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A hydrophobic 19.7-kDa amylase inhibitor (AI) was purified from corn kernels by 95% ethanol extraction and anionic exchange chromatography. The AI has an isoelectric point of 3.6 and was very stable at different pH values and high temperatures, maintaining 47.6% activity after heating to 94 degrees C for 60 min. Amino acid analysis indicated high valine, leucine, glycine, alanine, and glutamic acid/glutamine content, and especially high valine content (41.2 mol%). This inhibitor is not a glycoprotein. It required 30-min preincubation to maximize complex enzyme-inhibitor formation when the amylase from Fusarium verticillioides was tested. The optimal pH of interaction was 6.5. It showed broad-spectrum activity including the following amylases: human saliva, porcine pancreas, F. verticillioides, as well as those from some insects of agricultural importance (Acanthoscelides obtectus, Zabrotes subfasciatus, Sitophilus zeamais, and Prostephanus truncatus). This novel hydrophobic protein not only inhibited the amylase from F. verticillioides but also decreased the conidia germination. Thus, this protein represents an approach to decrease the production of fumonisin in corn, either by using it as a molecular marker to detect fungal resistance or through genetic engineering.

Alpha-amylase inhibitors have important roles in plant defense mechanisms, particularly against insects, and several of these inhibitors have been expressed in different crops to increase their resistance to particular insects. In this work, we report the cloning and expression of a gene encoding for a new alpha-amylase inhibitor (BIII) from rye (Secale cereale) seeds. The BIII gene contains 354 nucleotides that encode for 118 amino acids sequence. A 313 bp fragment of the gene was expressed in Escherichia coli and resulted in a functional inhibitor that reduced the activity of alpha-amylases of larvae of the coleopteran pests Acanthoscelides obtectus, Zabrotess subfasciatus and Anthonomus grandis. In contrast, the inhibitor did not inhibit the activity of porcine pancreatic alpha-amylase. Although the amino acid sequence of BIII showed high identity with those of bifunctional inhibitors, the recombinant protein was unable to inhibit trypsin-like serine proteinases. The effects of recombinant BIII were evaluated in vivo against A. grandis. When first instar larvae were reared on an artificial diet containing four different concentrations of BIII, a reduction in larval weight and a mortality of 83% were observed at the highest concentration.

A screening for specific amylase inhibitor levels against amylase from Fusarium verticillioides (Fusarium moniliforme), the most relevant mycotoxigenic fungus in corn, was conducted on 37 corn hybrids. The amylase inhibitor levels in these hybrids ranged from 5.5 to 16.0 amylase inhibitor units per gram of corn (AIU/g) in the MASTER and AG5011 hybrids, respectively. The hybrid with the maximum content of inhibitor was used as the source of this new protein. The inhibitor was partially purified using fractional precipitation, gel filtration on Sephadex G-75 column, high performance liquid chromatography (HPLC) Superose HR 10/30 column, and HPLC anion exchange chromatography, obtaining a 20.7-fold purification. Electrophoresis after denaturing and heating under reductive conditions showed an apparent 23.8 kDa molecular mass and an acidic isoelectric point of 5.4, which differs from previous molecular masses reported for other inhibitors present in corn seeds (14 and 22 kDa). This inhibitor showed activity against amylases from human saliva and pancreas, from the fungi F. verticillioides and Aspergillus flavus, and from the insects Acanthoscelides obtectus, Zabrotes subfasciatus, Tribolium castaneum, and Sitotroga cerealella. The mycoflora found in the corn grain indicated Fusarium sp. as the most prevalent fungi (81.1% of the samples), with a count ranging from 1.5 × 102 to 2.4 × 106 CFU/g of corn. The presence of fumonisins was detected in 21 out of the 37 hybrids studied, ranging from 0.05 to 2.67 μg of FB per gram of corn. No correlation could be established between this amylase inhibitor level in the corn seeds and the presence of Fusarium sp. or with the fumonisin content under the experimental conditions of the test.

Neotropical Entomology 34(3): 443-450 (2005) Amylase Inhibitors in Corn Hybrids as a Resistance Factor to Sitophilus zeamais (Coleoptera: Curculionidae)ABSTRACT -Sitophilus zeamais Motschulsky is one of the major pests of stored corn and it is mainly controlled by insecticides. However, due to the undesirable consequences of chemicals on the environment and human health other control measures have been investigated, especially the development of resistant plants. The presence of amylase inhibitors in corn kernels may interfere with the insect digestive process. This research was developed in order to evaluate resistance of corn hybrids to the attack of S. zeamais correlated with amylase inhibitors and nutritional composition of corn kernels. Adults of the maize weevil were exposed to grain samples of different corn hybrids at 27 ± 1°C and 75 ± 5% R.H. Biology, susceptibility index and grain consumption were evaluated. The results were correlated to the grain components (moisture content, ashes, lipids, proteins and carbohydrates) and with the amylase inhibitor activity. The amylase inhibitor activity was determined by incubating the amylase inhibitor with 0.2 units of amylase for 30 min at 25°C, and measuring the residual amylase activity determined by the iodine method. From the nutritional parameters evaluated, only the lipid content correlated significantly with the susceptibility index (r = 0.46). The amylase inhibitors obtained by the extraction with either ethanol or buffer were significantly and negatively correlated with the susceptibility index (r = -0.58), indicating that the inhibitors contribute to the resistance of corn hybrids to the attack of S. zeamais.KEY WORDS: Insecta, maize weevil, plant resistance, stored corn RESUMO -Sitophilus zeamais Motschulsky é uma das principais pragas do milho armazenado, sendo controlado, principalmente, com inseticidas. Porém, devido às conseqüências indesejáveis desses químicos sobre o meio ambiente e a saúde humana, outras táticas de controle têm sido investigadas, especialmente o desenvolvimento de plantas resistentes. A presença de inibidores de amilase nos grãos de milho pode interferir na digestão do inseto. Esta pesquisa foi desenvolvida com o objetivo de avaliar a resistência de híbridos de milho ao ataque de S. zeamais, correlacionada com a presença de inibidores de amilase e com a composição nutricional dos grãos. Adultos do gorgulho-do-milho foram expostos a amostras de grãos de diferentes híbridos a 27 ± 1 o C e 75 ± 5% de UR. Avaliou-se o ciclo biológico, peso dos adultos, índice de suscetibilidade e consumo dos grãos. Os resultados foram correlacionados com a composição dos grãos (teores de umidade, cinzas, lipídios, proteínas e carboidratos) e com a atividade de inibidores de amilase. A atividade inibidora de amilase foi determinada incubando-se o inibidor com 0,2 unidades de amilase durante 30 min. a 25°C, medindose posteriormente a atividade amilásica residual pelo método iodométrico. Dos parâmetros nutricionais avaliados, somente o conteúdo de ...