As dietas de vacas em lactação são compostas principalmente por carboidratos que não estão totalmente disponíveis para fermentação microbiana no rúmen, o que pode ser um fator crítico para a obtenção de energia em ruminantes. O objetivo do presente estudo foi avaliar o efeito da adição de enzima fibrolítica (Fibrozyme®, Alltech Inc., Springfield, KY) e amilolítica (Amaize, Alltech Inc., Springfield, KY) nas dietas sobre o consumo de nutrientes, índice de seleção de partículas, digestibilidade aparente total, fermentação ruminal, metabolismo de nutrientes, produção e composição do leite de vacas em lactação. Para o experimento, 32 vacas multíparas da raça Holandesa com 181.3 ± 35.3 (média ± SD) dias em lactação (DEL), 571 ± 72.7 kg de peso vivo (PV) e 29.6 ± 5.24 kg/d de produção de leite (PL), foram distribuídas em 8 quadrados Latinos 4×4. Os tratamentos foram obtidos por esquema fatorial 2×2 pela combinação de enzima fibrolítica e amilolítica, como segue: 1) Controle (CONT): dieta basal sem adição de enzimas exógenas; 2) Enzima fibrolítica (FIB): dieta basal com adição de 1 g/kg de Fibrozyme± no concentrado da dieta (51 UI de atividade xilanase/kg de matéria seca (MS) da dieta; Alltech, Nicholasville, KY, USA; batch#: 417990-2); 3) Enzima amilolítica (AMI): dieta basal com adição de 0,66 g/kg de Amaize no concentrado da dieta (203 FAU/kg MS da dieta; Alltech Nicholasville, KY, USA; batch: 432715-1); e 4) FIB+AMI: adição de 1 e 0,66 g/kg de Fibrozyme® e Amaize, respectivamente. As enzimas foram aplicadas para atender um consumo de 12 g/dia de Fibrozyme® e 8 g/dia de Amaize, de acordo com as recomendações do fabricante. A enzima foi adicionada ao concentrado durante a preparação na fábrica de ração (toda semana). Não foram observados efeitos das enzimas sobre o consumo e digestibilidade dos nutrientes. Contudo, houve efeito de interação entre FIB e AMI para o consumo de partículas entre 19 e 8 mm. A enzima amilolítica aumentou o consumo de partículas entre 19 e 8 mm nos animais que não recebiam FIB. Além disso, AMI reduziu o consumo de partículas maiores que 19 mm. Outro efeito observado foi a interação entre FIB e AMI sobre a concentração de butirato ruminal. A enzima amilolítica aumentou a concentração de butirato apenas nos animais tratados com FIB. Houve tendência de interação entre enzimas sobre a excreção de nitrogênio (N) no leite, em que FIB reduziu a excreção de N no leite apenas nos animais não tratados com AMI. Ainda, AMI reduziu a excreção de N na urina. Ainda, houve interação entre os efeitos de enzima fibrolítica e amilolítica sobre a concentração de colesterol e a atividade enzimática da aspartato aminotransferase (AST) e gama glutamiltransferase (GGT). A enzima fibrolítica reduziu a concentração de colesterol e aumentou a atividade da enzima GGT nos animais não tratados com enzima amilolítica. No entanto, a enzima amilolítica aumentou a concentração de colesterol e a atividade da AST nos animais alimentados com enzima fibrolítica. Houve interação entre enzimas exógenas sobre a produção de proteína e lactose no leite. A enzima fibrolítica reduziu a produção de proteína e lactose nos animais não alimentados com AMI. Não houve mudanças na produção de leite com a suplementação de enzimas exógenas. Em nosso estudo foram encontrados efeitos no consumo de partículas, fermentação ruminal, excreção de N e mudanças na composição do leite, contudo não foram observadas alterações no desempenho produtivo dos animais. / Lactating cows diets are comprised mostly of carbohydrates which are not fully available for microbial fermentation in the rumen, critical factor for to obtain energy in ruminants. The aimed of this study was to evaluate the effect of fibrolytic enzyme (Fibrozyme®, Alltech Inc., Springfield, KY) on nutrient intake, sorting index, total tract digestion, ruminal fermentation, nitrogen utilization, metabolic profile, milk yield and composition in diets with or without amylolytic enzyme (Amaize, Alltech Inc., Springfield, KY) of mid-lactating dairy cows. Thirty-two multiparous Hostein cows with 181.3 ± 35.3 (mean ± SD) days in milk (DIM), 571 ± 72.7 kg of body weight (BW) and 29.6 ± 5.24 kg/d of milk yield, were blocked and randomly allocated to a sequence of treatments in a 4 × 4 Latin square experimental design. Treatments were obtained in a 2 × 2 factorial arrangement, as follows: 1) Control (CONT), basal diet without exogenous enzymes; 2) Fibrolytic enzyme (FIB), provision of Fibrozyme® (batch#: 417990-2; Alltech, Nichollasvile, KY) at 1 g/kg of concentrate (51 IU of xylanase activity/kg diet DM); 3) Amylolytic enzyme (AMY), provision of AmaizeTM (batch#: 432715-1; Alltech) at 0.66 g/kg of concentrate (203 FAU/kg diet DM); and 4) Fibrolytic enzyme plus amylolytic enzyme (FIB+AMY), enzymes added at the same rate in FIB and AMY treatments. The enzymes were applied to meet the intake of 12 and 8 g/cow/day of Fibrozyme® and AmaizeTM, respectively. Enzymes products were added to concentrate during its preparation (once a week). Enzymes no effects on intake and digestibility of nutrients. However, there was FIB and AMY interaction effect on selection index of particle size between 19 and 8 mm. Amylolytic enzyme increase selection index of particles with 19 and 8 mm, only treatments without FIB. Furthermore, AMY decreased the sorting for feed with particle size greater than 19 mm. There was FIB and AMY interaction effect on butyric acid concentration. Amylolytic enzyme increased on butyrate concentrations in cows treated with fibrolytic enzyme. Fibrolytic and amylolytic enzyme interaction effect tended on N excreted in milk, in which FIB decrease N excreted in milk only on animals non-treated with AMY. Whereas AMY reduced urinary N excretion. There was interaction effects of fibrolytic and amylolytic enzymes on cholesterol concentration and the enzymatic activity of AST and GGT. The fibrolytic enzyme reduced cholesterol concentration and increased GGT enzyme activity in animals not treated with amylolytic enzyme. There was FIB and AMY interaction effect on lactose and protein production. Fibrolytic enzyme decreased lactose and protein production, only on animals non-treated with AMY. Exogenous enzymes had no impact on milk production of dairy cows. Enzymes exogenous affected on particle size greater, ruminal fermentation and milk composition. This study did not show evidences that fibrolytic and amylolytic enzymes can alter total tract nutrient digestibility and performance of mid-lactating cows.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-02T11:48:25Z No. of bitstreams: 1 2011 - Wellington Oliveira da Cruz.pdf: 7968929 bytes, checksum: 9dd68ee567386fa437d5f3438c9db428 (MD5) / Made available in DSpace on 2016-08-02T11:48:25Z (GMT). No. of bitstreams: 1 2011 - Wellington Oliveira da Cruz.pdf: 7968929 bytes, checksum: 9dd68ee567386fa437d5f3438c9db428 (MD5) Previous issue date: 2011-09-16 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior, CAPES. / CRUZ, Wellington Oliveira. Characterization of Alphitobius diaperinus (Coleoptera, Tenebrionidae) ?-amylase: Effect of Different Birds Diet and Inhibitors from Phaseolus vulgaris. 2011. 50 p Disserta??o (Mestrado em Qu?mica, ?rea de Concentra??o Bioqu?mica). Instituto de Ci?ncias Exatas, Departamento de Qu?mica, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. Alphitobius diaperinus known as mealworm of poultry litter, is one of the main poultry pests. It uses high protein and carbohydrates substrate of birds as a food source, colonizing all the production area finding a favorable environment for its proliferation. This insect causes sanitary and economic problems, affecting not only healthy and growth of the poultry but also acting as a microorganisms transmitter like bacterias, protozoas and virus. In this work it was identified an ?-amylase activity in crude extracts of A. diaperinus larvae and adults. This enzyme is responsible for starch degradation used as a energy source extremely important for survival of many insects. The ?-amylase was partially purified by ammonium sulfate precipitation (25-75%) in a chromatographic Sephadex column G (50-80), polyacrylamide gel revealed only one band with molecular weight of approximately 30 kDa, its purity was confirmed by the method of enzyme activity assay dinitrosalicylic 3.5-alkaline (DNS) was increased 7.2 times when compared with the crude extract. The ?-amylase assays presented a maximum activity at a pH 5.0 and pH 5.6 and an optimal activity temperature of 50 ? C. The maximum activity of ?-amylase A. diaperinus was achieved in the presence of 0.02 mM CaCl2, and inhibited by increasing this reaction concentration. The enzymatic activity of A. diaperinus ?-amylase was significantly inhibited by amylase inhibitor ?-AIs (lectin-like inhibitor) of Phaseolus vulgaris, suggesting that this inhibitors can be used as an important tool in biological control and development of these pests by reducing the digestion reducing amylase activity which plays an important role in the starch digestion / CRUZ, Wellington Oliveira. Caracteriza??o de ?-amilase de Alphitobius diaperinus (Coleoptera, Tenebrionidae): Efeito de Diferentes Ra??es de Aves e dos Inibidores de Phaseolus Vulgaris. 2011. 50 p Disserta??o (Mestrado em Qu?mica, ?rea de Concentra??o Bioqu?mica). Instituto de Ci?ncias Exatas, Departamento de Qu?mica, Universidade Federal Rural do Rio de Janeiro, Serop?dica, RJ, 2011. Alphitobius diaperinus, conhecido como cascudinho de cama avi?ria, ? uma das principais pragas av?colas. Ele utiliza o substrato das aves rico em prote?nas e carboidratos como fonte de alimento, colonizando toda a ?rea de produ??o, encontrando um ambiente favor?vel para sua prolifera??o. Este inseto causa problemas sanit?rios e econ?micos, afetando a sa?de e o crescimento das aves e atuando tamb?m como transmissor de microrganismos tais como baact?rias, protozo?rios e v?rus. Neste trabalho foi identificada a presen?a de uma ?-amilase no extrato bruto de larvas e adultos de Alphitobius diaperinus. Esta enzima digestiva ? respons?vel pela degrada??o de amido utilizado como combust?vel na obten??o de energia, sendo extremamente importante para sobreviv?ncia de muitos insetos. A ?-amilase foi parcialmente purificada por precipita??o em sulfato de am?nio (25-75 %) em coluna cromatogr?fica Sephadex G (50-80), revelando em gel de poliacrilamida uma ?nica banda com peso molecular de aproximadamente 30 KDa. Sua pureza enzim?tica foi confirmada atrav?s de ensaio de atividade pelo m?todo 3,5-dinitrosalic?lico alcalino (DNS), observando-se um aumento de 7.2 vezes em rela??o ao extrato bruto. A ?-amilase ensaiada apresentou uma atividade m?xima em pH 5.0 e pH 5.6 e uma temperatura de atividade ?tima de 50?C. A atividade m?xima da ?-amilase de A. diaperinus foi alcan?ada na presen?a de 0.02 mM de CaCl2, e inibida com o aumento da concentra??o deste reagente. A atividade enzim?tica da ?-amilase de A. diaperinus, foi significativamente inibida pelo inibidor ?-AIs, (inibidores tipo lectina) de Phaseolus vulgaris, sugerindo que estes podem ser utilizados como importantes ferramentas biol?gicas no controle e desenvolvimento de A. diaperinus atrav?s da redu??o da atividade digestiva destas pragas devido a atua??o desses inibidores sobre a a??o catalisadora de amilases, enzimas fundamentais para a digest?o de amido

O DNA de plasmídeos naturais de uma cepa de B. stearothermophilus foi clivado com a enzima de restrição Hind III e os fragmentos resultantes foram ligados com T 4 DNA ligase ao vetor pBR 322 (Bolívar et. al., 1977) que já havia sido previamente tratado com Hind III e fosfatase alcalina. A terça parte desta mistura de ligação foi usada para transformar células de E. coli HB 101. Foram obtidos cerca de 3.500 transformantes, dos quais 46% eram recombinantes. Duas cepas que mostraram caracter amilolítico consideravelmente maior que a doadora do gene continham o plasmídeo pBR 322 com uma inserção de 5.4 Kb. O mapa de restrição, tratamento com a enzima BAL 31 e, sucessivas subclonagens (Silva, E.F. et. al.,1986)mostraram que o gene que codifica e expressa a enzima amilolítica está contido em um fragmento de 2 Kb. A enzima produzida pelas células transformadas tem peso molecular de 60.000, é estabilizada por Ca+2, tem um ótimo de temperatura de 72ºC e retém 90% da atividade original após aquecimento a 85°C por 1 hora. Estes resultados, em conjunto com a análise dos produtos de hidrólise desta enzima em cromatografia de papel, sugerem que foi clonada a alfa - amilase de B. stearothermophilus em células de E. coli. Células de duas cêpas de B. subtilis, IA 289 e BD 241 foram transformadas respectivamente com os plasmídeos p USP 33.2 (Silva, E.F. et al., 1986;1987) e p BU 217 ami 2 (Silva, E.F. & Pueyo, M.T.,1988) para produzir em ambos os casos colônias fortemente amiloliticas. Os mecanismos pelos quais as 2 cêpas passaram a apresentar o fenótipo AMY + , são provavelmente diferentes. / The DNA of natural plasmids. from a B. stearothermophilus strain was cut with Hind III endonuclease and the resulting fragments were joined with T4 DNA ligase to the vector pBR 322 (Bolivar et al. , 1977), which had previously been treated with Hind III and alkaline phosphatase. One-third of the ligation mixture was used to transform E. coli HB 101 cells. It was obtained about 3.500 transformants, which included 46% recombinans. Two strains displayng amylolytic act ivity remarkably higher than the donor gene strain, harbored the plasmid pBR 322 with an insertion of 5.4 kb. The restriction map, Bal 31 treatment and successive subcloning (Silva, E.F, et al.,1986) showed that the entire gene which codifies and allows the expression of the amylolytic enzyme is contained in a 2 Kb fragment. The enzyme has a molecular weight of 60.000, is stabilized by Cata, has a temperature optimum at 72°C and retains 90% of the original activity after heating for 1h at 85°C. These features, together with the analysis of hydrolysis produts carried on paper chromatography , suggests that we succeded in cloning the amylase from B. stearothermophilus in E. coli cells. Cells from two B. subtilis strains, IQ 289 and BD 241 were transformed with the plasmid sp USP 33.2 (Silva E. F. et al., 1986 1987) and pBU 271 ami 2 (Silva, E. F. & Pueyo, M.T., 1988) , and produce in both strains, amyiolytic colonies. The methods in which the two strains have got the AMY + fenotype, may be very different.

Segundo a legislação brasileira, em uma cerveja, parte do malte de cevada pode ser substituído por cereais maltados ou não, e por carboidratos de origem vegetal, transformados ou não, conhecidos como adjuntos. Os adjuntos tem por finalidade contribuir como fonte alternativa de substrato, por geralmente terem preços inferiores ao malte de cevada e proporcionar à bebida características sensoriais peculiares. Este trabalho teve como objetivo utilizar a quirera de arroz preto (Oryza sativa L.), que não apresenta valor comercial, com o propósito de aumentar a contribuição de açúcares deste adjunto em 45 % no extrato de um mosto primitivo de uma cerveja. O arroz foi caracterizado em relação aos teores de amido total (83,20 % ± 1,41, b.s.) e suas frações, ao teor de metais e da temperatura de gelificação (78,68 °C). A ?-amilase termoestável utilizada no processo também foi caracterizada em relação ao teor de proteínas (20,5 ± 1,2 mg de proteína/mL de extrato), atividade amilásica (112 000 UI/mL de extrato) e atividade específica (5 463 UI/mg de proteína). Foram otimizadas as condições de hidrólise em relação ao tempo de processamento e concentração da enzima, segundo um planejamento experimental fatorial completo 22 com três pontos centrais e estudo rotacionado. A levedura (Saflager S-23) utilizada no processo foi selecionada por apresentar melhor desempenho fermentativo dentre 4 cepas avaliadas. Os ensaios de fermentação foram executados em escala de bancada e ampliados para a escala piloto, na qual foi obtida excelente eficiência de mosturação (73,71 % ± 4,69). A cerveja obtida foi avaliada do ponto de vista sensorial e físico-químico. O processo apresentou bom rendimento fermentativo (0,37 ± 0,04 g/g), eficiência de fermentação (72,48 % ± 7,61) e produtividade em álcool (0,32 g.L-1.h-1± 0,02). Concluiu-se que o processo pode resultar num produto com bom rendimento e características sensoriais muito intensas e agradáveis, similar a uma cerveja forte, consumida tipicamente durante o inverno. / As cited on Brazilian legislation, in a beer, part of the malt can be substituted by malted or non-malted cereals, and by carbohydrates from vegetal sources, called adjuncts. Adjuncts have the purpose of contributing as an altenative and cheaper source of sugars, when compared to barley malt, and provide peculiar sensory characteristics. This work aimed to use the black rice (Oryza sativa L.) grits, which does not have economic value, and use this raw material on the proportion of 45% on the primitive extract of a beer. On black rice, it was quantified starch (83,20 % ± 1,41, d.b.) and metal content, and measured the gelatinization temperature (78,68 °C). The enzyme (Brautec Alfa-TF) used in this work was characterized on protein content (20,5 ± 1,2 mg protein/mL de enzyme extract), activity (112000 IU/mL of extract) and specific activity (5463 IU/mg of protein). The enzymatic hydrolysis conditions were optimized using a full factorial 22 central composite design with star points aiming at it the sugar concentrations response on hydrolysate regarding the factors: enzyme concentration and processing time at 95 °C. The yeast strain was selected among 4 strains regarding the factors: alcohol yield (g/g), fermentation efficiency (%) and alcohol productivity (g.L-1.h-1). Assays were carried out on benchtop scale and scaled up to pilot scale, and showed excellent mashing efficiency (73,71 % ± 4,69). Beer was evaluated physico-chemically and sensorially and showed a good alcohol yield (0,37 ± 0,04 g/g), fermentation efficiency (72,48 % ± 7,61) and alcohol productivity (0,32 g.L-1.h-1 ± 0,02). It could be concluded that this process can result in product with good yield and very intense and pleasant sensory characteristics, similar to stronger beers, tipically consummed during the winter.

Pyrococcus furiosus extracellular &alpha / -amylase is a hyperthermostable glucosyl hydrolyzing enzyme which shows unique biochemical properties that may have impact on improving starch hydrolysis process / however, it is insignificantly expressed in its native archaeal host. In this study, it was aimed to express the P. furiosus extracellular alpha-amylase (PFA) in Pichia pastoris, which is a well-recognized overexpression host used in production of heterologous proteins. In this context, first, P. furiosus was grown under anaerobic conditions in capped bottles for t= 12 h at T=90&deg / C and then its genomic DNA was isolated. PFA coding cDNA frame was amplified using two specifically designed oligonucleotides and cloned into pPICZ&alpha / A expression vector. Then wild type P. pastoris X-33 cells were transfected with pPICZ&alpha / A::PFA construct. In shake flask production medium, existence of recombinant PFA activity was tested and biochemical characterization of the recombinant product was done. This was the first time PFA is expressed in an eukaryotic host. Optimum working temperature and pH of the rPFA were found to be 95 &deg / C and within the range of 4.5-6.5, respectively. rPFA is independent to metal ions and inhibition by production medium of P. pastoris was observed, in presence of divalent metal ions. Although Saccharomyces cerevisiae &alpha / -factor secretion signal was fused to the N terminal of rPFA, minute amount of extracellular secretion was detected but the majority of the enzymatic activity remained in the intracellular medium. The best producer strain was selected by measuring &alpha / -amylase activity in cell extracts by DNS method. Effects of pH on cell growth and recombinant protein production were determined by shake flask experiments and maximum of 4800 U/l rPFA was detected with 7.30 g/l wet cell density in pH=6 buffered medium. In order to achieve higher rPFA production, two bioreactor experiments were designed at two different pH operation conditions, namely pH=4 and pH=5, in a working volume of 1 L. The dissolved oxygen tension was kept over 20% and predetermined exponential methanol feeding strategy was employed in order to fix specific cell growth rate, &micro / , at 0.03 h-1. At pH=4 operation, maximum of 73,400 U/l &alpha / -amylase activity was detected at the t=27 h of production phase when the wet cell density was 209 g/l.

Diagnosis of acute pancreatitis (AP) is erroneous in up to one third of patients when based on clinical criteria and elevated serum amylase values. Furthermore, according to autopsy reports fatal pancreatitis remains clinically undiagnosed in 22 to 86 % of hospitalised patients. Consequently, search for better methods for the diagnosis of AP seems not only justified but urgent. The pancreas secretes an nonspecific lipase, the carboxylic ester hydrolase (CEH) with molecular properties different from other pancreatic secretory enzymes. These differences may imply that sites and rates of clearances from blood of pancreatic enzymes differ. Except for the pancreas this enzyme is secreted from the lactating mammary gland with milk. A sensitive and reproducible sandwich-ELISA for quantitative determination of CEH was developed. When establishing referent values it was noted that in individuals aged 20 to 65 years serum concentrations of CEH did not depend on age, gender, the time of the day or duration from food intake to blood sampling, or use of nicotine. The mammary gland did not contribute significantly to basal serum levels of CEH; enzyme levels in lactating women or women with mammary tumours were identical to those of the reference population. Seventy percent of patients with the diagnosis AP, based on elevated serum amylase levels and abdominal pain, had elevated CEH values. Among the patients with elevated amylase alone a probable cause of pancreatitis was lacking in the majority of patients. Contrastingly, a likely cause of AP could be identified in all patients presenting with abdominal pain and elevated CEH levels alone. These findings suggested that an elevated CEH level indicated AP more reliably than an elevated amylase level. In patients with AP diagnosed by contrast enhanced computed tomography (CECT) alone, or combined with histopathological diagnosis, serum CEH levels were elevated on admission in all but one patient, and in all within the next 24 h. Furthermore, in patients with severe pancreatitis CEH levels remained at a raised level from the second to at least the 10:th day following admission, whereas a significant decrease was noted in patients with mild pancreatitis. In contrast, serum amylase values were higher in patients with mild pancreatitis during the observation period than in those with severe pancreatitis. CEH levels were higher in patients with three or more Ranson signs than in those with less than three signs from the first day after admission. CEH levels were within referent range in 164 patients without known pancreatic disease admitted due to abdominal emergency conditions, or due to planned surgery for chronic extrapancreatic gastrointestinal diseases, and 16 patients having CECT without pathological findings in the pancreas. This suggests that AP can be excluded with very high degree of probability in presence of non-elevated CEH levels. A sandwich ELISA for determination of Guinea pig CEH and a model for graded pancreatitis in the same species were developed. CEH levels showed proportional to severity of inflammation, thus confirming previous clinical observations. CEH levels in bile were proportional to inflammation, while it was absent in urine. Amylase levels in urine were identical regardless of severity of inflammation, but low in bile. These results suggested differences in sites and rates of clearance between the two enzymes. Seemingly elevated CEH levels allowed identification of clinically significant pancreatitis following ERCP, which amylase levels did not. The presented studies have shown that quantitative determination in serum of CEH by the described method is a more reliable test for the diagnosis of AP than determination of amylase activity. The differences between CEH and amylase are, at least partly, due to differences in molecular properties determining rates and routes of clearances of the two enzymes from serum. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser.</p> / digitalisering@umu.se

Les enzymes pancréatiques de remplacement sont les plus utilisées dans le but d'améliorer la qualité de vie des personnes ayant un problème d'insuffisance pancréatique exocrine (IPE) relié aux maladies comme la pancréatite, la fibrose kystique et la stéatorrhée. Les enzymes pancréatiques de remplacement ont différentes origines et les plus utilisées proviennent du pancréas d'origine porcine (EPP), étudiées dans le présent travail. Dans le commerce on peut trouver des comprimés d'enzymes avec enrobages entériques. En effet, il y a plusieurs formes commerciales d'enzymes pancréatiques disponibles sur le marché et telles compositions peuvent être résistantes au milieu gastrique, mais le profil de libération n'est pas toujours satisfaisant. Comme un exemple, chez les patients atteints d'IPE la région supérieure du petit intestin est généralement acide ce qui a comme résultat que les préparations entériques normalement soient dissoutes trop tard dans l'intestin ce qui rendre indisponibles les enzymes dans le site approprié. Suite à la découverte de la capacité des comprimés faits d'enzymes pancréatiques de former une couche externe en milieu acide (FGS), l'objectif du présent travail a été d'élaborer des comprimés d'enzymes sans excipients additionnés et sans enrobage, capables de se protéger dans un milieu gastrique et de libérer son contenu dans l'intestin. Il semble que les enzymes ont une plus grande force de cohésion inter particulaire après leur compression et peuvent aussi être auto-assemblés en comprimés monolithiques. L'auto-assemblage est le résultat de plusieurs sortes d'interactions entre les chaines de protéines, comme les associations d'hydrogène (ex : entre lysine, histidine, tyrosine et sérine) et les interactions ioniques (ex : entre les -COO- et NH3+ impliquant glutamate-lysine et aspartate-lysine). Dans les comprimés obtenus, les protéines peuvent agir comme des tampons et de cette manière augmentent la stabilité gastrique. Dans le milieu intestinal, les groupes carboxyliques sont déprotonés et ionisés provoquant aussi l'hydratation, l'érosion et la désintégration des comprimés et la libération des enzymes thérapeutiques. Des études de stabilisation et de dissolution des comprimés d'EPP (lipase, amylase et protéase) ont été faites dans des milieux FGS et FIS. Suite aux résultats prometteurs du brevet « Compositions pharmaceutiques gastro-résistantes à base d'enzymes », le travail est approfondi sur des aspects structuraux en utilisant une préparation commerciale d'alpha-amylase. Les profiles FTIR et SDS/PAGE ont été utilisés pour étudier les interactions protéine-protéine dans des comprimés d'alpha-amylase et l'effet de la pression et du milieu gastrique sur la structure de cette enzyme. La nouveauté du concept est que les enzymes pancréatiques en plus d'être les principes actifs, agissent comme liants et comme agents chargés de générer et stabiliser une couche externe sensible au pH. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : enzyme, gastro-résistance, comprimés, pancréas, FTIR, alpha-amylase, lipase, protéase.

In this study, 11 bacterial isolates from Salt Lake,Turkey were identified by using fatty acid methyl ester (FAME) analysis. They were screened for production of industrially important enzymes xylanase, cellulase, &amp / #945 / -amylase and protease. These enzymes were characterized in terms of enzyme activity, stability, optimum temperature and optimum pH. One of the isolates was identified as Bacillus pumilus, and two of them were identified as Bacillus subtilis. Other isolates were determined to be Bacillus licheniformis. All the isolates were determined to produce xylanase. Optimum temperatures and optimum pH values of xylanases were 50-55 &deg / C and pH 7.0-8.0. Xylanases were quite stable up to pH 8.0 and 70 &deg / C. Isolates were not significant cellulase producers. Four of the isolates did not produce any cellulase enzyme and the rest produced negligible amounts of cellulase. Therefore, xylanases from the isolates were promising for pulp and paper industry, which requires cellulase free and stable xylanases. All the isolates produced appreciable quantities of &amp / #945 / -amylase. Optimum temperatures and optimum pH values of &amp / #945 / -amylases 60-80 &deg / C and pH 7.0-8.0. &amp / #945 / -Amylases were quite stable up to pH 9.0 and 80 &deg / C. &amp / #945 / -Amylases from the isolates were promising for starch processing industry, which requires &amp / #945 / -amylases stable at high temperatures and for detergent industry, which requires &amp / #945 / -amylases stable at alkaline pH values. Considerable protease productions were achieved by all the isolates. TTG 2 was the best protease producer with 271 U/ml. Optimum temperatures and optimum pH values of proteases were 50-60 &deg / C and pH 7.0-7.4. Proteases were quite stable up to pH 9.0 and 80 &deg / C. Proteases from the isolates were promising for detergent and leather industry, in which proteases must be stable at alkaline pH values.

Under certain environmental conditions, some varieties of wheat synthesize the enzyme alpha amylase late in grain ripening, even in the absence of rain or sprouting. The resulting grain has a sound appearance but can be unsuitable for end-product applications due to the presence of late maturity alpha amylase (LMA) activity. Reduction of LMA and the development of cultivars tolerant to boron toxic soils are high priority traits in the WA wheat breeding program and the use of molecular markers closely linked to these traits for marker assisted selection (MAS) is highly desirable. The aims of this study were to take a genomics approach to provide detailed structural information for the region on wheat chromosome 7BL in which quantitative trait loci (QTLs) for LMA and boron tolerance (Bo1) have been mapped. Once the structure had been determined, this then laid the foundation for further studies to investigate the function of putative candidate genes identified within this region. The research involved the use of bioinformatic tools and rice/wheat synteny to investigate the structure of this chromosome region, followed by the use of molecular probes to isolate genomic DNA clones (BAC clones) corresponding to this region. A two-step bioinformatics strategy was used, involving (1) alignment of portions of the wheat and rice genomes, to identify rice genomic regions syntenic to wheat group 7L and (2) selection of candidate genes from those regions of the rice genome. The selected candidate genes included an anion transporter, as a candidate gene for boron tolerance, and GAMYB-like genes, as candidate genes for LMA. The GAMYB class of transcription factors identified were of particular interest because of published literature indicating its importance in controlling ƒÑ-amylase levels in cereal grains. The key phenotype of interest in this thesis is LMA and different levels of expression of ƒÑ-amylase are a key feature of this phenotype. Molecular markers and candidate genes were then used to screen two BAC libraries, one derived from the French cultivar, ¡¥Renan¡¦ and the other derived from Aegilops tauschii (the source of the D genome of wheat). About 300 BAC clones corresponding to the chromosome region of interest were obtained. Of these, 8 BAC clones (6 chosen through hybridization to a GAMYB-like probe, and 2 from wheat ESTs anchored to the rice genome) were selected for sequencing, allowing for the development of new microsatellite and single-nucleotide polymorphism (SNP) markers and for the discovery of novel transposable elements that provide a rich source of polymorphism for the development of additional markers. Novel microsatellite and SNP markers that were identified from the BAC clone sequence were mapped on the Cranbrook/Halberd doubled haploid (DH) mapping population. Markers were located to chromosomes 7AL, 7BL and 7DL. New markers derived from the BAC sequence information were used to anchor the BAC clones to the genetic map and develop a framework physical-genetic map. An automated annotation pipeline has been established and was used to annotate selected contigs of the sequenced BAC clones. A new marker assisted selection strategy, termed Multiplex Trait Signature (MuTs) analysis, was developed and tested on 39 wheat cultivars of known LMA phenotype. MuTs provides a graphical genotype of individuals for a particular chromosomal region and is a convenient tool for interrogating genetic similarity in the individuals surveyed. Based on assays of 22 markers (12 spanning the LMA QTL on chromosome 7BL and 10 spanning the LMA QTL on chromosome 3BS) on these 39 wheat cultivars, it was found that the varieties can be grouped according to pedigree and provides a tool for interpreting LMA status for a variety. Validation of the 7BL LMA and boron tolerance (Bo1) QTL regions was achieved using a targeted mapping approach using the doubled haploid population Pastor/RAC891 using published molecular markers and markers developed in this thesis. The main outcome of this study is that the genomic organisation of this region on chromosome 7BL is complex, and that the identification of candidate genes in wheat controlling 1) tolerance of cultivars to boron toxic soils and 2) pathways regulating the expression of LMA, is likely to involve the interplay of a network of regulatory genes.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 6 uppsatser.