Construction of an RNAi vector for knockdown of GM-ACS genes in the cotyledonary nodes of soybean

Background: Ethylene plays an important role in the regulation of floral organ development in soybean, and 1-aminocyclopropane-1-carboxylate synthase (ACS) is a rate-limiting enzyme for ethylene biosynthesis. However, whether ACS also regulates floral organ differentiation in soybean remains unknown. To address this, we constructed an RNAi vector to inhibit ACS expression in cotyledonary nodes. Linear DNA cassettes of RNAi-ACS obtained by PCR were used to transform soybean cotyledonary nodes. Results: In total, 131 of 139 transiently transformed plants acquired herbicide resistance and displayed GUS activities in the new buds. In comparison to untransformed seedling controls, a greater number of flower buds were differentiated at the cotyledonary node; GM-ACS1 mRNA expression levels and ethylene emission in the transformed buds were reduced. Conclusion: These results indicate that the cotyledonary node transient transformation system may be suitable for stable transformation and that the inhibition of ACS expression may be an effective strategy for promoting floral organ differentiation in soybean.

Pedalitin from isodon japonica, an inactivation of soybean lipoxygenase-1 / Pedalitina de isodon japonica, un inactivador de la lipo oxigenasa-1 de poroto de soya

Pedalitin, isolated from the aerial part of Rabdosia japonica (Labiatae), inhibited soybean lipoxygenase-1 (EC 1.13.11.12, Type I) with an IC50 of 152.5 uM. The progress curves for an enzyme reaction, pedalitin inactivate the lipoxygenase-1 in a time dependent, irreversible manner, exhibiting kinetics with a kinact/KI of 59.6 +/- 10 mM-1min-1. In the pseudoperoxidase activity, pedalitin is very slowly oxidized by the soybean lipoxygenase-1 catalyzed decomposition of lipid hydroperoxides.

Pedalitina, aislada de las partes aereas de Rabdosia japonica inhibió a la lipooxigenasa-1 (EC 1.13.11.12 tipo I) con un IC50 de 152.5 uM. La curva de progreso para una acción enzimática, pedalitina inactivó a la lipooxigenasa-1 de una manera dependiente del tiempo, de una manera irreversible, exhibiendo una cinética con una kinact/KI de 59.6 +/- mM-1min-1. En la actividad pseudoperoxidasa, pedalitina es oxidada lentamente por la descomposición de la lípido hidroperóxido de la lipooxigenasa-1 de poroto de soya.

Produção de lacase de Pycnoporus sanguineus em meio de cultivo à base de melaço soja / Laccase production by Pycnoporus sanguineus in culture media with soybean molasses / Producción de lacasa de Pycnoporus sanguineus en medio de cultivo con melaza de soja

Lacases são polifenol oxidases que utilizam a capacidade redox de íons cobre para reduzir oxigênio a água e oxidar um substrato fenólico. A síntese e secreção de lacases de basidiomicetos dependem de vários fatores como os nutrientes presentes no meio de cultura. Visando à produção de lacase, Pycnoporus sanguineus foi cultivado em meio contendo melaço de soja como única fonte de carbono, ureia como fonte de nitrogênio suplementar em diferentes concentrações (0,6; 1,2; 2,4; 4,8 e 9,6 g/L de nitrogênio) e diferentes concentrações de CuSO4 (0, 150, 200, 250 e 300 µM). O extrato enzimático produzido nas melhores condições de cultivo foi utilizado para a descoloração dos corantes remazol azul brilhante R (antraquinona), amarelo 145, preto 5, vermelho 195 (azo) e verde malaquita (trifenilmetano). As concentrações de nitrogênio não afetaram a produção de lacase, exceto a maior concentração (9,6 g/L) que reduziu a atividade enzimática. A adição de cobre ao meio de cultivo (150 µM) aumentou a atividade de lacase em 112%. A maior atividade de lacase (~34300 U/L) promoveu a descoloração dos corantes remazol azul brilhante R (67,5%) e verde malaquita (28,3%) em 24h, sendo os corantes azo descoloridos apenas parcialmente. Concluiu-se que o melaço de soja é um resíduo agroindustrial adequado para produção de lacase de P. sanguineus com potencial para degradação de corantes.

Laccases are multicopper oxidases using the redox ability from copper ions to reduce oxygen to water, while oxidizing a phenolic substrate. Laccase synthesis and secretion in basidiomycetes depend on the conditions provided and on the nutrients present in the culture medium. Pycnoporus sanguineus was cultivated in medium containing soybean molasses as the sole carbon source, with urea as the source of supplemental nitrogen at different concentrations (0.6, 1.2, 2.4, 4.8 and 9.6 g/L nitrogen), and different CuSO4 concentrations (0, 150, 200, 250 and 300 µM). The enzymatic extract produced under the best cultivation conditions was used for the depigmentation of remazole brilliant blue R (anthraquinone), yellow 145, black 5, red 195 (azo) and malachite green (triphenylmethane). Nitrogen concentrations did not affect laccase production, except for the higher concentration (9.6 g/L) reducing enzymatic activity. The addition of copper to the culture medium (150 µM) increased laccase activity by 112%. The highest laccase activity (~34300 U/L) promoted the depigmentation of remazol brilliant blue R (67.5%) and malachite green (28.3%) dyes in 24 hours. Azo dyes were only partially discolored. Therefore, it can be considered that soybean molasses is an agro-industrial byproduct suitable for the production of P. sanguineus laccase with potential for dye degradation.

Lacasas son polifenoles oxidasas que utilizan la capacidad redox de iones de cobre para reducir el oxígeno del agua y oxidar un sustrato fenólico. La síntesis y secreción de lacasas de basidiomicetos dependen de las condiciones como los nutrientes presentes en el medio de cultura. Buscando la producción de lacasa, se cultivó Pycnoporus sanguineus en medio que contenía melaza de soja como única fuente de carbono, urea como fuente de nitrógeno suplementar a diferentes concentraciones (0,6, 1,2, 2,4, 4,8 y 9,6 g/L de nitrógeno) y diferentes concentraciones de CuSO4 (0, 150, 200, 250 y 300 µM). El extrato enzimático producido en mejores condiciones de cultivo ha sido utilizado para la decoloración de los colorantes remazol azul brillante R (antraquinona), amarillo 145, negro 5, rojo 195 (azoico) y verde malaquita (trifenilmetano). Las concentraciones de nitrógeno no afectaron la producción de lacasa, excepto la mayor concentración (9,6 g/L) que redujo la actividad enzimática. La adición de cobre al medio de cultivo (150 µM) aumentó la actividad de la lacasa en un 112%. La mayor actividad de lacasa (~34300 U/L) promovió la decoloración de los colorantes remazol azul brillante R (67,5%) y verde malaquita (28,3%) en 24h, siendo que los colorantes azoicos fueran parcialmente decolorados. Se concluye que la melaza de soja es un desecho agroindustrial adecuado para la producción de lacasa de P. sanguineus con potencial para degradación de colorantes.

Medium optimization of protease production by Brevibacterium linens DSM 20158, using statistical approach

Various cultivation parameters were optimized for the production of extra cellular protease by Brevibacterium linens DSM 20158 grown in solid state fermentation conditions using statistical approach. The cultivation variables were screened by the Plackett-Burman design and four significant variables (soybean meal, wheat bran, (NH4)2SO4 and inoculum size were further optimized via central composite design (CCD) using a response surface methodological approach. Using the optimal factors (soybean meal 12.0g, wheat bran 8.50g, (NH4)2SO4) 0.45g and inoculum size 3.50%), the rate of protease production was found to be twofold higher in the optimized medium as compared to the unoptimized reference medium.

Características de la soja como alimento / Characteristics of soy as a food

La soja es una leguminosa que forma parte de la alimentación tradicional en China y otros países asiáticos. En los países occidentales su incorporación a la dieta es reciente. La soja tiene un elevado contenido en proteínas de buena calidad, de gran importancia en las personas que siguen dietas vegetarianas. También presenta un contenido interesante en ácidos grasos poliinsaturados, isoflavonas y fibra. Existe evidencia sobre los efectos interesantes de las isoflavonas, aunque la evidencia disponible por el momento no es concluyente y existen algunas dudas sobre las dosis a ingerir como suplemento, tiempo de administración o posible riesgo de efectos secundarios. No obstante, es importante tener en cuenta que estas cuestiones no hacen referencia al consumo de la soja como alimento en el contexto de una dieta equilibrada (AU)

Soy is a legume that has been part of the traditional diet in China and other Asian countries. However, its use in Western countries is recent. Soy has a high content in quality protein, relevant for people under a vegetarian diet. It also has interesting content in polyunsaturated fatty acids, fiber and isoflavones. There is evidence on the effects of isoflavones on health, although the evidence available at the moment is not conclusive and there is some controversy about the amount of supplemental doses, time of administration and risk of potential side effects. However, it is important to note that these matters do not refer to the use of soybeans as food in the context of a balanced diet (AU)

La soja en la prevención de la enfermedad cardiovascular / Soy in the prevention of cardiovascular disease

Los alimentos de soja o aislados de proteína de soja contienen dos componentes bioactivos principales, las isoflavonas (ISF) asociadas con la proteína de soja y los péptidos generados a partir de las dos principales proteínas de la soja, B-conglicina y la glicina. Algunos componentes de la dieta con soja controlan las funciones de adhesión del endotelio vascular, mediante la regulación de los mecanismos claves de control de las moléculas de adhesión endoteliales, la función de la integrina en los monocitos, citoquinas y quimoquinas en el control del tráfico y migración de los monocitos. También podrían regular la oxLDL en la generación y regulación de la expresión de los recogedores de radicales libres. La dieta con soja y otras dietas con efectos antiinflamatorios pueden bloquear el proceso inflamatorio asociado con la aterogénesis, reduciendo así el riesgo de ECV. Se requieren de más estudios que aclaren tanto a nivel experimental como clínico los mecanismos de acción de las ISF en la protección cardiovascular (AU)

Soy food or soybean protein isolates contain two major bioactive components, isoflavones (ISF) associated with soy protein, and peptides generated from two major soybean protein, B-conglycinin and glycine. Some components of the diet with soybean control the functions of vascular endotelial adhesion, by regulating the control key mechanisms of endothelial adhesion molecules, integrin function in monocytes, cytokines and chemokines in the traffic control and migration of monocytes. OxLDL may also regulate the generation and regulation of expression of catchers of free radicals. The diet with soy and other anti-inflammatory diets can block the inflammatory process associated with atherogenesis, thus reducing the risk of CVD. Further studies are required to clarify both experimental and clinical mechanism of action of the ISF cardiovascular protection (AU)

Beneficios de la soja en la menopausia / Benefits of soy on menopause

Los datos epidemiológicos sugieren que el consumo de productos de soja está correlacionado con la mejora de los problemas asociados con la menopausia y la incidencia de enfermedades crónicas, como la osteoporosis y ciertos tipos de cáncer. Aunque la evidencia actual no es concluyente, sugiere que la soja podría constituir una opción alternativa a la terapia de sustitución hormonal (TSH) para aliviar la sintomatología menopáusica, considerando la posibilidad de emplearla únicamente en períodos de corta duración en mujeres sin antecedentes de cáncer. Son necesarios estudios bien diseñados para establecer, entre otros aspectos, la dosis óptima de consumo para conseguir los efectos beneficioso. La soja no es la única fuente de fitoestrógenos y su efecto podría deberse a la calidad de la dieta. Una dieta completa, variada y equilibrada es un pilar fundamental como prevención de la salud (AU)

Epidemiological data suggest that consumption of soy products is correlated with the improvement of the symptoms associated with menopause and the incidence of chronic diseases, such as osteoporosis and certain types of cancer. Although current evidence is not conclusive, suggests that soy could be an alternative to hormone replacement therapy (HRT) to relieve menopausal symptoms, considering the possibility of use only for short periods in women with no history of cancer. Well-designed studies are needed to establish, among other things, optimal dose to achieve the beneficial effects. Soy is not the only source of phytoestrogens and their effect may be due to the overall quality of the diet. A complete, varied and balanced diet is a mainstay in health prevention (AU)

Beneficios de la soja en la edad pediátrica / Benefits of soy in infancy and childhood

Las fórmulas infantiles de soja cumplen las recomendaciones nutricionales de la Sociedad Europea de Gastroenterología, Hepatología y Nutrición Pediátrica (ESPGHAN) y de la Academia Americana de Pediatría (AAP) para las fórmulas de inicio y continuación, así como las de otros órganos consultivos. Todas las fórmulas de soja están fortificadas con yodo, hierro, metionina, carnitina y taurina, y para contrarrestar su contenido en fitatos suelen llevar un 20% más de calcio y fósforo que los fórmulas a base de leche de vaca. Sus indicaciones médicas actuales se limitan al tratamiento de la galactosemia, la alergia a proteínas de leche de vaca sin sensibilización a la proteína de soja, además de aquellas situaciones en que por razones filosóficas o religiosas deban incluirse como parte de dietas vegetarianas. Tanto el Comité de Nutrición de la ESPGHAN y como el de la AAP y la European Society for Paediatric Allergology and Clinical Immunology desaconsejan su uso en niños menores de 6 meses. El Comité Asesor de Guías Dietéticas 2010 recuerda la importancia del consumo de proteínas de alta calidad en niños y recomienda basar la dieta en fuentes proteicas vegetales para reducir el riesgo de enfermedades crónicas y lograr un buen estado nutricional. La proteína de la soja puede considerarse única en la calidad de su composición dentro de las de origen vegetal, con una amplia disponibilidad y capacidad de producción. La soja constituye una fuente de proteínas de alto valor biológico libre de grasas saturadas (AU)

Soy formulas meet the nutritional recommendations of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) and the American Academy of Pediatrics (AAP), as well as other advisory bodies, for newborn and infant formulas. All soy formulas are fortified with iodine, iron, methionine, carnitine and taurine, and to counter act their phytate content typically carry 20% more calcium and phosphorus than formulas based on cow’s milk. Current medical indications for soy formulas are limited to treatment of galactosemia cow’s milk protein allergy without sensitization to soy protein, in addition to those situations where is included as part of vegetarian diets due to philosophical or religious reasons. The ESPGHAN Committee on Nutrition, the AAP and the European Society for Paediatric Allergology and Clinical Immunology advise against its use in children younger than 6 months. The Dietary Guidelines Advisory Committee 2010 recalls the importance of the consumption of high quality protein in children and recommended diet based on vegetable protein sources to reduce the risk of chronic disease and achieve good nutritional status. The soy protein can be considered as unique in its composition within the plant, with the widespread availability and capacity. Soy is a source of high biological value proteins free of saturated fats (AU)

Consejos prácticos para incluir la soja dentro de una dieta equilibrada / Tips to include soy in a balanced diet

La soja es una alimento interesante por su contenido en proteínas de alto valor biológico, grasas poliinsaturadas, e hidratos de carbono de absorción lenta, además de fitoquímicos como las isoflavonas. Las proteínas de la soja, incorporadas a una dieta baja en grasas saturadas, pueden reducir el nivel de colesterol, el riesgo de enfermedades cardiovasculares y el riesgo de padecer algunos tipos de cáncer como el de colon y próstata. También es interesante como sustitutiva en la intolerancia a la lactosa y en la alergia a la proteína de leche de vaca en el adulto, por sus efectos para una piel la saludable y por su posible papel en la prevención y/o tratamiento del cáncer de próstata. La evidencia disponible no es concluyente y es necesario seguir investigando con estudios bien diseñados sobre estos aspectos (AU)

Soybean is an interesting food because of its content in high biological value protein, polyunsaturated fats, and slow absorption carbohydrates, plus phytochemicals such as isoflavones. Soy proteins as part of a balanced diet low in saturated fat, can reduce the cholesterol level, the risk of cardiovascular disease and the risk of certain types of cancer such as colon and prostate cancer. It is also interesting as a substitute in lactose intolerance and allergy to cow’s milk protein in the adult, for its effects for a healthy skin and their possible role in the prevention and / or treatment of prostate cancer . The evidence available is not conclusive and further research is needed with well-designed studies on these aspects (AU)

Pectinolytic enzyme production by Colletotrichum truncatum, causal agent of soybean anthracnose

Antecedentes. Colletotrichum truncatum es el hongo patógeno más comúnmente asociado con la antracnosis de la soja, enfermedad de alta prevalencia en Argentina. Las enzimas pectinolíticas se relacionan con la patogenicidad de un amplio rango de hongos fitopatógenos. Objetivos. Investigar la producción de enzimas pectinolíticas por cepas aisladas de plantas de soja enfermas de diferentes regiones de nuestro país, y con ello contribuir a la caracterización fisiológica de dichos aislamientos como paso preliminar para esclarecer el aún desconocido rol biológico de las enzimas pectinolíticas en la interacción Colletotrichum spp-soja. Métodos. Se investigó la producción in vitro de enzimas pectinolíticas, en un medio sintético con pectina como fuente de carbono, de diez aislamientos de C. truncatum. Resultados. Todas las cepas crecieron en dicho medio, detectándose actividades polimetilgalacturonasa (PMG), poligalacturonasa (PG) y pectin liasa (PL). En general, el pico de galacturonasas precedió al día de máximo crecimiento, en cambio el de PL se registró posteriormente. La cepa BAFC 3097 (originaria de la Provincia de Santa Fe) produjo altos títulos de las tres enzimas tras 7–10 días: 1,08U/ml PG, 1,05U/ml PMG, 156U/ml PL. C. truncatum, cultivado en un medio con glucosa como fuente de carbono, produjo PG y PMG (pero no PL), aunque su síntesis disminuyó marcadamente sugiriendo que estas enzimas son constitutivas. Conclusión. La disparidad registrada en la producción enzimática entre cepas no puede atribuirse al crecimiento fúngico; tampoco se corresponde con su distribución geográfica; pero podría relacionarse con diferencias en su virulencia, que aún no se han investigado(AU)

Background. Colletotrichum truncatum is the most common pathogenic fungus associated with soybean anthracnose, a prevalent disease in Argentina. Pectinolytic enzymes are involved in the pathogenicity of a wide range of plant pathogenic fungi. Objectives. To explore pectinolytic enzyme production in Argentinian Colletotrichum strains isolated from diseased soybean plants from different geographic locations, as a preliminary step to establish the biological role of the pectinolytic enzymes in the Colletotrichum spp.–soybean system, yet unknown. Methods. Ten strains were screened for in vitro pectinolytic enzyme production on a defined medium based on pectin as carbon source. Results. All isolates were able to grow in this medium and polymethylgalacturonase (PMG), polygalacturonase (PG) and pectin lyase (PL) activities were detected. On the whole, the peak of polygalacturonases activities preceded the day of maximum growth, while PL activity reached its highest level afterwards. Strain BAFC 3097 (from Santa Fe province) yielded high titles of the three enzymes (1.08U/ml PG, 1.05U/ml PMG, 156U/ml PL), after a short incubation period (7–10 days). Low synthesis of polygalacturonases in cultures containing glucose as unique carbon source suggests that these enzymes are constitutive in contrast with PL, which was not detected. Conclusions. The disparity observed in enzyme production among strains cannot be related to fungal growth, since no major differences in mycelial yield were found; it was not connected with their geographic origin, but might be associated with differences in virulence among strains not yet evaluated(AU)

Avaliação de equivalência substancial e potencial de alergenicidade de cultivares de soja tolerantes ao herbicida glifosato / Evaluation of substantial equivalence and potential of allergenic reactions of soybean cultivars tolerant to the glyphosate herbicide

Os parâmetros de avaliação de segurança de alimentos geneticamente modificados fundamentam-se na comparação de equivalência substançial entre as variedades e pela inocuidade de proteínas da planta GM com as proteínas encontradas nas plantas convencionais. O objetivo deste trabalho foi avaliar a segurança alimentar de três cultivares de sojas geneticamente modificadas para tolerarem o herbicida glifosato através da determinação da equivalência substancial e do potencial alergênico das mesmas quando comparadas às suas respectivas parentais isogênicas. Seis amostras de soja foram analisadas, sendo três convencionais parentais e três GM, referentes ao cultivo de 2004-2005, em Goiás. Para a composição química foram realizadas análises em triplicata de proteínas, lipídeos, umidade, minerais e fibra alimentar. Análises complementares para determinação de aminoácidos, ácidos graxos, isoflavonas e ácido fítico também foram realizadas. O potencial de alergenicidade foi avaliado em extratos protéicos brutos de três cultivares convencionais e suas correspondentes GM. Os mesmos extratos protéicos foram fracionados para obter as globulinas 7S e 11S por precipitação e posterior purificação em coluna de bioafinidade Sepharose 4B...

Qualidade protéica de linhagens de soja com ausência do Inibidor de Tripsina Kunitz e das isoenzimas Lipoxigenases / Protein quality of soy lines lacking Kunitz's Trypsin Inhibitor and Lipoxygenase isozymes

OBJETIVO: Avaliar a qualidade protéica de farinhas de soja, que diferem entre si com relação à presença de lipoxigenases e/ou Inibidor de Tripsina Kunitz. MÉTODOS: Procedeu-se aos ensaios biológicos com ratos, em que foram avaliados a Razão da Eficiência Protéica, Razão Protéica Líquida, Utilização Protéica Líquida e a digestibilidade. Determinou-se a composição aminoacídica das farinhas e cálculo do Escore Químico Corrigido pela Digestibilidade. RESULTADOS: Os valores de Razão da Eficiência Protéica, Razão Protéica Líquida, Utilização Protéica Líquida para as variedades de soja foram inferiores aos valores obtidos para caseína. Para as farinhas sem Inibidor de Tripsina Kunitz foram obtidos valores de digestibilidade maiores que para as farinhas com Inibidor de Tripsina Kunitz, e esses foram bem próximos aos da caseína. Com relação ao teor aminoacídico, constatou-se que o aminoácido limitante dessa soja é lisina e não metionina, ao contrário do que aponta a literatura. CONCLUSAO: A eliminação genética do Inibidor de Tripsina Kunitz melhora consideravelmente a digestibilidade da proteína de soja. Os resultados do Escore Químico Corrigido pela Digestibilidade não indicaram diferença entre as farinhas sem Inibidor de Tripsina Kunitz das farinhas derivadas de linhagens com Inibidor de Tripsina Kunitz, como foi observado pelos resultados da digestibilidade in vivo.

Degradação da clorofila durante a maturação e secagem de sementes de soja / Chlorophyll degradation during maturation and drying of soybean seeds

A presença de clorofila em óleos provenientes da extração de grãos verdes reduz sua qualidade oxidativa, confere coloração escura indesejável e diminiui a velocidade do processo de hidrogemação, sendo que sua remoção eleva o custo da refinação. Da mesma forma, a presença do pigmento reduz o valor comercial dos grãos, levando a prejuízos na comercialização, frente ao enorme volume de exportação brasileira. A retenção da clorofila em sementes oleaginosas ocorre em condições de colheita prematura acoplada a estresse hídrico no campo, ou secagem rápida em estufa, porém, o mecanismo de degradação ainda não foi bem esclarecido. Neste trabalho investigou-se a degradação da clorofila e o aparecimento...

Actividad ureásica en productos de soja. Propuesta de un nuevo método / Ureasic activity in soy products. Proposal of a new method

The evaluation of residual ureasic activity in soy products is usually employed as an indicator of the efficiency of the inhibition treatments. The purpose of this study is to compare the AACC method (22-90), based on differences of pH, with an assay where ureasic activity is measured by its hydrolytic action on urea and quantification of the ammonium produced with Berthelot reaction. Twenty different samples of soy bean flour with and without thermal inactivation treatments were assessed with the two methods. The new method has a good correlation with that of the AACC--r = 0.9416 (p < 0.0001). It also presents a better specificity because it measures the concentration of the reaction product and shows a more amplified answer than the increase of pH.

Inactivación de lipoxigenasas e inhibidoeres de tripsina en un proceso de obtención de leche de soja por molienda directa y ultra alta temperatura (MD-UAT) / Lipoxygenases and trypsin inhibitors inactivation in a soymilk processing by direct milling and ultra high temperature (DM-UHT)

La elaboración de leche de soja mediante un nuevo proceso de Molienda Directa de sémola de soja y Ultra Alta Temperatura (MD-UAT), ha sido objeto de estudios de laboatorio en sus etapas de molienda y tratamientos térmicos, a los efectos de evaluar la extracción de sólidos y proteínas, como así también la inactivación de lipoxigenasas e inhibidores de tripsina (IT). Para la determinación de IT en extractos y leche de soja se modificó el método clásico de Kakade (17). Los mayores rendimientos de extracción en la molienda se obtienen a 70 grados Celsius durante 2 minutos. La inactivación de lipoxigenasas es notablemente más eficiente durante la molienda si se utiliza solución de carbonato de sodio 0,01 M en lugar de agua como medio dispersante (actividad residual de 14 por ciento en carbonato frente a 46 por ciento en agua), reduciéndose así la generación de sustancias indeseables. Se completa la destrucción de lipoxigenasas con un breve calentamiento de 30 segundos con vapor desde 70 grados Celsius hasta la temperatura de ebullición a 96 grados Celsius. Por otra parte, los inhibidores de tripsina no son adecuadamente inactivados en la molienda ni tampoco en el tratamiento con vapor a 96 degrees Celsius durante varios minutos. Posteriormente, en la etapa de UAT a 139 grados Celsius durante dos minutos se logra la inactivación de los IT hasta los valores considerados aceptables, posibilitando la destrucción simultánea de microorganismos. Estas condiciones se logran sin dificuldad en el proceso MD-UAT, cuya flexibilidad permite adecuar temperaturas y tiempos a requerimentos específicos.

Inhibition of aminopeptidase activity by aromatic and other cyclic compounds

The effect of 2-naphthylamine, p-nitroaniline, o-phenanthroline, sodium deoxycholate and hydrocortisone succinate on the activity of human urine aminopeptidase, rat kidney methionyl and arginyl aminopeptidase, soybean and Enterolobium contortisiliquum seed aminopeptidase was studied using aminoacyl-2-naphthylamide and L-Leu-p-nitroanilide as substrates. Ki values ranged from 10 microM to 2.7 mM. On the basis of Ki and Km values, and catalytic efficiency for each enzyme, it is clear that the aminopeptidases from human urine and from soybean seed should be assayed with both substrates, whereas L-Leu-p-nitroaniline is a more appropriate substrate for the rat kidney aminopeptidases. Sodium deoxycholate is a better inhibitor than hydrocortisone succinate. Non-competitive inhibition was observed in all cases except for E. contortisiliquum seed aminopeptidase

ELISA na determinaçäo quantitativa e análise genética nas lipoxigenases L1 e L2 em soja / ELISA in the quantification and genetic analysis of lipoxygenases L1 and L2 from soybeans

A soybean breeding program has been undertaken at the Federal University of Viçosa aiming to reduce lipoxygenase lelvels ib the seeds of commercial varieties. A direct ELISA (Enzyme Linked Immunosorbent Assay) procedure has been developed to quantify lipoxygenases L1 and L2 in soybean seeds. Polyclonal antibodies were reised in rabbits against pure L1 and L2 and purified further by affinity chromatography. The best conditions to perform the analaysis were determined as foloow: coating antibody dilution 1:100, crude soybean protein extract 1:2 x 105 and conjugate 1:100 for both L1 and L2 isozymes. The method proved to be sensible enough to identify homozygous and heterozygous genotypes in the breeding program performing single non destructive analysis of seeds or even cotileldones right after germination. Besides, L1 was quantified in about 120 soybean Brazilian cultivars an L2 in some of them. The results showed a significant variability in the level of lipoxygenase-1 among the cultivars analysed. With respect to L2, it was found that the level of this isozyme was significantly greater in the seeds than observed for L1, in spite of its lower "in vitro" specific activity

Dupla difusäo radial na análise genética das lipoxigenases L1 e L2 em soja / Double gel immunodiffusion in the genetic analysis of lipoxygenases L1 and L2 from soybeans

In the effort to genetically manipulate soybean seeds aiming to improve flavor quality toward human consuption, lipoxygenase null alleles are being transferred to a brazilian commercial variety. Immunochemical approach has been taken to perform analysus of hundred of seeds derived from the breeding program. For that reason lipoxygenases L1 and L2 were puridied to hemogeneity by gel filtration and affinity chromatography. Policlonal antibodies were prepared in rabbits and purified further by affinity chromatogrphy. By using the purified anti-L1 and anti-L2 an Ouchterlony double immunodifusion method has been developed in our lab to detect the presence/absence of the isozymes simultaneously in soybean seeds. The method proved to be highly specific and efficient enought to perform single non destructive seed analysis