Tesi sul tema "Amylolytic enzymes"

A purification scheme has been developed for the extracellular carbohydrases; aglucosidase and a-amylase, secreted by the ascosporogenous soil yeast Lipomyces starkeyi NCYC 1436. Growth and optimum enzyme yield conditions were determined with cultures of L. starkeyi grown at 30°C on a medium containing 2% soluble starch, 1% yeast extract and 1% Bactopeptone, appropriate enzyme assay procedures having been devised. Both enzymes were initially precipitated from the cell free supernatant by an 85%(w/v) ammonium sulphate precipitation. Many different chromatographic media were then assessed, but the choice of a hydrophobic (Phenyl Sepharose CL-4B) column had the advantage of utilizing the ammonium sulphate precipitate with minimum sample preparation. The pellet was adjusted to 1M salt and adsorbed to the hydrophobic column. Elution of the two activities was carried out by a series of decreasing salt washes. Although fractionation of the two enzymes was not complete, the column was effective in eliminating a substantial quantity of inactive material, whilst maintaining good recoveries of approximately 59% for cc-glucosidase and 72% for a-amylase. Both enzymes were substantially purified using ion exchange chromatography (QSepharose fast flow medium), with a-glucosidase being apparently close to electrophoretic homogeneity; elution of the two enzyme activities was carried out at 4°C, using a linear gradient with sodium acetate buffer, pH 5.0. Individual HIC fractions purified by ion exchange showed between a 0.3 to 24.6-fold increase in purity for a-glucosidase, and between a 2.8 to 7.6 increase in purity for aamylase. However, by selecting the most active a-glucosidase fraction isolated after running the 0.01M HIC fraction on ion exchange, the purification factor rose to 83.4. Several properties of the purified a-glucosidase enzyme were investigated. The molecular weight of the a-glucosidase was determined by electrophoresis under denaturing and non-denaturing conditions, after ion exchange chromatography. A single major band was detected by SDS-PAGE with an estimated molecular weight of 93,000 ± 5,000 Daltons, and under native conditions, the molecular weight was estimated at 162,000 Daltons. Specific enzyme activity staining of the native gel confirmed that the single band was an a-glucosidase. However, isoelectric focusing of the purified Lipomyces starkeyi a-glucosidase, detected three bands - within the pi range of 4.6-5.0. Further characterisation studies revealed the pH and temperature optimum of the purified L starkeyi a-glucosidase at 4.5 and 55°C respectively. Km and Fmax measurements using a range of substrates suggested that the aglucosidase had a broad substrate specificity but showed a marked preference for substrates with a-1,4 linkages such as soluble starch, PNPG, and a-1,3 linkages, such as nigerose SDS and native gel electrophoresis (combined with specific activity staining) revealed 3 distinct a-amylase activities, and the molecular weights were determined as 82,000, 68,000 and 48,500 Daltons.

A germinação do milho (Zea mays L.) é usada no processamento de alimentos pela ativação de enzimas endógenas em benefício das propriedades sensoriais, nutricionais e funcionais. Dados sobre alterações composicionais em sementes germinadas são escassas, sobretudo em sementes geneticamente modificadas. Neste estudo foram avaliadas quatro variedades de milho, duas geneticamente modificadas (G1 e G3) e suas genitoras convencionais (C1 e C3), durante as duas primeiras fases da germinação, enfocando a composição química, o desenvolvimento de atividades amilolíticas, as mudanças na concentração e morfologia do amido, e o perfil das proteínas remanescentes durante um período de 72h. As sementes germinaram rapidamente ocorrendo a primeira protrusão radicular às 12 h após a embebição, ao atingirem um teor de umidade de aproximadamente 20%. A atividade de α-amilase alterou-se após 24 h, seguida por acentuada elevação até o tempo 72h. A atividade β-amilásica foi detectada desde o início dos ensaios de germinação, caracterizada por incremento constante até 48h entre as sementes convencionais e até 72h entre as sementes modificadas. Os níveis de atividade amilolítica total permaneceram elevados no decorrer da germinação, com maior intensidade nas variedades GM, porém com os perfis das hidrólises semelhantes. A redução dos teores de amido foi observada após as 12h e 24h, indicando o início da mobilização de reservas de carboidratos. As alterações morfológicas no grânulo de amido nas diferentes etapas da germinação foram acompanhadas por microscopia eletrônica de varredura, com resultados semelhantes nas quatro variedades investigadas. Diferenças significativas foram observadas apenas nas composições químicas e nos perfis proteicos entre os dois grupos de sementes de milho (C1-G1 e C3-G3), indicando variação normal entre as variedades convencionais genitoras, sem comprometer a segurança dos milhos GM, nem tampouco o emprego em processos fermentativos. Ao contrário, as sementes de milho GM apresentaram mobilização mais rápida do amido nas primeiras duas fases da germinação.
The maize (Zea mays L.) germination activates endogenous enzymes that is useful on food processing to develop sensorial, nutritional, and functional properties. Data about germinated seeds are scarce, especially in genetically modified seeds. In this study we evaluated four maize varieties, two genetically modified (G1 and G3) and their conventional parents (C1 and C3) during the first two steps of germination, focusing on the chemical composition, changes in the starch concentration and morphology, the development of amylolytic activity and the profile of the proteins remaining during a period of 72h. The seeds germinate quickly occurring the first roots protrusion at 12 hours after imbibition, to reach a moisture content of approximately 20%. The activity of αamylase changed after 24 h, followed by marked elevation until 72 hours. The activity of β-amylase was detected since the beginning of the tests of germination, characterized by the constant increase until 48 hours by the conventional seeds up to 72h by the modified seeds. The levels of total amylolytic activity stay high in the course of germination, with a greater intensity in the GM varieties, however with the similar hydrolysis profiles. The reduction of the levels of starch was observed after 12h and 24h, indicating the beginning of the mobilization of the carbohydrate reserves. The morphological changes in the starch granules at the different stages of germination were observed by scanning electron microscopy, with similar results in the four varieties investigated. Significant differences were observed only in chemical compositions and in profiles the protein between the two groups of the corn seeds (C1-G1, and C3-G3), indicating normal variation between conventional varieties, without compromising the safety of the GM maize, or their employment in fermentative processes. In contrast, GM maize seeds showed faster mobilization of starch in the first two stages of germination.

Description des methodes de criblage des champignons thermophiles ayant des activites polyoside hydrolases. Caracterisation des enzymes produites par la souche selectionnee trichoderma reesei: enzymes cellulolytiques, beta -1,3-glucanase et exo-1,4-alpha -d-glucosidase thermostables. Utilisation de ces enzymes pour la saccharification a 60-70 **(o)c de substrats bruts cellulosiques ou amylaces, tels que des ordures menageres et des farines de ble. Comparaison des performances de la souche avec celles de trichoderma reesei

A mandioquinha-salsa (Arracacia xanthorrhiza Bancroft.) possui um período de conservação pós-colheita de cerca de uma semana, que contrasta com o seu ciclo de cultivo, em torno de 10 meses. Os mecanismos de deterioração das raízes ainda não são conhecidos e tampouco foram estudadas as principais vias enzimáticas de degradação do amido, sua principal reserva energética. O objetivo deste trabalho foi de identificar e caracterizar bioquimicamente o extrato enzimático dessas raízes, que possuem atividade amilolítica. Os resultados mostraram que as enzimas apresentam pH e temperatura ótimos de atividade enzimática em torno de 6,2 e 46°C, respectivamente, confirmados por Análise de Superfície de Resposta. Foram detectadas três bandas com atividade amilolítica em gel de poliacrilamida. Os valores de Ea, Km e Vmáx aparentes encontrados para o extrato enzimático foram de 7,53 kcal/mol, 0,41 mg/mol e 1,11 mg/mL/min, respectivamente. A hidrólise do amido aumenta 95% na presença de Ca+2 e a enzima mantém apenas cerca de 47% da sua atividade original na presença de EDTA. Os resultados mostraram a presença de duas possíveis isoformas de α-amilase e uma β-amilase no extrato enzimático in vitro, podendo estar ativas na raiz íntegra. Um ensaio de acompanhamento da atividade amilásica das raízes durante o armazenamento por 9 dias mostrou um aumento na atividade de água acompanhado de perda de textura e de oscilação na atividade hidrolítica. No entanto, tais alterações devem ocorrer também devido a ação de enzimas exógenas de mofos, que participam do processo de deterioração da mandioquinha-salsa.
Roots of Peruvian carrot (Arracacia xanthorrhiza Brancroft) have a post-harvest conservation time of approximately one-week, a very short period related to its long cultivation cycle, around 10-11 months. The mechanisms of post-harvest deterioration of the roots are unknown as well as the main enzymatic pathways such as the starch degradation, have not been studied. The purpose of this investigation was to identify and characterize the amylolitic activity of this root. The results showed that the optimum enzymatic conditions for the maximum activity are 46°C and pH 6.2 confirmed by Surface Ternary Plots. Three bands with amylolitic activity were detected in native PAGE. The kinetic values were Ea 7.53 kcal/mol, Km 0.41 mg/mol and Vmáx 1.11 mg/mL/min. The starch hydrolysis speed increases almost twice when Ca+2 is present and remained only 20% on the presence of EDT A. The results showed two possible isoforms of α-amylase and one β-amylase in the crude extract. An essay following the amylolitic activity during a 9-day storage period showed an increase of water activity associated to a loose of texture and a oscillating starch hydrolytic activity, although these behavior may have the contribution of exogenous enzymes from molds on the deteriorative process of the Peruvian carrots.

Este trabalho teve como principais objetivos verificar as alterações na atividade de enzimas amilolíticas, pectinolíticas e celulásicas em raízes de mandioquinha-salsa durante o período pós-colheita, sob diferentes condições de armazenamento, visando avaliar os mecanismos de deterioração das raízes, bem como identificar o microrganismo possivelmente responsável pela alta perecibilidade das raízes. Além disso, foram estudadas características físico-químicas e reológicas do amido de mandioquinha extraído em laboratório. Para a detecção de atividade pectinesterásica (PE) e poligalacturonásica (PG) nas raízes, os parâmetros de extração destas enzimas foram otimizados através de metodologia de superfície de resposta (MSR). As enzimas apresentaram pH ótimo de extração de 7,5 e 4,0 para a PE e PG, respectivamente, sendo os outros parâmetros, como tempo de extração e concentração de NaCI, considerados como não-significativos pelo modelo. As enzimas pectinolíticas parecem estar relacionadas à deterioração das raízes durante o armazenamento, associadas ao amolecimento das raízes. Devido à alta produção de gás, sob condições de temperatura e embalagem, a presença de bactérias produtoras de enzimas pécticas e causadoras de podridão-mole pode ser uma das principais causas da alta perecibilidade das raízes. A atividade amilolítica apresenta um importante papel na deterioração da mandioquinha uma vez os açúcares redutores provenientes da hidrólise do amido podem vir a ser substrato para o ataque de microrganismos oportunistas. Representa um dos principais aspectos metabólicos em um tubérculo rico em amido. A atividade celulásica foi praticamente nula durante o armazenamento. A melhor forma de conservação das raízes ocorreu à temperatura de refrigeração, sob acondicionamento a vácuo. O microrganismo isolado das raízes foi identificado através de provas bioquímicas como Erwinia carotovora subsp. odorífera. Em relação às características físico-químicas, o amido de mandioquinha apresentou um teor de amilose abaixo dos valores determinados para cereais e raízes convencionais (12,1 %) e uma boa estabilidade durante a cocção, analisada através da determinação de viscosidade. A turbidez das suspensões de amido também apresentou estabilidade durante o armazenamento, assim como a sinerese, que.em condições ótimas de preparação do gel apresentou 2,53 % de expulsão de água. Ainda sobre a relação água/gel, o pH da solução e a concentração de amido influenciam significativamente a capacidade de retenção de água, enquanto a temperatura de formação do gel não se mostrou um parâmetro significativo. A textura das preparações de amido foi influenciada significativamente pela temperatura de formação do gel e pela concentração de amido utilizada na preparação. O conhecimento de algumas características reológicas e de propriedades físico-químicas do amido de mandioquinha, visando a aplicação como ingrediente alimentício, pode ser uma alternativa a utilização das raízes in natura, principalmente no que diz respeito ao excedente de safra e ao aproveitamento de raízes com baixo valor comercial.
The aim of this work was to verify the changes in amylolytic, pectinolytic and cellulasic activity in Peruvian carrot roots after harvest, under different storage conditions, in order to evaluate the deteriorative mechanisms of the roots, as well as to identify the microorganism possible responsible to its low conservation time. In addition, physico-chemical and rheological characteristics of Peruvian carrot starch were studied. For pectinesterase (PE) and poligalacturonase (PG) detection on the roots, the extraction parameters of both enzymes were optimized by response surface methodology. The enzymes presented the optimum pH values at 7.5 and 4.0 for PE and PG, respectively. Extraction time and NaCI concentration were considered non-significant by the model. Pectic enzymes seams to be related to the deterioration process of Peruvian carrot, that is associated to the root softening. Considering the high volume of gas under specific packing and temperature, the presence of microorganisms soft rot promoters could be the main cause of the high perecibility of the roots. The amylolytic enzymes present an important role on Peruvian carrot deterioration related to the starch hydrolysis and the releasing of reducing sugars, substrate for opportunistic microorganisms. The cellulasic activity was not significant during storage time. Best conditions for roots conservation occurred at 4°C and under vacuum package. The bacteria isolated from the roots were identified by biochemical reactions as Erwinia carotovora subsp. odorifera. Peruvian carrot starch presented low values of amylose content (12.1 %) in comparison to other starch sources and a good stability during cooking, analyzed by viscoamylogram Brabender. The turbidity of starch suspensions presented good stability during storage, as well as water holding capacity, which presented 2.53% in optimal conditions of gel preparation. Syneresis is positively influenced by the pH of past solution and starch concentration whereas temperature of gel formation was not significant. Texture of starch preparations was significant influenced by temperature of gel formation as well as starch concentration. The knowledge of some rheological and physico-chemical properties of Peruvian carrot starch can be useful focusing its use as a food ingredient, which can be an alternative to the consumption of the roots in nature, especially considering crop excesses and low commercial value roots.

A maca (Lepidium meyenii Walpers) é uma planta herbácea bienal da família Brassicae, cultivada principalmente na região dos Andes da América do Sul. A parte subterrânea vem sendo consumida por muito tempo devido a seu valor nutricional e energético, mas é mais conhecida no mercado peruano e internacional por alegadas propriedades terapêuticas. Esta raiz apresenta até 76% de carboidratos, dos quais 30% é amido. Este trabalho teve como objetivos estudar: as propriedades físico-químicas e funcionais do amido isolado; os parâmetros enzimáticos durante o armazenamento e a purificação parcial de enzimas amilolíticas. Em relação às propriedades do amido, este apresentou um teor de amilose de 20% valor semelhante aos encontrados em raízes e tubérculos similares. A turbidez das suspensões de amido apresentou estabilidade durante o armazenamento. A temperatura de gelatinização e a viscosidade da pasta foram a 45,7° e 46°C, respectivamente. Com base nos dados obtidos, o amido de maca seria indicado para alimentos que requeiram temperaturas moderadas no processamento, não sendo apropriado para o emprego em alimentos congelados. Os parâmetros enzimáticos medidos tais como teor de amido total, teor de açúcares solúveis, atividade amilolítica total, atividade de α e β amilases, não mostraram diferenças significativas entre as medidas durante um período de armazenamento de 16 dias. As microscopias eletrônicas de varredura (MEV) dos grânulos de amido mostraram grãos íntegros com superfícies lisas, com algumas depressões ao redor dos grânulos os quais poderiam indicar o inicio de ataque enzimático, ou fraturas na purificação. Em relação à purificação de enzimas amilolíticas, foi possível separar uma fração ativa com a carboximetilcelulose (CMC) seguida de cromatografia liquida de alta resolução (CLAE) que permitiu a separação de duas frações protéicas, analisadas por eletroforese SDS-PAGE e eletroforese bidimensional (2D). Os polipeptídeos identificados no gel 2D apresentaram massa molecular semelhante entre 22 a 27 kDa, e os pontos isoelétricos entre 4,8 e 7,3.
Maca (Lepidium meyenii Walpers) is a biennial herbaceous plant from Brassicae family, grown mainly in the Andes of South America. The underground part has been consumed for a long time due to its nutritional value and energy, but is best known in the Peruvian and international market for alleged therapeutic properties. This root has up to 76% carbohydrates, of which 30% is starch. This work aimed to study: the physico-chemical properties of isolated starch, the enzymatic parameters during storage and partial purification of amylases. In relation to the properties of starch, the amylose content showed a 20% value similar to those found in roots and tubers alike. The turbidity of starch suspensions was stable during storage. The gelatinization temperature and viscosity of the paste were 45.7 ° and 46 ° C, respectively. Based on data obtained from the starch of litter would be given to foods that require moderate temperatures in processing and is not suitable for use in frozen foods. The enzymatic parameters measured such as total starch content, soluble sugars, total amylolytic activity, activity of α and β amylases, showed no significant differences between the measures over a storage period of 16 days. Electronic microscopy (SEM) of starch granules showed grains with smooth surfaces, with some depressions around the granules which could indicate the beginning of enzymatic attack, or fractures in the purification. Regarding the purification of amylases was possible to separate an active fraction with carboxymethylcellulose (CMC) followed by high-resolution liquid chromatography (HPLC) which allowed the separation of two protein fractions, analyzed by SDS-PAGE and two-dimensional electrophoresis (2D ). The polypeptides had a molecular mass between 22 and 27 kDa and isoelectric points ranging from 4.8 to 7.3.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo silagem não inoculada e suplementados com amilase apresentaram alta proporção de ácido propiônico e baixa de ácido acético, e consequentemente baixa relação de aceitoc:propiônico. A síntese de proteína microbiana tendeu a ser maior (P=0,097) nos carneiros alimentados com silagem não inoculada e suplementados com amilase e também nos que receberam dieta contendo silagem inoculada sem suplementação com amilase (8,01; 8,05 g/d, respectivamente). Entretanto, nenhum efeito foi verificado na eficiência de síntese de proteína microbiana. No segundo estudo, cordeiros alimentados com silagem inoculada apresentaram maior consumo de FDN (P=0,019) do que aqueles alimentados com silagem não inoculada (266,5 vs. 245,0 g/d). Cordeiros que receberam dieta contendo silagem inoculada apresentam maior ganho de peso diário (P=0,019) quando comparados àqueles alimentados com silagem de milho não inoculada (232,5 vs. 211,5). A inoculação da silagem aumentou (P<0,05) o conteúdo de ácidos graxos saturados (AGS) e diminuiu (P<0,05) o conteúdo de ácidos graxos insaturados (AGI) (47,55 vs. 46,21% e 52,44 vs. 53,79%, respectivamente), e consequentemente, diminuiu a relação UFA:SFA. A suplementação com amilase no momento da alimentação tendeu (P<0,10) a diminuir a relação AGPI:AGS (0,14 vs. 0,16). O uso de amilases em dietas contendo silagem de milho inoculada não resultou em respostas positivas na digestibilidade e síntese de proteína microbiana de carneiros, bem como não alterou as características de carcaça e qualidade de carne de cordeiros. O uso de dietas contendo silagem de milho inoculada com L. plantarum e B. subtilis e não suplementadas com amilase, aumentou o ganho de peso de cordeiros.
This trial aimed to evaluate the effects of diets containing corn silage inoculated with Lactobacillus plantarum and Bacillus subtilis and supplemented or not with amylase on the apparent digestibility, ruminal fermentation and microbial protein synthesis of wethers as well as, the growth performance and meat quality of lambs. For that, two studies were carried out and in both studies the animals received one of four treatments (diets): 1) Corn silage uninoculated and without amylase added to TMR; 2) Corn silage uninoculated and amylase added to TMR; 3) Corn silage inoculated with 1×105 CFU LP [MA 18/5U] and 1×105 CFU BS [AT553098] without amylase added to TMR; 4) Corn silage inoculated with 1×105 CFU LP [MA 18/5U] and 1×105 CFU BS [AT553098] and amylase added to TMR. The enzyme utilized was amylase at the rate of 2 g of the product / kg of dietary dry matter (DM) (602 dextrinizing unit (DU)/kg of dietary DM). Amylase supplementation on the diet containing uninoculated silage increased (P=0.045) dry matter (DM) intake of wethers compared with wethers fed uninoculated silage without amylase supplementation (1,311 vs. 1,066 g/d), but not differed from others treatments. The apparent digestibility of DM, OM, CP, NDF and GE increased (P<0.01) in wethers fed with inoculated silages or supplemented with amylase, without interaction among inoculants and amylase. Wethers fed diets containing uninoculated silage and supplemented with amylase showed higher propionic acid and lower acetic acid proportion, with low acetic:propionic acid ratio, consequently. Microbial N supply tended to be higher (P=0.097) in wethers fed uninoculated silage with amylase supplementation and inoculated silage without amylase (8.01; 8.05 g/d). However, no effect was verified on the efficiency of microbial N synthesis. In the second study, lambs fed inoculated silage had higher NDF intake (P=0.019) than lambs fed uninoculated silage (266.5 vs 245 g/d). Lambs fed inoculated silage had higher average daily gain (P=0.019) when compared with lambs fed uninoculated silages (232.5 vs. 211.5). The inoculation of silage increased (P<0.05) the content of saturated fatty acid (SFA) and decreased (P<0.05) the unsaturated fatty acid (UFA) (47.55 vs. 46.21% and 52.44 vs. 53.79%, respectively) and consequently decreased the UFA:SFA ratio. The amylase supplementation at moment of feeding trended (P<0.10) to decrease the values of PUFA:SFA ratio (0.14 vs. 0.16). The association of amylase in diets containing inoculated silage did not provided positive responses on the digestibility and microbial N supply of wethers and did not alter the carcass and meat quality of lambs. Inoculation of silage with L. plantarum and B. subtilis improved the average daily gain of lambs when was not associated with amylase supplementation.
CNPq: 141008/2014-8

Orientador: Ricardo Andrade Reis
Coorientador: Juliana Duarte Messana
Banca: Marcia Helena Machado da Rocha Fernandes
Banca: Giovani Fiorentini
Banca: Thiago Fernandes Bernardes
Banca: Odilon Gomes Pereira
Resumo: Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Objetivou-se neste trabalho avaliar os efeitos de dietas contendo silagem inoculada com Lactobacillus plantarum e Bacillus subtilis e suplementadas ou não com amilase sobre a digestibilidade aparente, fermentação ruminal e síntese de proteína microbiana em carneiros, assim como o desempenho e qualidade de carne de cordeiros. Para tanto, dois estudos foram conduzidos, no quais os animais receberam um dos quatro tratamentos (dietas): 1) silagem de milho não inoculada sem adição de amilase na mistura total da ração (MTR); 2) silagem de milho não inoculada e amilase adicionada na MRT; 3) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis, sem adição de amilase; 4) silagem de milho inoculada com 1 × 105 UFC de L. plantarum e 1 × 105 UFC de B. subtilis e amilase adicionada na MRT. A enzima utilizada foi a amilase numa taxa de aplicação de 2 g de produto / kg de matéria seca (MS) da dieta (602 unidade dextrinizante (UD) / kg de MS da dieta). A suplementação com amilase em dietas contendo silagem não inoculada aumentou (P=0,045) o consumo de matéria seca dos carneiros quando comparados com aqueles alimentados com silagem não inoculada sem suplementação com amilase (1,311 vs. 1,066 g/d), mas não diferiu dos outros tratamentos. A digestibilidade aparente da MS, MO, PB, FDN e EB aumentou (P<0,01) nos carneiros alimentos com silagem inoculada ou suplementados com amilase, sem interações entre os tratamentos. Os animais alimentados com dietas contendo silagem não inoculada e suplementados com amilase apresentaram alta proporção de ácido propiônico e baixa de ácido acético, e consequentemente baixa relação de aceitoc:propiônico. A síntese de proteína microbiana tendeu a ser maior (P=0,097) nos carneiros alimentados com silagem não inoculada e suplementados com a... (Complete abstract click electronic access below)
Doutor

The initial action of porcine pancreatic a-amylase (PPA, EC 3.2.1.1) on various native and hydrothermally treated starches was investigated, in order to understand how structural differences in the substrate influence starch digestion. Initial reaction rates at various starch concentrations were measured and fitted to the Michaelis-Menten equation to obtain enzyme kinetic parameters, KM, kcat and kcalKM. The KM is a measure of the accessibility of the enzyme towards its substrate and kcalKM is an index of catalytic efficiency by which the effectiveness towards different substrates can be compared. The kcst is the dissociation rate of the enzymesubstrate (ES) complex. Starches from common food sources were used as substrates. Physical and chemical data on the characteristics of the starches was obtained, using methods such as scanning electron microscopy and differential scanning calorimetry. The kcab kcalKM ratio increased and KM value decreased following hydrothermal treatment of all starches at 100°C, but the effect was variable, depending on the source. Potato starch was the most favoured substrate for PPA in the heat-treated state and heat-treated rice starches were the worst substrate for PPA. The kinetic data, coupled with the results of physico-chemical investigations, indicated that the polymorph type, amylose content, the amount of amorphous material and granule size are important determinants of the amylolysis rate of native starches. Heat treatment at 100°C of the starches decreased the KM for all starches, which suggested that the enzyme attack is facilitated by the induced disorder (i.e. increase in the amount of amorphous material). In addition, the increase in kcat parameter suggests that the dissociation of the ES complex occurs more readily for hydrothermally treated starches. The effects of a wider range of hydrothermal treatments have been studied using wild-type and mutant pea starches. The kClIlKM ratio increased as the pre-treatment temperature increased, but each starch showed a distinct trend in this relationship and this was reflective of their gelatinisation behaviour.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1997.
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Biofilms are characterized by surface attachment, structural heterogeneity; genetic diversity; complex community interactions and an extracellular matrix of polymeric substances (EPS). Biofilms deposit and adhere to all surfaces that are immersed in aqueous environments. EPS serves many functions including: facilitation of the initial attachment of bacterial cells to a surface; formation and maintenance of the micro colony; enables the bacteria to capture nutrients; causes biofouling; cell-cell communication and enhances bacterial resistance antimicrobial agents. EPS also function as a stabilizer of the biofilm structure and as a barrier against hostile environments. Extracelullar polymeric substances are composed of a wide variety of materials including polysaccharides, proteins, nucleic acid, uronic acid, DNA, lipid and even humid substances. EPS can be hydrophilic or hydrophobic depending on the structural components making up such EPS and the environmental conditions were the biofilms are developing. The exopolysachharides (EPS) synthesized by microbial cells vary greatly in their composition and in their chemical and physical properties within the bacterial strains. Due to variety in the structural components of the bacterial EPS, removal of biofilms by compounds that have no effects on the biofilm EPS would be difficult. Enzymes are proven to be effective in degrading biofilm EPS. The manner in which enzymes degrade the biofilm EPS is through binding and hydrolysis of the EPS components (proteins and carbohydrates) molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. The objectives of this study were to grow Pseudomonas fluorescens and mixed bacterial species biofilms in nutrient rich and nutrient limited medium conditions; to determine the EPS, protein and carbohydrate concentrations of the biofilm grown in rich and in limited nutrient conditions and to test the efficiency of protease and amylase enzymes for the degradation of the EPS and biofilm removal. In the results, there was a slight difference in the number of viable cells grown in biofilms that were fed than the cells of the unfed biofilms. As a result, the EPS, protein and carbohydrate concentrations were higher in the fed biofilms than the unfed biofilms. There are contradictory reports about the composition of EPS especially with the ratio of carbohydrate to protein. Some of these reports indicate that certain biofilms EPS have bigger proportion of proteins and some found polysaccharides to be the dominant composition of the EPS of the biofilms. Nonetheless, the quantity and the composition of the EPS produced by bacterial biofilms depend on a number of factors such as microbial species, growth phase and the type of limiting substrate. Enzymes were tested individually and in combination for the degradation of biofilm EPS. For efficient removal of biofilm, it is important that the structural components of the biofilm EPS should be known before application of the relevant enzymes. In this study, the test enzymes were effective for the degradation of the biofilm EPS except for the protease Polarzyme which had no activity. The reason for the inefficiency of Polarzyme may be due to its incompatibility with the specific protein structural components of the biofilm EPS tested in this study. The manner in which the enzymes degrade the biofilm EPS is through binding and hydrolysis of the protein and carbohydrate molecules and converting them into smaller units that can be transported through the cell membranes and then be metabolized. In addition, the mode of enzymatic action will depend on the specific EPS components and this in turn will determine its efficacy. The protease enzymes tested individually and in combination were most effective for EPS degradation. The efficiency of the proteases may be due to their broad spectrum activity in degrading a variety of proteins acting partly as the multi structural components of Pseudomonas fluorescens and mixed bacterial species biofilm EPS. On the other hand, amylase enzymes tested individually and in combination was less effective for the EPS degradation. The structures of polysaccharides synthesized by microbial cells vary. Microbial exopolysaccharides are comprised of either homopolysachharides or heteoropolysaccharides. A number of lactic acid bacteria produce heteropolysaccharides and these molecules form from repeating units of monosaccharides including D- glucose, D- galactose, L- fructose, L- rhamnose, D- glucuronic acid, L- guluronic acid and D- mannuronic acid. The type of both linkages between monosaccharides units and the branching of the chain determines the physical properties of the microbial heteropolysaccharides. Due to a wide range of linkages and the complexity of polysaccharides structures, it would therefore be difficult for the amylases to break down the bond linkages and the monomers making up polysaccharides which determine the physical and chemical structure of the EPS. It was therefore not surprising that the amylase enzymes tested for the degradation of Pseudomonas fluorescens and mixed bacterial species biofilms, were less effective than the proteases. Hence, when the amylase enzymes were tested in combination with the protease enzymes, efficiency improved. It was therefore concluded that the protease enzymes were the primary remedial compounds and the amylase enzymes were the secondary remedial compounds. Conclusion If a compound or compounds capable of destroying all the structural components of different EPS that are produced by different biofilms growing under different conditions is found then the “city of microbes” (biofilms) would be destroyed permanently. If only an enzyme or enzymatic mixture capable of shutting down or deactivating the quorum sensing systems of different biofilm EPS could be found, then there would not be any formation of biofilms. In this study, protease enzymes tested individually and in combination were the most effective in the degradation of biofilm EPS than the amylase enzymes resulting in the reduction of large population of the biofilm cells attached on the substratum. Recommendation Amylase enzymes tested individually and in combination were less efficient for the degradation of the biofilm EPS and biofilm removal. This may be due to the complex structure of the exopolysaccharides synthesized by different biofilms. Also, the bond linkages between monosaccharides units and the branching of the chain complex the structures and as a result confer in the physical properties of the microbial biofilms. Hence, when the amylase enzymes were tested in combination with the protease enzymes, activity improved. For efficient degradation of biofilm EPS, it is therefore recommended that, protease and amylase enzymes should be tested in combination. In addition, the structure of the biofilm EPS should be investigated so that relevant enzymatic mixtures are tested for biofilm removal.
Thesis (PhD)--University of Pretoria, 2010.
Microbiology and Plant Pathology
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Thesis (Ph. D.)--University of Wisconsin--Madison, 1990.
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碩士
國立師範大學
生物學研究所
82
A.espejiana Bal 31 野生型菌株及其突變株之澱粉水解酵素純化及物理， 化學性質探討為本研究之主題。利用硫酸銨沉澱，膠體過濾層析和親和性 管柱層析等方法將釋出於培養基的澱粉水解酵素加以純化分離。得到野生 型菌株澱粉水解酵素的純化倍率為 18 倍，突變株澱粉水解酵素則為 7 倍。兩者之分子量差異不大約為 80 KD。依酵素對可溶性澱粉之水解產物 分析結果推論，兩者皆為"liquefying".alpha. 澱粉水解酵素。 且其作 用最適溫度與最適 PH 值分別為攝氏 40 度及 7.0。所純化酵素對熱十分 敏感，突變株之澱粉水解酵素於攝氏 45 度作用 10 分鐘幾乎完全喪失活 性，野生型菌株澱粉水解酵素於攝氏45 度反應 10 分鐘，活性僅剩下約百 分之四十；若於 55 度作用 15 分鐘後即測不到活性。兩者作用的活化能 都有偏低的趨勢，二者分別為每莫耳 3.52 仟卡（ 野生型 ）及 4.01 仟 卡（ 突變株 ）。鈣離子可增加兩者對熱的耐受性，而鈉離子和氯離子對 突變株澱粉水解酵素之 耐熱性有提高的作用；若兩種離子同時添加則效果 更好，但野生型菌株澱粉水解酵素則否。然而兩者的活性均會受到 SDS、 尿素、papain 及 EDTA 的抑制，唯突變株澱粉水解酵素之耐受性均比野生 型菌株澱粉水解酵素高。Triton X 100及一般常見之金屬離子對酵素活性 並不具有明顯的抑制作用。兩者胺基酸的組成中，小分子厭水性胺基酸所 佔比例很高且均缺少 cysteine 和 arginine；這些特殊的胺基酸組成可能 可以增加蛋白質分子結構之彈性，使其得以適應溫度較低的海洋環境。 The properties of secreted amylolytic enzyme from A. espejiana Bal 31 and its mutant were investigated。The enzymes in caltured medium were purified by ammonium sulfate precipitation, gel filtration chromatography and affinity chromatography.The molecular weight of both enzymes were close and estimated to be about 80KD by SDS-PAGE. According to the substrate specificity and the hydrolytic productsanalysis, we interfer both enzymes are deduced to be the liquefyingtype of α-amylase. Both anzymes exhibit the same optimum pH andtemperature of 7.0 and 40℃ respectively. Relatived lower activationenergy and heatlabile of the enzymes were observed. Addition of calciumcould increase the thermostability of enzymes, but sodium and cholride ions showed more effective to the mutant's enzyme on the protection against heat. The tolerances of enzymes to the inactivators, such as urea,SDS,papain and EDTA were also different. However,triton X 100 and metal ions in our testing concentrations showed no obvious effects to enzyme activity. The amino acid composition of the enzymes displayed the unusual fraction of hydrophobic amino acids and the lacking of cysteine and arginine residues. This characteristic constitution might enhance the structural flexibility of enzyme protein to adapt to the marine enviroment.