Dissertations / Theses on the topic 'Fungal amylase'

The food industry has responded to the American Heart Association's overwhelming concerns about the complications of obesity with an array of fat reduced products that maintain the functionality of fat in given systems. In baked products, it appears that no one single ingredient effectively mimics these functions. The present study investigated the effect of liquid or dry honey as a partial replacement for sugar on the baking and keeping qualities of fat reduced muffins. The fat reduced muffins also utilized a hydrocolloid fat replacer, bacterial and fungal amylases, and an emulsifier (DATEM). Results showed that both liquid and dry honey significantly (p<0.05) increased crust and crumb color at all replacement levels, however the use of 25% liquid honey was shown to favorably increase the crust color of fat reduced muffins. Volume was not significantly (p>0.05) affected but appeared to decrease with the addition of honey due to either premature starch gelatinization or a decrease in batter pH. The addition of honey increased moisture content, and decreased water activity, but did not decrease firmness or staling rates especially after prolonged frozen storage. Sensory panelists noted that the addition of liquid or dry honey increased the cohesive forces and decreased tenderness. The addition of moisture to the fat reduced system did not appear to improve the perceived moistness of the product.
Master of Science

Orientador: Márcia Rossini Justino Mutton
Banca: Eduardo da Silva Martins
Banca: João Martins Pizauro Júnior
Resumo: A utilização de fermentação em estado sólido a partir de subprodutos agroindustriais possibilita a obtenção de várias biomoléculas de interesse industrial. As amilases são enzimas com grande aplicação nas indústrias têxtil, farmacêuticas, alimentícias e sucroalcooleira, representando aproximadamente 25% do mercado mundial. Neste trabalho foram isoladas três linhagens fúngicas (Rhizopus oryzae, Malbranchea pulchella e Chrysosporium zonatum), que apresentaram potencial amilolítico, quando cultivadas em três resíduos agroindustriais (quirera de arroz e de milho e farelo de trigo). Para a cepa que apresentou maior atividade enzimática, avaliou-se melhor substrato, tempo de cultivo, teores de umidade, fontes suplementares de nitrogênio, pH e temperatura de incubação, objetivando otimizar as condições de cultivo. Observou-se que a maior atividade enzimática foi obtida com a cepa de R. oryzae em 24 horas de fermentação, em meio de cultura contendo farelo de trigo como substrato, em temperatura de 35°C, utilizando solução de sais composta de NH4NO3 a 0,1% e MgSO4.7H2O a 0,1% como fonte de nitrogênio. Alterações de pH entre 4,0 e 6,0 não afetaram significativamente a síntese da enzima. A condições ótimas de atuação foram temperatura de 75°C e pH de 4,5. As enzima manteve-se estável a 75°C na ausência de substrato por 25 minutos
Abstract: The use of fermentation in a solid state from agroindustrial byproducts permits the obtaining of several biomolecules of interest the industry, like the enzymes. The amylases are enzymes with great application on the textile, pharmaceutical and food industries, representing around 25% of the worldwide market. In this work three ungal strains (Rhizopus oryzae, Malbranchea pulchella and Chrysosporium zonatum) were isolated. They presented amylolytic potential when cultivated in three agroindustrial by products (brewers rice, corn grits and wheat bran). To the strain that presented the greatest enzyme activity, it was evaluated the best substrate, cultivation time, moisture content, additional sources of nitrogen, pH and incubation temperature, aiming the optimization of cultivation conditions. I was observed that the greatest enzyme's activity was obtained with 24 hours of fermentation, in R. orizae in a culture medium that contained wheat bran as the substrate, at 35°C, using salt solution made with NH4NO3 0.1%, MgSO4.7h2O 0.1% and (NH4)2SO4 0.1% as nitrogen source. pH alterations between 4.0 and 6.0 didn't change significantly the enzyme's activity. The optimum conditions of performance of the enzyme were temperature of 75°C and pH 4.5. The enzyme kept stable in the lack of substrate for 25 minutes in 75°C
Mestre

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A utilização de fermentação em estado sólido a partir de subprodutos agroindustriais possibilita a obtenção de várias biomoléculas de interesse industrial. As amilases são enzimas com grande aplicação nas indústrias têxtil, farmacêuticas, alimentícias e sucroalcooleira, representando aproximadamente 25% do mercado mundial. Neste trabalho foram isoladas três linhagens fúngicas (Rhizopus oryzae, Malbranchea pulchella e Chrysosporium zonatum), que apresentaram potencial amilolítico, quando cultivadas em três resíduos agroindustriais (quirera de arroz e de milho e farelo de trigo). Para a cepa que apresentou maior atividade enzimática, avaliou-se melhor substrato, tempo de cultivo, teores de umidade, fontes suplementares de nitrogênio, pH e temperatura de incubação, objetivando otimizar as condições de cultivo. Observou-se que a maior atividade enzimática foi obtida com a cepa de R. oryzae em 24 horas de fermentação, em meio de cultura contendo farelo de trigo como substrato, em temperatura de 35°C, utilizando solução de sais composta de NH4NO3 a 0,1% e MgSO4.7H2O a 0,1% como fonte de nitrogênio. Alterações de pH entre 4,0 e 6,0 não afetaram significativamente a síntese da enzima. A condições ótimas de atuação foram temperatura de 75°C e pH de 4,5. As enzima manteve-se estável a 75°C na ausência de substrato por 25 minutos
The use of fermentation in a solid state from agroindustrial byproducts permits the obtaining of several biomolecules of interest the industry, like the enzymes. The amylases are enzymes with great application on the textile, pharmaceutical and food industries, representing around 25% of the worldwide market. In this work three ungal strains (Rhizopus oryzae, Malbranchea pulchella and Chrysosporium zonatum) were isolated. They presented amylolytic potential when cultivated in three agroindustrial by products (brewers rice, corn grits and wheat bran). To the strain that presented the greatest enzyme activity, it was evaluated the best substrate, cultivation time, moisture content, additional sources of nitrogen, pH and incubation temperature, aiming the optimization of cultivation conditions. I was observed that the greatest enzyme’s activity was obtained with 24 hours of fermentation, in R. orizae in a culture medium that contained wheat bran as the substrate, at 35°C, using salt solution made with NH4NO3 0.1%, MgSO4.7h2O 0.1% and (NH4)2SO4 0.1% as nitrogen source. pH alterations between 4.0 and 6.0 didn’t change significantly the enzyme’s activity. The optimum conditions of performance of the enzyme were temperature of 75°C and pH 4.5. The enzyme kept stable in the lack of substrate for 25 minutes in 75°C

"This thesis is based on the following articles, referred to in the text by the Roman numerals given below. In addition some unpublished results are presented. I. Caiyan Wu ... [et al] Improvement of the secretion of extracellular proteins and isolation and characterization of the amylase I (amyI) gene from Ophiostoma floccosum [pub. in ] Gene 384: 96-103 -- II. Caiyan Wu ... [et al.] Activity-based identification of secreted serine proteases of the filamentous fungus Ophiostoma. Accepted by Biotechnology letters DOI 10.1007/s10529-007-9333-6 -- III. Caiyan Wu ...[et al.] Expression of a thermostable bacterial xylanase in the filamentous fungus Ophiostoma floccosum. Submitted to Letters in applied microbiology in July 2007." - leaf 9.
Thesis (PhD)--Macquarie University, Division of Environmental and Life Sciences, Dept. of Chemistry & Biomolecular Sciences, 2007.
Bibliography: leaves 100-123.
Introduction -- Materials and methods -- Results and discussion -- Conclusion and future aspects -- References -- Publications I, II and III.
Ophiostoma spp. belong to the Ophiostomataceae family, a large group of ascomycetes, which are the most frequent blue stain fungi isolated from stained wood. Most Ophiostoma species do not compromise the strength properties of wood, but do reduce the aesthetic quality of timber and therefore decrease the economic value of lumber. Some albino variants of O. floccosum and O. piliferum have been used as biological control agents to prevent blue staining. This successful whole organism approach plus the added capability of extracellular protein secretion makes Ophiostoma spp. attractive for industrial application. In addition, Ophiostoma produces only a small range of abundantly secreted proteins in liquid culture, which can facilitate downstream purification of any recombinant gene product introduced into the system. Genes encoding efficiently secreted proteins provide a potential souce for strong promoters for high-level gene expression. These characteristics provide an excellent starting point for the development of a novel expression system.
In this study, UV-mutagenesis was applied to improve protein secretion in Ophiostoma floccosum. Amylase activity was used as an indicator for enhanced protein secretion after repeated rounds of mutagenic treatment. Several mutants of O. floccosum derived by UV mutagenesis were isolated and the total amount of secreted protein was increased by 4 to 6 times. The amylase activity in the culture supernatant of the best mutant (MQ.5.1) was increased by more than 240-fold compared to the initial parental strain. At the same time, the amount of total secreted protein was about six times greater to that of the parental strain. Proteinase profiles in the culture supernatants of several key mutants were characterized for the future matching of an expression host with a particular gene product. N-terminal sequencing of the five dominant proteins separated by SDS-PAGE from the culture supernatant was conducted. Two of the proteins identified were subtilisin-like proteinases and one was a pepsin-like proteinase. In addition, one protein was identified as an_-amylase and one remained unidentified. A 6.5 kb DNA fragment was isolated by Genomic Walking PCR using primers based on the _-amylase amino acid sequence. The amplified fragment contained the entire gene encoding_-amylase (amyl) and its regulatory sequences. Analysis showed that multiple transcripts were generated from the single _-amylase gene locus.
A series of expression vectors containg the _-amylase regulatory sequences and partial amyl gene were constructed. Several selection markers were screened and the hph gene conferring hygromycin resistance under the regulation of the Aspergillus nidulans gpd promoter was chosen and inserted into the amyl expression vectors. The gene encoding a red fluorescent protein DsRed-E5 was used as a reporter gene to test the expression system using mutant MQ.5.1 as host. However, no transformants were obtained by either biolistic transformation or protoplast transformation. Subsequently, an alternative strategy was developed using a thermostable xylanase B as a reporter. Thermostable xylanase activity was detected in the culture supernatants of several transformants. Production of xylanase by transformant SS41 which exhibited high secreted xylanase activity was investigated. Xylanase activity in the culture supernatant of SS41 was visualized by a zymogram gel assay. Two active proteins with molecular masses of around 27 and 30 kDA, which were larger than the predicted Mr of 25 kDA were detected. This is the first report describing successful expression of a recombinant thermostable bacterial enzyme in Ophiostoma.
Mode of access: World Wide Web.
158 leaves col. ill

O Brasil apresenta cerca de 10 a 17,6% da biodiversidade mundial e apenas uma fração dela é conhecida. Os fungos filamentosos são bons produtores de enzimas e despertam um grande interesse biotecnológico. O amido é o principal carboidrato de reserva das plantas. Dentre as enzimas amilolíticas estão as glucoamilases, que catalisam a hidrólise das ligações -1,4 e -1,6 das extremidades da cadeia do amido liberando glucose. Neste trabalho foram isolados 25 fungos filamentosos de amostras de materiais em decomposição da Mata Atlântica. Dos micro-organismos com alta atividade amilolítica foram selecionados e identificados Aspergillus brasiliensis e Rhizopus oryzae. Foi realizada a otimização do cultivo e caracterização das amilases do extrato bruto de ambos os fungos. Após a obtenção destes dados foi selecionado A. brasiliensis, pois, sua amilase é mais termoestável e ainda não reportada na literatura. Após purificação a enzima foi identificada como glucoamilase, a qual é monomérica com 69 kDa e contém aproximadamente 21% de carboidratos. Apresenta um domínio de ligação ao amido na porção terminal e estrutura secundária rica em -hélice. Sua atividade ótima ocorre em pH 4,5 a 60°C, seu pI é de 3,21, pode ser ativada com a adição de Mn2+, e é inibida por glucose em concentrações maiores que 0,1 M. A glucoamilase apresenta excelente estabilidade ao pH e boa estabilidade a temperatura (a 50°C mantém 67% de atividade após 7 horas; a 55°C a meia vida é de 147 minutos). Com amido de batata a enzima apresentou as seguintes constantes cinéticas (km 2,21 mg/mL; Vmáx 155 U/mg; kcat 179 s-1; kcat/km 81,06). A glucoamilase foi imobilizada em DEAE-PEG com ativação de 12 vezes e possibilidade de reuso de 10 vezes com perda de apenas 31% de atividade. O derivado demostrou maior facilidade para hidrolisar a amilopectina do que à amilose. Também foi realizada uma análise de neighbor joining, que agrupou a glucoamilase de A. brasiliensis próxima às glucoamilases de espécies de Aspergillus, que são consideradas as mais derivadas.
Brazil holds about 10-17.6% of the world\'s biodiversity and just a percentage of it is known. Filamentous fungi are enzyme producers that have great biotechnological application. Starch is the main reserve carbohydrate in plants. Among the amylolytic enzymes there are the glucoamylases, that catalyze the hydrolysis of -1,4 and -1,6 linkages of the end of starch chains, and releases glucose. In this research 25 filamentous fungi from Atlantic forest decaying material samples were isolated. Among microorganisms with high amylolytic activity Aspergillus brasiliensis and Rhizoupus oryzae were selected and identified. The cultivation parameters were optimized and the enzymes of crude extract were characterized. Considering the previous data Aspergillus brasiliensis was selected because its amylases are more thermostable and it has not been described in the literature yet. After purification the enzyme was identified as a glucoamylase, which is monomeric with 69 kDa and about 21% of carbohydrates in its composition. The enzyme has a starch binding domain in the terminal position and its secondary structure is rich in -helix. The optimum pH for glucoamylase activity is 4.5, the temperature is 60ºC and its pI is 3.21. The enzyme can be activated by the addition of Mn+2, and inhibited in concentrations above 0,1M glucose. The glucoamylase has an excellent pH stability and a good temperature stability (at 50ºC 67% of the activity was retained after 7 hours; at 55°C its half-life was 147 minutes). The best kinetic values were obtained with potato starch (km 2.21 mg/mL; Vmax 155 U/mg; kcat 179 s-1; kcat/km 81,06). The glucoamylase was immobilized on DEAE-PEG, with an activation of 12 times and enzyme reuse 10 times with just 31% loss of its activity. The immobilized enzyme has a greater activity on amylopectin than amylose. A neighbor joining analysis with glucoamylases from filamentous fungi species was made and Aspergillus brasiliensis glucoamylase was grouped close to the glucoamylases of Aspergillus species, which are considered the most derivative.

A knowledge of molecular properties and structure of heat-stable enzymes is important for the understanding of basic principles governing thermo stability of proteins and evolution of life at high temperatures. Information on functional characteristics of thermo stable enzymes is necessary also for improving existing biotechnologies and developing new ones. Because of these reasons enzymes from thermophilic organisms are being exploited. In this context, amylolytic enzymes represent a useful choice for investigation from both basic and applied points of view. a-Amylase and glucoamylase hydrolyse starch into oligosaccharides and glucose, respectively. In the present study a thermophilic fungus, Thermomyces lanuginosus, was selected as a source of thermostable starch-degrading enzymes. The main objectives of this research were to understand the physicochemical properties, mechanism of starch utilization by T. lanuginosus and effect of heat, on amylo1ytic enzymes. Purification of amylolytic enzymes A strain of T, lanuginosus, IlSc 91, isolated from a manure heap in our laboratory was found to produce higher levels of extra cellular amylolytic enzymes than strains obtained from culture collection in U.S.A, and Europe. This strain produced 4 units of glucoamylase and 40 units of a-amylase per ml of culture filtrate when grown on 2% starch at 50 C. Culture filtrate was used as the starting material for purification of these enzymes. Glucoamylase and a-amylase were purified by ultrafiltration and a combination of ion exchange and gel-filtration chromatography 93- and 112-fold with 30 and 41% recovery, respectively; Homogeneity of purified enzymes was established by the criteria of native- PAGE, SDS-PAGE, gel-filtration on HPLC and N-terminal amino acid analysis. Some of the physicochernical properties of these enzymes were studied. Physicochemical characteristics Glucoernylase is a monomeric glycoprotien (carbohydrate content 11 %, w/w) and has a molecular weight, of 45 kDa. It produces only glucose from starch. Km and Vmax, for soluble potato starch are 0.04 mg ml-1 and 666 p mole glucose min-1 mg protein-1, respectively. The enzyme is optimally active at 70 C at pH 6.0. Its activation energy is 14.0 kCal mole-1. It has melting temperature of 73 C. Molar extinction coefficient of glucoamylase is 5.5 x 104 mole-1 cm-l. It is stable at 60°C for > 7h. The enzyme is rich in alanine, serine and aspartate/ asparagine. Glucoamylase contains alanine as the N-terminal amino acid. It does not contain cysteine. Purified a-amylase is a homodimeric protein of 40 kDa and contains 5% (w/w) carbohydrate. It liberates oligosaccharides from starch with maltose being the principle product of hydrolysis. The Km for soluble starch is 2.5 mg ml-1. A high Vmax, of 8000 mg starch min-1 mg protein-1 was found. The enzyme is optimally active at 65°C at pH 5.6. The activation energy is 10.9 kCal mole-1. At 50DC, which is the optimal temperature of growth of T. lanuginosus, purified a-amylase is completely stable for over 6 h. Ca2+ increases the melting temperature of a-amylase from 66°C to 73°C. a-Amylase requires Ca2+ for its activity and structural stabi1it.y The molar extinction coefficient of the enzyme is 4.7 x 10' mole-1 cm-1 a- Amylase is rich in aspartate / asparagine, glutamatme /glutamine, alanine, glycine and leucine. It does not contain cysteine. a- Amylase contains alanine as the N-t.ermina1 amino acid. Hydrolysis of starch by a-amylase and glucoamylase Experiments were done to understand the role of a-amylase and glucoamylase in the utilization of starch by T. lanuginosus. Crude and purified amylase preparations hydrolyse raw potato starch slightly more efficiently than soluble potato starch. The extent of starch hydrolysis by a mixture of a-amylase and glucoamylase is equal to that by culture filtrate containing the same amount of enzyme activities, Electrophoresis of crude culture filtrate proteins on native-PAGE and activity staining on gel showed the presence of one species each of a-amylase and glucoamylase. This suggests that in T. lanuginosus hydrolysis of starch is mediated by one species each of extracellular a-amylase and glucoamylase. The hydrolysis of starch by a mixture of a-amylase and glucoamylase is equal to the arithmetic sum of hydrolysis by individual enzyme showing that the enzymes do not act synergistically. a-Amylase is the main starch depolymerizing enzyme. Conversion of starch into glucose by glucoamylase does not require the presence of a-amylase. Starch is hydralysed to a maximum of 72 and 97% by glucoamylase and a-amylase, respectively. Effect of heat on a-amylase The effect, of heat, on a-amylase and glucoamylase was studied with the view to obtain information on the thermal inactivation of these proteins. Five-min heat treatment of the native a-amylase (40 kDa) results in the specific conversion of all protein molecules into partially active (approximately 50% residual activity) and SDS-undissociable dimer of 45 kDa. a-Amylase (45 kDa) after 5-min heat treatment. is partially active and can he rendered completely active by incubation at 37°C for 3 h. This altered form of a-amylase is not due to the formation of disulfide linkage in protein because the enzyme does not contain cysteine and b mercaptoethanol does not prevent heat-induced structural change. Heat, treatment, for 20 min or more results in further structural changes which result in the irreversible inactivation of the enzyme. Prolonged heating (>40 min) probably causes the degradation of protein. Reactivation of 20-min heat-inactivated a-amylase occurs specifically at 37°C or 50°C within 3 h but not at lower temperatures (0°C or 4°C). Native-PAGE analysis of the native and 20-min heated-reactivated a-amylase shows that the reactivated sample is comprised of two protein species of different charge and/or mass. Activity staining shows that only one of these protein band is active and it has electrophoretic mobility identical to that of the native enzyme. Native and the active fraction of 20-min heated-reactivated a-amylase possess similar specific activity. This suggests that it is cat8alytmically and perhaps structurally similar to the native enzyme. The native and the reactivated a-amylase are resist ant to trypsin digestion. However, heat- inactivated a-amylase is degraded into low molecular weight, peptides. These observation suggest that heat-inactivated a-amylase is partially unfolded, Unlike the native, the heat-treated (94"C, 5 min) a-amylase can not be stained with AgN03 while both forms can be stained with Coomassie brilliant blue R and by Schiff's base. On the basis of these observations a tentative model was proposed for the effect of heat on a-amylase (Fig.) Staining by + + Staining by AgNO3, + + Staining by Schiff 's base + + Sensitivity to trypsin + + Figure : Schematic represent ation of heat-induced changes in a-amylase

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Avaliou-se o efeito de uma solução de amilase produzida por Aspergillus awamori sobre a digestibilidade in vitro da matéria seca (DIVMS) de milho. Foram realizados dois experimentos, onde o primeiro a solução de enzima amilase foi aplicada por pulverização em 24g de milho moído (2 mm) e o segundo a solução de enzima amilase foi aplicado no fluido ruminal. Os tratamentos foram: controle (0 enzima), T1 (5Ml de enzima) e T2 (10Ml de enzima) para cada experimento. O ensaio da DIVMS foi obtido usando a técnica de rúmens artificiais adapatada durante os períodos de 15 ; 1,30 , 3, 6, 12 e 24 horas. Para a coleta de líquido ruminal foi utilizado um bovino de peso aproximado de 380 kg. O animal foi mantido em baia e adaptado a dieta durante um período de 10 dias antes do recolhimento do líquido ruminal com acesso livre à água e sal mineral. Para os dois experimentos foi adotado o delineamento inteiramente casualizado, em esquema de parcelas subdivididas 3 x 6, com quatro repetições (jarros). As parcelas foram constituídas por milho tratado com três diferentes níveis de enzima e as subparcelas por seis momentos de digestão. Para enzima amilase aplicada no líquido ruminal o resultado de DIVMS para os três tratamentos nos períodos de 3, 6 e 12 horas não diferiram estatisticamente entre si. Entre o tratamento controle e T1 houve diferença significativa nos tempos 15 e 1,30 horas. Foi observado maior DIVMS para o tratamento controle, em relação ao T1, com valores de 54,54% e 49,05 , e não houve diferença nos tempos 3, 6, 12 e 24 horas. Entre o tratamento controle e T2 não houve diferença no tempo 15 e 24 horas. O controle foi superior a T2 28,74% e 10,53%, respectivamente. A DIVMS foi superior para o tratamento controle, indicando que os níveis de 5 e 10 ml de enzimas injectados no fluido ruminal não aumentaram a DIVMS. Para amilase aplicada por pulverização em 24g de milho moído, no tempo de 15 , observou-se que o tratamento controle e T1 não diferiram. No entanto, o T2 melhorou a DIVMS em 55,54%, comparado ao grupo controle. O tratamento T1 aumentou a DIVMS apenas em tempos de 3 e 24 horas de incubação, em relação ao controle. Com a aplicação de 10 ml de enzima, a DIVMS aumentou em todos os tempos de incubação, em comparação com o controle.

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O conhecimento da atividade das enzimas hidrolíticas é essencial para compreender as transformações dos nutrientes no solo. O milho é uma das principais culturas sob o ponto de vista econômico. O objetivo deste estudo foi avaliar a influência do crescimento dos fungos em dois tipos de solos, na presença e ausência de glicose, no desenvolvimento das plântulas de milho, no padrão isoenzimático dos coleóptilos, nos teores de carboidratos totais residuais, nas atividades da desidrogenase e amilase. Utilizou-se delineamento inteiramente casualizado com esquema fatorial. A adição de glicose no meio de cultura aumentou a velocidade de crescimento de A. flavus e Penicillium sp., mas não de F. verticillioides. Na presença de glicose, a produção de esporos aumentou de 1,2 (F. verticillioides) e 8,2 vezes (A. flavus), e reduzido de 3,5 vezes com Penicillium sp. A redução dos teores de carboidratos totais ajustou-se significativamente às equações de 1º e 2º grau. A.flavus e Penicillium sp., apresentaram maior consumo de carboidratos totais que o solo sem inoculação ou inoculado com F. verticillioides. A adição de glicose no solo favoreceu o consumo de carboidratos residuais, provavelmente devido ao estímulo do crescimento dos fungos. Exceto com F. verticillioides, a atividade da desidrogenase aumentou em média de 1,5 a 1,8 vez (p<0,05) nos solos com glicose em relação aos solos sem glicose. À atividade da amilase, aumentou 1,3 a 1,5 vez por efeito da adição de glicose no solo. Maior atividade da amilase foi observada no solo Latossolo Vermelho distrófico (LVd) com glicose e inoculado com A.flavus e Penicillium sp. em relação ao controle. As dimensões das plântulas como altura dos coleóptilos, comprimento das raízes e massa seca das plântulas de milho dos solos adicionados de glicose e/ou inoculados com os fungos foram diminuídas. A expressão genética...
The knowledge about hydrolytic enzymes activity is essential to understand the nutrients transformations in soil. Corn is one of the main crops from the economic view in the whole world. The objective of this study was evaluate the fungi growth in two soil types, in the presence and absence of glucose, coleoptiles development, roots and weight of corn seedlings, coleoptiles isoenzyme pattern, content of total residual carbohydrates, enzymes dehydrogenase and amylase activity. Was used a completely randomized design with factorial arrangement. The glucose addition in culture medium increased the growth rate of A. flavus and Penicillium sp., but not F. verticillioides. In the presence of glucose, the spores number was increased from 1.2 (F. verticillioides) and 8.2 times (A. flavus), but was reduced 3.5 times with Penicillium sp. Reducing the levels of total carbohydrates, significantly adjusted to the equations of 1st and 2nd grade. A.flavus and Penicillium sp., showed higher carbohydrates consumption that the soil control or inoculated with F. verticillioides. The glucose addition in soil, favored the use of residual carbohydrates, probably due fungi growth stimulation. Except F. verticillioides, dehydrogenase activity increased in the range 1.5 to 1.8 times (p<0.05) in soils with glucose compared to glucose free soil. Amylase activity increased 1.3 to 1.5 times by glucose addition effect in soil. Increased amylase activity was observed in the Distrophic Red Latosol (DRL) with glucose and inoculated with A.flavus and Penicillium sp., compared to control. The seedlings dimensions as coleoptiles height, root length and dry weight of maize seedlings of the soils added with glucose and inoculated with fungi were decreased. Gene expression of maize seed was changed due probable by fungi inoculated infection in soil. The coleoptiles isoenzymes pattern was amended as early as 5 incubation... (Complete abstract click electronic access below)

Orientador: Ranulfo Monte Alegre, Vanildo Luiz Del Bianchi
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O fungo Metharhizium anisopliae tem sido empregado no biocontrole de insetos em diferentes regiões do Brasil, com aplicação principal em lavouras de cana-de-açúcar. O processo de biocontrole envolve a adesão dos esporos do fungo à superfície do inseto, a destruição do mesmo através da pressão mecânica das hifas, além de processos de digestão enzimática de proteínas, quitinas e lipídeos. Por esse motivo, estudos indicam a viabilidade da produção de amilases, proteases, lipases e quitinases pelo fungo. Além disso, este fungo tem sido objeto de estudos como produtor de swainsonine, um alcalóide com ação potencial na inibição da metástase celular e do crescimento primário de tumores, sendo promissora a sua utilização em tratamentos contra o câncer e AIDS. Sendo assim, este trabalho teve como objetivo estudar o potencial amilolítico de linhagens de Metarhizium anisopliae. Para a produção das enzimas utilizou-se fermentação em meio sólido. Para tanto, o microrganismo mantido em meio BDA foi inoculado através da suspensão de células na concentração de 107 esporos/g em 10 g de meio farelo e casca de arroz, em proporções de 7/3 (p/p), 6/4 (p/p) e 8/2 (p/p). A umidade inicial dos meios de cultivo variou em 47%, 65% e 74%. A fermentação ocorreu em sacos de polipropileno, os quais foram mantidos a 28ºC durante 480 horas, sendo retiradas amostras a cada 24 horas. No extrato enzimático obtido foram determinadas as atividades de a-amilase e de amiloglicosidase. Para o estudo da influência da interação de fatores como meio de cultivo, umidade, pH e concentração de esporos na produção das enzimas em estudo, foi utilizado um planejamento experimental 24-1, onde se observou que o pH apresenta influência na produção de a-amilase. Para a produção de a-amilase e amiloglicosidase, observou-se que o melhor meio de cultivo foi o constituído por farelo e casca de arroz na proporção 6/4 (p/p), com umidade de 47%. O pH ótimo para a atividade de a¿amilase e de amiloglicosidase foi de 5 - 5,5, apresentando estabilidade numa faixa de 5 ¿ 6,5 para a a¿amilase e de 5,5 ¿ 6 para a amiloglicosidase. A temperatura ótima para a atividade enzimática de a¿amilase foi de 70ºC e de 60ºC para a amiloglicosidase, apresentando estabilidade numa faixa de 10 a 50ºC. A atividade enzimática de a¿amilase foi influenciada positivamente pelos íons Mg+2, Mn+2, K+, Ca+2 e EDTA, enquanto o íon Zn+2 influenciou negativamente a atividade da enzima. Para a amiloglicosidase, todos os íons testados apresentaram efeito negativo, com destaque para o Zn+2 que apresentou a maior redução na atividade enzimática
Abstract: The fungus Metharhizium anisopliae has been used as biocontrol agent, actuating on insects in different areas of Brazil, with main application in sugarcane farmings. Such a biocontrol process involves the adhesion of the fungus spores to the surface of the insect, destruction of the same through the mechanical pressure of the hyphal, as well the processes of enzymatic digestion of proteins, chitins and lipids. For that reason, studies indicate the viability of the production of amylases, proteases, lipases and chitinases by this fungus. Besides, it is object of studies as producer of swainsonine, an alkaloid with potential action in the metastasis cellular and in the primary growth of tumors, being promising its use in treatments against the cancer and AIDS. The objective of this work was to evaluate the amylolytic potential of the strains of Metharizium anisopliae. For the enzymes production, it was utilized solid state fermentation. For that, the microorganism maintained in PDA media was inoculated through the suspension of cells in the concentration of 107 spores/g in 10 g of media constituted by bran and rice chaff, in proportions of 7/3 (p/p), 6/4 (p/p) and 8/2 (p/p). The initial moisture content of fermentation was varied on 47%, 65% and 74%. The experiments were carried in polypropylene bags, which were incubated at 28ºC for 480 hours of fermentation, being removed from it, samples every 24 hours. From the crude enzymatic solution, there were determined the activities of a-amylase and the amiloglucosidase. For the study of the interaction of factors such as cultivation medium, moisture, pH and inoculum size in the enzymes production, a statistical design 24-1 was used, where it was observed that the pH presents influence in the production of a-amylase. For the production of a-amylase and amiloglicosidase, it was concluded that the best cultivation medium is constituted by bran and rice chaff in the proportion 6/4 (p/p), with a moisture content of 47%. The optimum pH for a-amylase and amiloglicosidase was of 5 - 5,5, presenting stability in a pH range from 5 to 6,5 for the a- amylase and 5,5 to 6 for the amiloglicosidase activity. The best temperature for a-amylase activity was 70ºC and 60ºC for the amiloglicosidase, presenting stability in a range from 10 to 50ºC. The a-amylase activity was influenced positively by the ions Mg+2, Mn+2, K+, Ca+2 and EDTA, while the ion Zn+2 influenced negatively the enzyme activity. For the amiloglicosidase, all of the tested ions presented negative effect, with prominence for Zn+2 that presented the largest reduction in the enzymatic activity
Mestrado
Mestre em Engenharia de Alimentos

Orientador: Ely Nahas
Banca: José da Cruz Machado
Banca: Antônio Carlos Monteiro
Resumo: O conhecimento da atividade das enzimas hidrolíticas é essencial para compreender as transformações dos nutrientes no solo. O milho é uma das principais culturas sob o ponto de vista econômico. O objetivo deste estudo foi avaliar a influência do crescimento dos fungos em dois tipos de solos, na presença e ausência de glicose, no desenvolvimento das plântulas de milho, no padrão isoenzimático dos coleóptilos, nos teores de carboidratos totais residuais, nas atividades da desidrogenase e amilase. Utilizou-se delineamento inteiramente casualizado com esquema fatorial. A adição de glicose no meio de cultura aumentou a velocidade de crescimento de A. flavus e Penicillium sp., mas não de F. verticillioides. Na presença de glicose, a produção de esporos aumentou de 1,2 (F. verticillioides) e 8,2 vezes (A. flavus), e reduzido de 3,5 vezes com Penicillium sp. A redução dos teores de carboidratos totais ajustou-se significativamente às equações de 1º e 2º grau. A.flavus e Penicillium sp., apresentaram maior consumo de carboidratos totais que o solo sem inoculação ou inoculado com F. verticillioides. A adição de glicose no solo favoreceu o consumo de carboidratos residuais, provavelmente devido ao estímulo do crescimento dos fungos. Exceto com F. verticillioides, a atividade da desidrogenase aumentou em média de 1,5 a 1,8 vez (p<0,05) nos solos com glicose em relação aos solos sem glicose. À atividade da amilase, aumentou 1,3 a 1,5 vez por efeito da adição de glicose no solo. Maior atividade da amilase foi observada no solo Latossolo Vermelho distrófico (LVd) com glicose e inoculado com A.flavus e Penicillium sp. em relação ao controle. As dimensões das plântulas como altura dos coleóptilos, comprimento das raízes e massa seca das plântulas de milho dos solos adicionados de glicose e/ou inoculados com os fungos foram diminuídas. A expressão genética... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The knowledge about hydrolytic enzymes activity is essential to understand the nutrients transformations in soil. Corn is one of the main crops from the economic view in the whole world. The objective of this study was evaluate the fungi growth in two soil types, in the presence and absence of glucose, coleoptiles development, roots and weight of corn seedlings, coleoptiles isoenzyme pattern, content of total residual carbohydrates, enzymes dehydrogenase and amylase activity. Was used a completely randomized design with factorial arrangement. The glucose addition in culture medium increased the growth rate of A. flavus and Penicillium sp., but not F. verticillioides. In the presence of glucose, the spores number was increased from 1.2 (F. verticillioides) and 8.2 times (A. flavus), but was reduced 3.5 times with Penicillium sp. Reducing the levels of total carbohydrates, significantly adjusted to the equations of 1st and 2nd grade. A.flavus and Penicillium sp., showed higher carbohydrates consumption that the soil control or inoculated with F. verticillioides. The glucose addition in soil, favored the use of residual carbohydrates, probably due fungi growth stimulation. Except F. verticillioides, dehydrogenase activity increased in the range 1.5 to 1.8 times (p<0.05) in soils with glucose compared to glucose free soil. Amylase activity increased 1.3 to 1.5 times by glucose addition effect in soil. Increased amylase activity was observed in the Distrophic Red Latosol (DRL) with glucose and inoculated with A.flavus and Penicillium sp., compared to control. The seedlings dimensions as coleoptiles height, root length and dry weight of maize seedlings of the soils added with glucose and inoculated with fungi were decreased. Gene expression of maize seed was changed due probable by fungi inoculated infection in soil. The coleoptiles isoenzymes pattern was amended as early as 5 incubation... (Complete abstract click electronic access below)
Mestre

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Sabe-se que uma variedade de microrganismos produzem um ou mais tipos de amilases para degradar os amiláceos presentes no ambiente e que as amilases produzidas por microrganismos termofílicos apresentam características mais termoestáveis do que aquelas produzidas por mesófilos. Essas amilases termoestáveis são de grande interesse na indústria de processamento de amido, uma vez a temperatura do gelatinização do mesmo, etapa importante do processo, fica em torno de 70ºC. Além disso, os processos que ocorrem em altas temperaturas, têm menor risco de contaminação por mesófilos e a diminuição da viscosidade do meio permite trabalhar com elevadas concentrações de substrato. No presente trabalho foram isoladas, a partir de amostras de solos, compostagens e resíduos agro-industriais, 339 linhagens microbianas termofílicas (326 bacterianas e 13 fúngicas) capazes de crescer a 55ºC em meio contendo amido como única fonte de carbono. As 163 linhagens (162 bactérias e 1 fungo) que se destacaram quanto à produção de amilases e à velocidade de crescimento em meio sólido, foram selecionadas e submetidas às fermentações submersa (meio nutriente) e semi-sólida (farelo de trigo). As atividades enzimáticas foram determinadas pelo método dextrinizante (a- amilase), pela quantificação da glicose (glucoamilase) e de açúcares redutores liberados a partir da hidrólise do amido. Das linhagens testadas, nove mostraram-se boas produtoras de a-amilase em fermentação submersa e/ou semi-sólida, fato pelo qual foram selecionadas para produção de amilases em meios formulados a partir de resíduos líquidos da extração de mandioca e milho (fermentação submersa) e sólidos, constituídos de farelos de mandioca, milho e trigo (fermentação semi-sólida). Quatro linhagens se destacaram, sendo suas enzimas brutas caracterizadas quanto às propriedades físico-químicas...
It is known that some microorganisms produce one or more amylase types that degrade organic compound present in the environment. The amylases produced by thermophilic microorganisms present characteristics more thermostable than those produced by mesophilics. Since starch gelatinization temperature is around 70ºC, the thermostable amylases are very important to starch processing industry. Besides, the processes that happen in high temperatures have less risk of contamination for the mesophilic microorganisms, the decrease of the viscosity of the media and allows to work with high substrate concentrations. In the present work thermophilic microorganisms (326 bacteria and 13 fungi) were isolated from soil samples, decaying agricultural waste and agriculture-industrial residues. They are able to grow at 55ºC in media containing starch as only carbon source. 163 strains (162 bacteria and 1 fungus) that showed high amylase production and fast growth in solid media were selected and submitted to the submerged (nutrient media) and semi-solid (wheat bran) fermentations. The enzymatic activities were determined by dextrinogenic method (a-amylase), by the quantification of the glucose (glucoamylase) and of the reducing sugars released from the starch hydrolysis. Among the tested strains, nine have shown high a-amylase production in submerged and/or semi-solid fermentations and because of this they were selected for amylase production in medium formulated with liquid wastes of extraction starch from the cassava and corn (submerged fermentation), and solid wastes constituted of cassava, corn, and wheat brans (semi-solid fermentation). Among these strains, four were selected and their crude enzymes were characterized regarding to their physicochemical properties. The strains Bacillus sp A13-22 and Mucor sp A13-36 produced amylases with optimum temperature of 70 and 80 ºC...(Complete abstract click electronic access below)

Resumo: Sabe-se que uma variedade de microrganismos produzem um ou mais tipos de amilases para degradar os amiláceos presentes no ambiente e que as amilases produzidas por microrganismos termofílicos apresentam características mais termoestáveis do que aquelas produzidas por mesófilos. Essas amilases termoestáveis são de grande interesse na indústria de processamento de amido, uma vez a temperatura do gelatinização do mesmo, etapa importante do processo, fica em torno de 70ºC. Além disso, os processos que ocorrem em altas temperaturas, têm menor risco de contaminação por mesófilos e a diminuição da viscosidade do meio permite trabalhar com elevadas concentrações de substrato. No presente trabalho foram isoladas, a partir de amostras de solos, compostagens e resíduos agro-industriais, 339 linhagens microbianas termofílicas (326 bacterianas e 13 fúngicas) capazes de crescer a 55ºC em meio contendo amido como única fonte de carbono. As 163 linhagens (162 bactérias e 1 fungo) que se destacaram quanto à produção de amilases e à velocidade de crescimento em meio sólido, foram selecionadas e submetidas às fermentações submersa (meio nutriente) e semi-sólida (farelo de trigo). As atividades enzimáticas foram determinadas pelo método dextrinizante (a- amilase), pela quantificação da glicose (glucoamilase) e de açúcares redutores liberados a partir da hidrólise do amido. Das linhagens testadas, nove mostraram-se boas produtoras de a-amilase em fermentação submersa e/ou semi-sólida, fato pelo qual foram selecionadas para produção de amilases em meios formulados a partir de resíduos líquidos da extração de mandioca e milho (fermentação submersa) e sólidos, constituídos de farelos de mandioca, milho e trigo (fermentação semi-sólida). Quatro linhagens se destacaram, sendo suas enzimas brutas caracterizadas quanto às propriedades físico-químicas...(Resumo completo, clicar acesso eletrônico abaixo)
Abstract: It is known that some microorganisms produce one or more amylase types that degrade organic compound present in the environment. The amylases produced by thermophilic microorganisms present characteristics more thermostable than those produced by mesophilics. Since starch gelatinization temperature is around 70ºC, the thermostable amylases are very important to starch processing industry. Besides, the processes that happen in high temperatures have less risk of contamination for the mesophilic microorganisms, the decrease of the viscosity of the media and allows to work with high substrate concentrations. In the present work thermophilic microorganisms (326 bacteria and 13 fungi) were isolated from soil samples, decaying agricultural waste and agriculture-industrial residues. They are able to grow at 55ºC in media containing starch as only carbon source. 163 strains (162 bacteria and 1 fungus) that showed high amylase production and fast growth in solid media were selected and submitted to the submerged (nutrient media) and semi-solid (wheat bran) fermentations. The enzymatic activities were determined by dextrinogenic method (a-amylase), by the quantification of the glucose (glucoamylase) and of the reducing sugars released from the starch hydrolysis. Among the tested strains, nine have shown high a-amylase production in submerged and/or semi-solid fermentations and because of this they were selected for amylase production in medium formulated with liquid wastes of extraction starch from the cassava and corn (submerged fermentation), and solid wastes constituted of cassava, corn, and wheat brans (semi-solid fermentation). Among these strains, four were selected and their crude enzymes were characterized regarding to their physicochemical properties. The strains Bacillus sp A13-22 and Mucor sp A13-36 produced amylases with optimum temperature of 70 and 80 ºC...(Complete abstract click electronic access below)
Orientador: Eleni Gomes
Coorientador: Célia M. L. Franco
Banca: Cláudio Cabello
Banca: Gustavo Orlando Bonilla Rodriguez
Mestre

Thesis (M.Sc. (Microbiology)) -- University of Limpopo, 2018
Beer is one of the most widely consumed alcoholic beverages in the world. The brewing process is based on natural enzymatic activities that take place during the malting of barley grain, mashing of grist and fermentation of wort. Insufficient malt enzyme activity during the mashing process leads to high levels of barley β-glucan, arabinoxylan (AX) and dextrins in the wort as well as in the final beer. It was reported that high levels of β-glucan and AX increase wort and beer viscosity which lower the rate of beer filtration and this negatively affect the production rate in the brewery. During beer fermentation, brewing yeast catalyses the conversion of wort sugars to ethanol, carbon dioxide and other metabolic products. However, non-fermentable carbohydrates i.e., limit dextrins remain in the wort and final beer. These non-fermentable carbohydrates are known to contribute to the caloric value of beer which might lead to weight gain in consumers. The objectives of this study were to evaluate the effect of recombinant yeast strains expressing an endo-β-1,4-glucanase or an endo-β-1,4-xylanase on beer viscosity (as an indicator of filterability) and an α-amylase on residual sugars levels. The effect of the above mentioned enzymes on the aroma, appearance, flavour, mouth-feel and overall quality of the beer was also determined. Wort was produced in the University of Limpopo micro-brewery and the wort was pitched with different recombinant strains. The wild-type strain served as control. The results obtained showed that the xylanase expressing strain produced a measurable decrease in viscosity over the course of the fermentation, but endo-glucanase did not have any effect on the beer viscosity. The α-amylase producing strain, did not show a measurable reduction of residual sugars in the final beer probably as a result of very low activity on α-1,6 glycosidic bonds in dextrins during fermentation. The xylanase and α-amylase producing strain fermented effectively with good attenuation (decrease in wort specific gravity). The beer produced by the α-amylase and control strains were preferred in terms of taste and had similar qualities. The secreted amylolytic activity was not sufficient to significantly reduce residual sugar in the final beer. Although the xylanase secreting strain produced a beer with lower viscosity, the enzyme had a negative impact on the taste of the beer. Key words: Brewer’s yeast, beer fermentation, low calorie beer, amylase, xylanase, endo-glucanase.