A mandioca (Manihot esculenta Crantz) é uma raiz originária e cultivada na América do Sul, com alta perecibilidade no período pós-colheita. Seus principais processos de deterioração envolvem reações enzimáticas, oxidativas e microbiológicas. Neste trabalho foram estudadas raízes de mandioca da variedade Zolhudinha catalogada pela EMBRAPA como IM-158, provenientes da região amazônica, que se destacam pela alta atividade amilolítica. Foram analisadas as condições físico-químicas junto com o isolamento e purificação da α-amilase da raiz e a possível participação desta enzima no processo deteriorativo pós-colheita. Por ser uma variedade de mandioca não comercial, o tempo de cocção foi em média de 4,30 h, teor de umidade em tomo de 64 % e porcentagem de amido de cerca de 30 %. A atividade amilásica decai em 1/3 de sua intensidade no quinto dia pós-colheita, em contraponto a formação de açúcares redutores, cuja concentração aumenta cinco vezes. A purificação foi obtida com duas etapas cromatográficas, com DEAE-celulose e Sephacryl S-200, revelando duas isoenzimas de α-amilase treze vezes mais purificadas, com recuperação protéica de 7,5 % e com pesos moleculares entre 14 e 19 kDa. / Cassava (Manihot esculenta Crantz) is a root from a native plant, cultivated in South América, that is hightly perish on the post-harvest time. The mechanisms of the root deterioration are due to enzymatic and oxidative reactions as well as the microbiological attack. In this work were studied roots of Zolhudinha variety, EMBRAPA - IM 158, cultivated in Amazonian area, which distinguishes from others varieties by its higher amylase activity. Physicochemical properties were analyzed during the post-harvest time, the purification of α-amylase were performed to establish a possible involvement on the deteriorative process. As a non-commercial variety, the cooking time of the roots was 4.30 hours on average, with 64 % of water content and 30 % of starch. The amylase activity during the post-harvest decrease 1/3 from the original at the day 5, that matches with the reducing sugar in roots by increase of five times. The purification was achieved by two chromatography steps on DEAE-cellulose and Sephacryl S-200, providing two isozymes of α-amylase, thirteen times more purified with a recovery of 7.5 % of the protein fraction, the estimated molecular weights were between 14 and 19 kDa.

Orientador: Jaime Aparecido Cury / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T16:20:44Z (GMT). No. of bitstreams: 1 Botelho_JulianaNunes_D.pdf: 1920977 bytes, checksum: 99c53e9c14bfc15c6b8859cce1fe5dec (MD5) Previous issue date: 2014 / Resumo: Sacarose é o carboidrato mais cariogênico da dieta e o amido é considerado não cariogênico para esmalte e moderadamente cariogênico para dentina. Por outro lado, a combinação de amido e sacarose (amido+sacarose) tem sido considerada mais cariogênica que apenas sacarose, mas esse ainda é um assunto em debate. Além do mais, o efeito do dentifrício fluoretado na cariogenicidade dessa combinação é desconhecido. Assim, com o objetivo de estudar esse assunto três experimentos foram conduzidos: (i) o primeiro avaliou efeito de amido+sacarose na desmineralização de esmalte e dentina, usando um modelo de biofilme de S. mutans modificado pela adição de saliva para simular a ação da amilase, (ii) o segundo avaliou in situ o efeito do dentifrício contendo 1.100 µg F/g (DF) na progressão da desmineralização da dentina radicular, e o terceiro (iii) avaliou in situ o efeito do fluoreto no potencial cariogênico de amido+sacarose na desmineralização de esmalte e dentina. In vitro, biofilmes de S. mutans foram formados sobre blocos de esmalte e dentina radicular, por 5 e 4 dias respectivamente, em meio de cultura contendo saliva e expostos a um dos seguintes tratamentos: amido a 1%, sacarose a 10% ou de sua combinação (8x/dia). Os biofilmes foram analisados quanto às suas composições bioquímicas e microbiológicas, e a desmineralização dos blocos foi avaliada. Biofilmes expostos à combinação foram mais acidogênicos (p<0,0001) e provocaram maior desmineralização (p<0,0001) no esmalte e dentina que o efeito dos carboidratos isolados. In situ, o efeito do DF foi testado em um estudo piloto, cruzado no qual sacarose a 10% foi aplicada extraoralmente 8x/dia em 2 fases de 14 dias. Após 10 e 14 dias em cada fase, a desmineralização da dentina foi avaliada. O efeito do dentifrício foi significativo (p<0,0001), mas o efeito do tempo não (p>0,05). Esses resultados sugerem que o DF com 1.100 µg F/g é capaz de diminuir a cárie dentinária mesmo sob alto desafio cariogênico de acúmulo de biofilme e exposição à sacarose. In situ, o efeito dos tratamentos (água, amido a 2%, sacarose a 10% e amido+sacarose) e o efeito do dentifrício (não fluoretado e fluoretado) foram testados em um estudo cruzado, cego, boca-dividida em 4 fases de 14 dias. Os voluntários usaram dois dos tratamentos 8x/dia e um dos dentifrícios 3x/dia. O efeito dos fatores (dentifrício e tratamentos) foram significativos (p<0,05) para esmalte e dentina, mas a interação não (p>0,05). Os resultados sugerem que, independente do desafio cariogênico provocado pelos diferentes carboidratos da dieta testados, o dentifrício fluoretado é efetivo na redução da desmineralização de esmalte e dentina. Em conclusão, os resultados sugerem que amido deve aumentar o potencial cariogênico da sacarose mas que fluoreto de dentifrício é capaz de reduzir a desmineralização tanto do esmalte quanto da dentina provocada pela combinação desses carboidratos / Abstract: Sucrose is the most cariogenic dietary carbohydrate while starch is considered non-cariogenic for enamel and slightly cariogenic for dentine. The combination starch and sucrose (starch+sucrose) has been considered more cariogenic than sucrose alone but this subject remains debatable. Also, the effect of fluoride toothpaste on the cariogenicity of this combination is unknown. The aims of this study were to evaluate: (i) the effect of starch+sucrose on enamel and dentine demineralization using an S. mutans biofilm model modified by adding human saliva to simulate amylase action; (ii) the in situ effect of fluoride toothpaste (FT) containing 1100 µg F/g on dentine demineralization progression; and (iii) the in situ effect of fluoride on the cariogenic potential of starch+sucrose on enamel and dentine demineralization. In vitro, S. mutans biofilms were grown on enamel and root dentine slabs for 5 and 4 days, respectively, in a saliva-containing medium and exposed to the following treatment: 1% starch; 10% sucrose; or starch+sucrose (8x/day). Biofilms were then analyzed for their biochemical and microbiological compositions, and dental demineralization was evaluated. Biofilms exposed to starch+sucrose were more acidogenic (p < 0.0001) and caused higher demineralization (p < 0.0001) on either enamel or dentine than those exposed to each carbohydrate alone. The in situ effect of FT on dentine demineralization was tested in a pilot crossover study, in which 10% sucrose was applied extra-orally to the slabs 8x/day in 2 phases of 14 days each. At days 10 and 14 of each phase, dentine demineralization was evaluated. The effect of toothpaste was significant (p<0.0001), but the effect of time was not (p>0.05). The results suggest that FT at 1100 µg F/g can reduce dentine demineralization even under high cariogenic challenges - biofilm accumulation and sugar exposure. The in situ effect of the treatments (water, 2% starch, 10% sucrose and starch+sucrose) and that of the toothpastes (non-FT and FT) were tested in a crossover, single-blind and split-mouth study conducted in 4 phases of 14 days each. The volunteers used two of the treatments 8 times/day and one of the toothpastes 3 times/day. The effect of the factors (toothpaste and treatments) was significant (p<0.05) for enamel and dentine, but not (p>0.05) for the interaction. The findings suggest that, regardless of the cariogenic challenge provoked by the different sources of the dietary sugars tested, fluoride toothpaste is effective in reducing enamel and dentine demineralization. In conclusion, the results suggest that starch may enhance the cariogenic potential of sucrose and fluoride from toothpaste reduces enamel and dentine demineralization caused by the combination of these carbohydrates / Doutorado / Cariologia / Doutora em Odontologia

The aim of this study was to determine the mean lethal concentration (96 h) for waterborne copper (LC50), as well as the effect of the exposure to copper on growth, metabolic parameters (glycogen, glucose, lactate, and protein) in some tissues, activity of acetylcholinesterase (AChE) (brain and muscle), amylase and maltase (intestine) in silver catfish (Rhamdia quelen). The LC50 for copper was 0.4 mg/L. On growth experiments fish were exposed to 10 and 20% LC50, i.e., 0.04 and 0.08 mg/L respectively. Exposure to copper did not change growth parameters (weight, length and biomass). In the liver, lactate levels increased in juveniles exposed to 0.04 mg/L and decreased in those maintained at 0.08 mg/L, while protein levels decreased in those exposed to both concentrations compared to unexposed specimens. Glycogen levels in the muscle were lower in fish exposed to both concentrations, glucose and lactate were higher in those exposed to 0.04 mg/L and decreased in juveniles maintained at 0.08 mg/L, while protein was higher in those exposed to 0.08 mg/L. Glucose and lactate plasma levels were higher in juveniles exposed to 0.04 mg/L, but protein levels were lower in those maintained at both copper concentrations. Amylase activity was lower in juveniles exposed to both concentrations, but maltase was higher in those exposed to 0.04 mg/L than control group. Brain AChE activity was lower in fish exposed to both concentrations while muscle AChE activity was not affected after 45 days of exposure. It can be concluded that copper can change several metabolic parameters and enzymes of toxicological and feeding interest even at sublethal concentrations. / O objetivo deste estudo foi verificar a concentração letal média em 96 h (CL50) para o cobre, bem como o efeito da exposição ao cobre sobre o crescimento, parâmetros metabólicos (glicogênio, glicose, lactato e proteína) em alguns tecidos (fígado, músculo e cérebro) e a atividade da acetilcolinesterase (AChE) (cérebro e músculo), amilase e maltase (intestino) em jundiás (Rhamdia quelen.). A CL50 para o cobre foi 0,4 mg/L. Nos experimentos de crescimento os peixes foram expostos durante 45 dias a 10 e 20% da CL50, ou seja, 0,04 e 0,08 mg/L respectivamente. A adição de cobre não alterou os parâmetros de crescimento avaliados (peso, comprimento e biomassa). No fígado, os níveis de lactato aumentaram nos exemplares expostos a 0,04 mg/L e diminuíram nos mantidos em 0,08 mg/L, enquanto que os níveis de proteína diminuíram em ambas as concentrações em relação ao grupo controle. No músculo houve redução na atividade do glicogênio nos exemplares mantidos nas duas concentrações testadas, a glicose e o lactato aumentaram nos expostos a 0,04 mg/L e diminuíram nos expostos a 0,08 mg/L, e a proteína aumentou nos mantidos em 0,08 mg/L. Os níveis de glicose e lactato no plasma foram maiores nos exemplares mantidos em 0,04 mg/L e diminuíram os níveis de proteína nos expostos a ambas as concentrações de cobre. A atividade da amilase foi menor nos juvenis expostos a ambas as concentrações, enquanto a da maltase foi maior em 0,04 mg/L quando comparada ao grupo controle. A atividade da AChE cerebral foi menor nos exemplares expostos a ambas concentrações, enquanto que a AChE muscular não sofreu alterações após os 45 dias de exposição. Conclui-se que o cobre mesmo em concentrações subletais pode alterar diversos parâmetros metabólicos e enzimas de interesse toxicológico e alimentar.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The potato (Solanum tuberosum) is important raw material for agro-industrial production of hydrolysates. The lack of information about the process of hydrolysis of potato starch industry hampers the use of regional raw material and promote the use of crops such as maize, cassava and sugarcane to produce ethanol. The objective of this study was to determine the optimum range of the effects of different concentrations of substrate, concentration of enzyme amyloglucosidase (AMG) and reaction times, reduce process time, enzymes and material to be hydrolyzed. This was a central composite experimental design with three independent variables namely: [1] substrate concentration (3-7%) [2] enzyme dosage (0.36 to 1.0 μL.g MS-1) [ 3] time of action of enzymes computed after the addition of AMG in the middle (4 to 48 hours). The α-amylase concentration remained constant at 0.8 μL/g starch. The experiments were conducted in the bioreactor followed by metabolic bath. The dependent variables analyzed were the concentration of reducing sugars (RS) and the efficiency of hydrolysis of starch. The data were processed by Statistica 8.0, to generate predictive models at 95% confidence. The larger fraction of RA was reached in the middle (35.55 g/L), with time (26 hours) and dose of enzyme (0.68 mL) and substrate concentration at the maximum level (7%). The efficiency of hydrolysis (125%) occurred with the minimum ratio of substrate (3%) and enzyme dosage (0.68 mL) and the average time (26 hours). To reduce costs of enzyme (0.36 mL AMG), working with the same concentration of substrate, the model estimated that the hydrolysis for 39 hours was able to release 33.17 g/L of the AR 128, 62% efficiency. The use of different enzyme concentrations showed no significant increase in responses, and the time factor is set lower than recommended by the manufacturer of the enzymes. The concentration of substrate influenced more significantly than other factors, for both dependent variables. The performance presented demonstrates that this process is an efficient alternative to the industry, emerging as an important source this material for the production of hydrolysates. / A batata (Solanum tuberosum) é importante matéria-prima agroindustrial para produção de hidrolisados. A falta de informações sobre o processo de hidrólise do amido de batata na indústria dificulta a utilização desta matéria-prima regional e favorece o uso das culturas como: milho, mandioca e cana de açúcar para produção de álcool etílico. O objetivo deste trabalho foi apurar a faixa otimizada dos efeitos de diferentes concentrações de substrato, concentração de enzima amiloglicosidase (AMG) e períodos reacionais, na redução de tempo do processo, enzimas e material a ser hidrolisado. Foi realizado um desenho experimental central composto, com três variáveis independentes sendo elas: [1] concentração de substrato (3-7%), [2] dosagem enzimática (0,36-1,0μL/g de MS), [3] tempo de ação das enzimas computado após a adição da AMG no meio (4 a 48 horas). A concentração de α-amilase manteve-se constante em 0,8μL/g de amido. Os experimentos foram conduzidos em bioreator seguido de banho metabólico. As variáveis dependentes analisadas foram a concentração de açúcares redutores (AR) e a eficiência de hidrólise do amido. Os dados foram tratados pelo Statistica 8.0, para gerar modelos preditivos a 95% de confiança. O teor maior de AR foi alcançado no meio (35,55g/L), com tempo (26 horas) e dosagem de enzima (0,68μL) e concentração de substrato no nível máximo (7%). A maior eficiência de hidrólise (125%) ocorreu com a razão mínima de substrato (3%) e dosagem de enzimas (0,68μL) e tempo no nível médio (26 horas). Para a redução dos custos com enzima (0,36μL/g MS de AMG), trabalhando-se com a mesma concentração de substrato, o modelo estimou que a hidrólise por 39 horas era capaz de liberar 33,17 g/L de AR , com 128,62% de eficiência. O uso de diferentes concentrações de enzimas não apresentou incremento significativo nas respostas, sendo que o fator tempo se estabeleceu abaixo do recomendado pelo fabricante das enzimas. A concentração de substrato influenciou de forma mais significativa que os demais fatores, para ambas variáveis dependentes. O rendimento apresentado demonstra que este processo é uma alternativa eficiente à indústria, despontando este material como fonte importante para produção de hidrolisados.

Cette étude vise à produire deux enzymes amylolytiques (α-amylase et pullulanase) thermostables par des levures contaminant le blé récolté dans des zones semi arides et arides (Biskra - Sahara, Sud Algérien) et capables d’hydrolyser à la fois les liaisons α-1-4 et α-1-6 de polysaccharides comme l’amidon et le pullulane, molécules d’intérêt industriel. Après isolement et caractérisation de colonies levuriennes, la méthode de plate-test-agar permet d’isoler des souches amylolytiques et de montrer que la souche L7 est la plus performante dans la production enzymatique parmi une douzaine de souches de levures productrices d’α-amylase et de pullulanase thermostables. L’identification des souches, basée sur les caractères morphologiques, les tests biochimiques et la biologie moléculaire a permis de répartir la population comme suit : 50% Clavispora lusitaniae (forme anamorph Candida lusitaniae), 25%, Pichia guilliermondii, 8% Pichia carribbicca, 8% Meyerozyma guilliermondii et 8% Rhodotorula rubra. Par sa richesse en amidon, le biotope du blé est favorable à la survie des levures amylolytiques. La majorité de ces souches dont la souche L7 est productrice de pseudo ou vrai mycélium et est tolérante à certains paramètres comme la température, la salinité, les stress osmotique et éthanolique. La souche de levure L7, Clavispora lusitaniae ABS7, semble être la plus performante dans la production d’enzymes thermostables. Son identification moléculaire a montré deux bandes avec l’endonucléase HAE III alors que les autres souches de la même espèce de Clavispora lusitaniae (L5, L9, L10, L11 et L12) présentent une seule bande. En conditions optimales (agitation 136,56 rpm, température 54,14°C, amidon 2,66g/l, extrait de levure 0,365g/l, sels 8, 75ml/l et oligo-éléments 4,3ml/l en erlenmeyers de 250 ml), la production maximale atteint les valeurs suivantes : 13456,36±300 UI pour l’ α-amylase et 12611, 6±154 UI pour la pullulanase. Ces performances sont en accord étroit avec la prédiction du modèle statistique évaluée à 13231UI pour l’α-amylase et 12825,5 UI pour la pullulanase. La production optimisée a pratiquement doublé par rapport à la production avant l’optimisation (6639,16 UI pour l’α-amylase et 6308,5 UI pour la pullulanase). En conditions optimales et en fermenteur de 2 L, la production maximale pour les deux enzymes de la levure Clavispora lusitaniaeABS7 est obtenue au bout de 28 h avec un optimum de croissance obtenu à 40 heures. La production des deux enzymes n’est donc pas associée à la croissance. La production maximale des deux enzymes s’effectue à pH 8. pic protéique. L’élution sur DEAE-cellulose confirme la présence des deux activités dans la même fraction. Les deux enzymes sont donc présentes sur la même molécule. L’α-amylase et la pullulanase sont purifiées avec un taux de purification de 50,5 et 44,6 respectivement et des rendements respectifs de 23,9% et 21,1%. L’extrait purifié montre une seule bande sur le gel de SDS-PAGE avec un poids moléculaire estimé à 75KDa et une activité amylolytique contenant à la fois les activités α-amylasique (indépendante de Ca2+) et pullulanasique (une métalloenzyme à calcium). La souche de la levure Clavispora lusitaniae ABS7 possède donc une enzyme amylolytique avec deux sites actifs. La CCM révèle une enzyme qui hydrolyse l’amidon en maltose et glucose et le pullulane en maltotriose, maltose et glucose, ce qui montre que l’enzyme est saccharifiante et correspond à une pullulanase de type II (amylopullulase). L’optimisation de l’immobilisation de l’enzyme a permis l’amélioration de l’activité: α-amylasique à 4907,75 UI (rendement 72,3 %) et celle de la pullulanase à 4491,83 UI (rendement 70,1%) avec un pH optimum de 8,5. Il ressort de notre étude que l’amylopullulanase type II libre de Clavispora lusitaniae ABS7 est thermostable puisqu’elle résiste à un traitement thermique de 75°C pendant 3 heures d’incubation et conserve 88% de son activité initiale. / This study aims to produce two amylolytic enzymes (α-amylase and pullulanase) by thermostable wheat contaminant yeast harvested in semi arid and arid zones (Biskra, Sahara, Algeria SUD) and capable of hydrolyzing both the α links 1-4 and 1-6 of polysaccharides such as starch and pullulan, molecules of industrial interest. After isolation and characterization of levuriennes colonies, the test method of agar-plate allows to isolate amylolytic strains and show, that the L7 strain is the most effective, in the enzymatic production of the 12 yeast strains producing α-amylase and pullulanase the thermostable. The identification of strains, based on morphological, biochemical tests and molecular biology has helped spread the population as follows: 50% Clavispora lusitaniae (anamorph form Candida lusitaniae), 25%, Pichia guilliermondii, 8% carribbicca Pichia, 8% Meyerozyma guilliermondii and 8% Rhodotorula rubra. By its high starch, the wheat biotope is favorable to the survival of amylolytic yeasts. Most of these strains, including the strain L7, is producer, pseudo or true mycelium and is tolerant to certain parameters such as temperature, salinity, osmotic stress and ethanolic stress. The yeast strain Clavispora lusitaniae ABS7 (L7) seems to be the most efficient in the production of thermostable enzymes. Its molecular identification showed two bands with the endonuclease HAE III while other strains of the same species Clavispora lusitaniae (L5, L9, L10, L11 and L12) have a single band. In optimal conditions (agitation 136.56 rpm, temperature 54.14 ° C, starch 2,66g / l, yeast extract 0,365g / l, salts 8 75ml / l and trace elements 4,3ml / liter Erlenmeyer flasks into 250 ml), the maximum production reached: 13456.36 ± 300 IU for the α-amylase and 12611, 6 ± 154 IU for pullulanase. This performance is in close agreement with the prediction of the statistical model 13231UI evaluated for α-amylase and 12825.5 IU for pullulanase. The optimized production almost doubled compared to production before optimization (6639.16 IU for the α-amylase and pullulanase for 6308.5 IU). In optimal conditions, and 2 L fermenter, the maximum production for the two enzymes of Clavispora lusitaniae ABS7 obtained after 28 hours, with an optimum of growth obtained at 40 hours. The production of both enzymes is thus not associated with growth. The maximum production of both enzymes is obtained at pH 8. The kinetics are characterized by an increase in carbohydrate and a substance spooning the wall of the fermenter, probably an exo-polysaccharide. The chromatographic profile on Sephacryl S200 reveals two α-amylase and pullulanase activities eluted along with the protein peak. Elution DEAE cellulose confirms the presence of both activities in the same fraction. Both enzymes are present on the same molecule. The α-amylase and pullulanase were purified with a purification rate of 50.45 and 44.59 respectively and respective yields of 23.88% and 21.11%. The purified enzyme showed a single band on SDS-PAGE gel with a molecular weight estimated at 75 KDa and an amylolytic activity containing both the α-amylase activities (independent of Ca2+) and pullulanase (a calcium metalloenzyme). The strain of the yeast Clavispora lusitaniae ABS7 therefore has an amylolytic enzyme with two active sites. TLC reveals an enzyme which hydrolyzes starch into maltose and glucose and pullulan into maltotriose, maltose and glucose, which shows that the saccharifying enzyme, and corresponds to a pullulanase type II (amylopullulase). The optimization of the immobilization of the enzyme enabled the improvement of the activity: α-amylase to 4907.75 IU (yield 72.3%) and pullulanase to 4491.83 IU (yield 70, 1%) with a pH optimum of 8.5. It appears from our study that amylopullulanase type II free is thermostable to heat treatment of 75 ° C for 3 hours of incubation, and retains 88% of its original activity.

Um dos eventos mais importantes durante o amadurecimento da banana é a degradação do amido, concomitante com o acúmulo de açúcares solúveis. Várias enzimas, que supostamente atuam na degradação do amido, já tiveram sua atividade/proteína específica detectadas nesta fase na banana. Entre elas a &#945;-amilase, a &#946;-amilase, as amido-fosforilases, as &#945;-glicosidases e as isoamilases. A síntese do amido e, normalmente, sua degradação, ocorrem dentro do amiloplasto, que possui duas membranas a serem transpostas antes do acesso ao grânulo de amido ou aos produtos da ação de outras enzimas. Uma das isoformas da &#946;-amilase em banana possui um peptídeo de transporte predito em sua seqüência, necessário para transpor estas membranas e entrar no amiloplasto. Uma maneira de contornar a dificuldade em estabelecer a real importância de cada enzima na degradação do amido é isolar os grânulos e as enzimas e submetê-lo à atividade seqüencial das enzimas supostamente responsáveis pela degradação. O ideal é utilizar a enzima endógena, mas o processo de purificação de enzimas em frutos é demorado e nem sempre bom em termos de pureza, quantidade e atividade. Estudos baseados na expressão heteróloga de genes da &#946;-amilase permitiriam melhor compreender os mecanismos de atuação dessa enzima presente na polpa da banana. Assim, foram feitos ensaios de expressão heteróloga em Pichia pastoris na tentativa de produzir essa enzima em quantidade suficiente para purificação, aplicação nos grânulos de amido e produção de anticorpos policlonais. Foram testadas várias condições de indução da proteína, tais como aeração, temperatura, pH, concentração de metanol e tempo de indução, bem como a montagem de uma nova construção gênica com tag de histidina no vetor de expressão pPICZ&#945;A com confirmação do fenótipo dos transformantes positivos. Porém, a obtenção de &#946;-amilase recombinante com atividade não foi bem sucedida, necessitando talvez de alterações nesses padrões de indução. / One of the most important events that occurs during ripening of banana is the starch degradation concomitantly with the accumulation of soluble sugars. Several enzymes, known by acting on starch degradation, had their activity and/or specific protein detected at this stage of banana. These include the &#945;-amylase, the &#946;-amylase, the starch-phosphorylases, the &#945;-glucosidase and the isoamilases. The synthesis and starch degradation occur inside of the amyloplast that contain two membranes which has to be reach before accessing the starch granule or the products of the other enzymes. One of the isoforms of &#946;-amylase in bananas has a transit peptide predicted in the sequence, required to access the amyloplast. To establish the real importance of each enzyme in the starch degradation it is necessary to isolate the granules and enzymes and submit them to the sequential activity to confirm the supposed degradation. The idea is to use the endogenous enzyme, but the process of purification of the enzyme in fruits demands a lot of time and the results of purity, quantity and activity are not guaranteed. Studies based on heterologous expression of the &#946;-amylase genes allow us to understand the mechanisms of action of this enzyme present in the pulp of banana. Thus, tests were carried out with heterologous expression in Pichia pastoris in order to produce this enzyme in sufficient quantity to purification, and then, applied on starch granules and produce a polyclonal antibody. We tested different conditions of protein induction such as aeration, temperature, pH, methanol concentration and induction time, as well as the new genic construction with the histidine tag with an expression vector pPICZ&#945;A confirming the phenotype of positive transformants. However, recombinant &#946;-amylase with activity was not obtained successfully, necessitating changes in these patterns of induction.

The aim Master thesis was to evaluate the influence of malting technology on the content of hydrolytic enzymes -amylase and -amylase. Another aspect of this thesis was to select a wheat variety with the highest activity of enzymes -amylase and -amylase. The first part of the practical part of my work is shown measuring enzyme malt model varieties of wheat set (Triticum aestivum), where individual samples differ technological processing of malting and the second part is focused on the application of efficient malting conditions on other varieties from different locations in the Czech Republic in 2016 . All malt samples were analysed to -amylase and -amylase activity by assay Ceralpha method and Betamyl-3 method made by Megazyme company. After measuring the first part, the most effective malting condition was evaluated, such as a 7 day malting time, a 45% water content, a germination temperature of 18 ° C, and a drying temperature of 50 ° C. In the second part, the varieties are so malted and the highest content of enzyme -amylase was Rumor, it had averafe contanin -amylase 277 U/g, Bonanza – 255 U/g and Izzy –254 U/g. The biggest contain of enzymes -amylasehad varieties Matchball – 37,9 U/g , Fakir – 35,6 U/g, and Bernstein – 35,3 U/g.

<p>  </p> <p>Pacheco, Kathryn Nichole. M.S. Purdue University, December 2022. Snacking Interventions Differentially Influence Saliva, Salivary Alpha Amylase Activity, and Sensation. Major Professor Dr. Cordelia A. Running.</p> <p>Human saliva contains the enzyme alpha amylase, which greatly influences many facets of human health such as digestion, absorption of nutrients, and the sensory perception of certain foods. However, the complex relationships between chewing behavior, food texture preference, and salivary amylase require further investigation. In this study, we aim to observe salivary alpha amylase through a simple assay using pudding, and to examine whether salivary amylase activity relates to diet, the sensory properties of starchy foods, or mouth behavior. We hypothesized that the pudding/salivary amylase activity assay would show more activity (less pudding remaining) 1) at the end of the high dietary starch intervention week, with little or no change from baseline to the end of the low dietary starch intervention week and 2) for people with greater baseline starch consumption compared to less baseline starch consumption. A counter-balanced, crossover design was implemented for the study. 34 participants (11 Men, 23 Women, 0 Other) completed study tasks, consisting of a 3-day dietary recall, 2 separate weeks of dietary intervention consisting of high starch or low starch snacks, and 4 research visits. These research visits included participant taste and smell acuity assessments, sensory ratings of the study foods, a mouth behavior typing test, and our salivary amylase activity assay that determined flow rate of a mixture of participant saliva and starch-containing ready-to-eat pudding. After our higher and lower starch snack interventions, we saw minimal evidence of changes to salivary amylase activity in our assay; the only trend we observed was opposite our expectation (less amylase activity after the low starch intervention). However, we did observe mouth behavior grouping tended to associate with sensory ratings that validate the premise of the mouth behavior typing tool we utilized. Ultimately, more work on the consistency and usefulness of the salivary amylase activity assay will need to be conducted if it is to be utilized for research purposes, but our data do help validate the concept that different people prefer foods due to their preferred methods of orally manipulating foods. r.  </p>

Mapeamento dos subsítios de &#945;-amilase de Xanthomonas axonopodis pv. Citri envolvidos na interação com o substrato A família das enzimas &#945;-amilases é um modelo experimental interessante para o estudo das interações entre os aminoácidos e seus ligantes, já que estas enzimas apresentam especificidade variável, são frequentemente alvos de estudos por mutagênese e há estruturas cristalinas disponíveis para alguns membros da família. A proposta deste trabalho foi o mapear subsítios da &#945;-amilase de Xanthomonas axonopodis pv. citri (AXA) envolvidos na interação com substratos, através de comparações estruturais, mutagêneses sítio-dirigidas, análises de parâmetros cinéticos sobre amido e do padrão de clivagem sobre p-nitrofenil malto-oligossacarideos (PNPG7, PNPG5, PNPG4). Foi criado um modelo estrutural para AXA a partir da estrutura tridimensional da &#945;-amilase de Alteromonas haloplanctis (Aghajari et al., 1998). O modelo de AXA foi sobreposto na estrutura da &#945;-amilase pancreática de porco (Qian et al., 1994) e 11 resíduos foram selecionados e mutados para alanina. As &#945;-amilases recombinantes mutantes e selvagem foram secretadas pela levedura Pichia pastoris GS115, apresentando uma massa molecular aparente de 45 kDa. Todos os mutantes analisados reduziram em maior ou menor grau a atividade catalítica da enzima sobre amido e p-nitrofenil maltooligossacarideos. Mutações dos resíduos H88, F136, D196, E223, D295 e N299, deletaram a atividade enzimática, indicando que suas cadeias laterais são essenciais para o desempenho catalítico da enzima. As análises cinéticas e estruturais sugerem fortemente que D196, E223 e D295 são os resíduos catalíticos. Substituições das cadeias laterais de C157, H200, G227, T230 e H294 reduziram a eficiência catalítica (kcat/Km) da &#945;-amilase sobre o substrato amido para, respectivamente, 28%, 41%, 84%, 81% e 51%. As mutações em G227 e T230 foram menos importantes para a atividade da enzima e afinidade pelo amido, entretanto, estes resíduos mostraram-se importantes para a estabilização de complexos com substratos curtos (pNPG4). Os resultados indicam que o sítio ativo de AXA é formado por, no mínimo, seis subsítios. As interações dos anéis de glicose com os subsítios +2 e -2 são favorecidas em relação às interações nos subsítios -3 e +3, respectivamente, e a interação do anel de glicose no subsítio -3 é favorecida em relação à interação no subsítio +3. A enzima selvagem diva preferencialmente a terceira ligação glicosídica de p-nitrofenil maltooligossacarideos. Como produtos de hidrólise a enzima libera maltopentaose, maltotetraose, maltotriose, maltose e glicose. / The &#945;-amylase family is an interesting group for structure/function relationship investigation, as this family exhibits a variable deavage patterm, several crystal structures are available, and its members were studied by mutagenesis. The aim of this study was the mapping of Xanthomonas axonopodis pv. Citri &#945;-amylase (AXA) subsites involved in substrate binding, using structural comparison, site-directed mutagenesis and lcinetics analyses. A structural model for AXA was created from the three-dimensional structure of the &#945;-amylase from Alteromonas haloplanctis (Aghajari et al., 1998). This model was superimposed on the structure ofthe pig pancreatic &#945;-amylase, PPA (Qian et. al., 1994), and 11 residues were selected and changed to alanine. Wild type and mutant AXA were secreted by Pichia pastoris strain GS115 cells and showed apparent molecular mass of 45 kDa. All mutants have reduced &#945;-amylase activity on starch and 4-nitrophenyl maltooligosaccharides (pNPG7, PNPG5 and PNPG4) at different levels. Mutation of residues H88, F136, D196, E223, D295 and N299 indicate their essential role by complete loss of activity. Kinetic and structural analyses strongly suggested that D196, E223 and D295 are the catalytic residues. The substitution of the side chain of C157, H200, G227, T230 and H294 reduced the catalytic efficiency (kcat/Km) of &#945;-amylase on starch to respectively 28%, 41%, 84%, 81% and 51%. Although G227 and T230 were not much important for activity and binding on starch, these residues were important for stabilization of complexes with short substrates (PNPG4). The results indicate that AXA\'s active site is composed of at least six sugar binding subsites. The binding of the glucoses at subsites +2 and -2 are favored against binding at subsites -3 and +3, respectively. The binding of glucose at subsite -3 is favored against binding at subsite +3. The wild type enzyme primarily hydrolyzes the third glucosidic bond in PNPG7, PNPG5 and PNPG4 and the products of hydrolysis were maltopentaose, maltotetraose, maltotriose, maltose and glucose.

科学研究費補助金 研究種目:総合研究(A) 課題番号:03303014 研究代表者:山根 隆 研究期間:1991-1992年度