 1471-1481.
  21. Melo, F. R., Sales, M. P., Pereira, L. S., Bloch Jr., C., Franco, O. L. and Ary, M. B. (1999) ${\alpha}-Amylase$ inhibitors from cowpea seeds. Protein Pept. Lett. 6, 385-390
  22. Mirkov, T. E., Whalstrom, J. M., Hagiwara, K., Finardi-Filho, F., Kjemtrup, S. and Chrispeels, M. J. (1994) Evolutionary relationships among proteins in the $phytohemagglutinin-arcelin-{\alpha}-amylase$ inhibitor family of the common bean and its relatives. Plant Mol. Biol. 26, 1103-1113.
  23. Monnerat, R., Dias, S. C., Oliveira-Neto, B., Nobre, S. D. and Grossi-de-Sa, M. F. (1999) Criacao de bicudo do algodoeiro Anthonomus grandis em dieta artificial e estabelecimento para bioensaios com Bacillus thuringiensis. IV Congresso Brasileiro de Algodao, p. 214-216, Ribeirao Preto, Brazil.
  24. Mundy, J., Hejgaard, J. and Svendsen, I. (1984) Characterization of a bifunctional wheat inhibitor of endogenous ${\alpha}-amylase$ and subtilisin. FEBS Lett. 67, 210-214.
  25. Murad, A. M., Laumann, R. A., Lima, T. A., Sarmento, R. B., Noronha, E. F., Rocha, T. L., Valadares-Inglis, M. C. and Franco, O. L. (2006) Screening of entomopathogenic Metarhizium anisopliae isolates and proteomic analysis of secretion synthesized in response to cowpea weevil (Callosobruchus maculatus) exoskeleton. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 142, 365-370.
  26. Nielsen, P. K., Bonsager, B. C., Fukuda, K. and Svensson, B. (2004) Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering. Biochim. Biophys. Acta 1696, 157-164.
  27. Paes, N. S., Gerhardt, I. R., Coutinho, M. V., Yokoyama, M., Santana, E., Harris, N., Chrispeels, M. J. and Grossi-de-Sa, M. F. (2000) The effect of arcelin-1 on the structure of the midgut of bruchid larvae and immunolocalization of the arcelin protein. J. Insect Physiol. 46, 393-402.
  28. Payan, F. (2004) Structural basis for the inhibition of mammalian and insect ${\alpha}-amylases$ by plant protein inhibitors. Biochim. Biophys. Acta 1696, 171-180.
  29. Pelegrini, P. B. and Franco, O. L. (2005) Plant g-thionins: novel insights on the mechanism of action of a multi-functional class of defense proteins. Int. J. Biochem. Cell Biol. 37, 2239-2253.
  30. Pelegrini, P. B., Murad, A. M., Grossi-de-Sa, M. F., Mello, L. V., Romeiro, L. A., Noronha, E. F., Caldas, R. A. and Franco, O. L. (2006) Structure and enzyme properties of Zabrotes subfasciatus ${\alpha}-amylase$. Arch. Insect Biochem. Physiol. 61, 77-86.
  31. Pereira, P. J. B., Lozanov, V., Patthy, A., Huber, R., Bode, W., Pongor, S. and Strobl, S. (1999) Specific inhibition of insect ${\alpha}-amylase$ in complex with the Amaranth ${\alpha}-amylase$ inhibitor at 2.0 resolution. Structure 7, 1079-1088.
  32. Richardson, M. (1990) Seeds storage proteins: the enzyme inhibitors, In: Methods in Plant Biochemistry 5, 261-307.
  33. Sales, M. P., Andrade, L. B., Ary, M. B., Miranda, M. R., Teixeira, F. M., Oliveira, A. S., Fernandes, K. V. and Xavier-Filho, J. (2005) Performance of bean bruchids Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) reared on resistant (IT81D-1045) and susceptible (Epace 10) Vigna unguiculata seeds: relationship with trypsin inhibitor and vicilin excretion. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 142, 422-426.
  34. Strobl, S., Maskos, K., Wiegand, G., Huber, R., Gomis-Ruth, F. X. and Glockshuber, R. (1998) A novel strategy for inhibition of ${\alpha}-amylase$ in complex with the Ragi bifunctional inhibitor at 2.5 resolution. Structure 6, 911-921
  35. Suzuki, K., Ishimoto, M. and Kitamura, K. (1994) cDNA sequence and deduced primary structure of an alpha-amylase inhibitor from a bruchid-resistant wild common bean. Biochem. Biophys. Acta 1206, 289-291.
  36. Thomma, B. P., Cammue, B. P. and Thevissen, K. (2002) Plant defensins. Planta 216, 193-202.
  37. Vallee, F., Kadziola, A., Bourne, Y., Juy, M., Rodenburg, K. W., Svensson, B. and Haser, R. (1998) Barley alpha-amylase bound to its endogenous protein inhibitor BASI: crystal structure of the complex at 1.9 ${\AA}$ resolution. Structure 6, 649-659.
  38. Yamada, T., Hattori, K. and Ishimoto, M. (2001) Purification and characterization of two ${\alpha}-amylase$ inhibitors from seeds of tepary bean (Phaseolus acutifolius A. Gray). Phytochem. 58, 59-66.

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