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

Inibidores de alfa amilase de feijão (Phaseolus vulgaris) foram caracterizados. O inibidor da variedade Jalo apresentou peso molecular de 50kDa (por filtração em gel), ponto isoelétrico de 4,75 e 9,6% de carboidratos. O inibidor da variedade Argentino apresentou peso molecular de 48kDa, ponto isoelétrico de 4,90 e 7,6% de carboidratos. Ambos inibidores apresentaram pH ótimo de interação com alfa amilase pancreática de porco de 5,0 e, a pH 6,9, a complexação com a mesma enzima se deu na proporção de 1:1. Esses resultados, mais a composição de aminoácidos, indicaram que as características desses inibidores são semelhantes às de outros já purificados de diferentes variedades de feijão. As principais diferenças entre o IA do Jalo e do Argentino, foram observadas através de termogramas, do teor de carboidratos e dos ensaios imunológicos. Além disso, são sintetizados entre 20 e 30 dias após a floração, sendo que o processo ocorre simultaneamente ao da síntese das proteínas de reserva do grão. Inibidores purificados das variedades Jalo, Argentino e Rico 23, em diferentes fases do desenvolvimento, indicaram ausência de grandes alterações estruturais durante esse processo. / &#945;-amylase inhibitors of bean (Phaseolus vulgaris) were characterized. The amylase inhibitor from Jalo had molecular weight of 50 kDa (by gel filtration), an isoeletric point of 4.75 and a carbohydrate content of 9.6%. Argentino presented molecular weight of 48 kDa, an isoeletric point of 4.90 and a carbohydrate content of 7.6%. The optimum pH for inhibition of porcine pancreatic &#945;-amylase for both inhibitors was about 5.0 and they formed 1:1 stoichiometric complex. These results and the amino acid composition indicated that these inhibitors have characteristics similar to others already purified from beans. They were synthesized between 20 and 30 days after anthesis and this process occurred simultaneously to the storage proteins synthesis. Purified inhibitors from seeds in different phases of development indicated lack of great structural changes during this process.

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.

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.

The stress system comprises the hypothalamic-pituitary-adrenal (HPA) and the sympathetic-adrenal-medullary (SAM) axes. The two operate synergistically to regulate metabolic and biological processes, maintain homeostasis, and manage physiological responses towards various environmental challenges, known as stressors. Numerous studies have observed impaired HPA response among White breast cancer survivors followed by an eventual normalization of the HPA axis, but little is known about SAM functioning, the complementary relationship between physiological and psychological stress, and the influence of culturally related factors. This dissertation aims to address these gaps in the literature via three studies. In Study 1, we examined the diurnal and reactive patterns of salivary alpha-amylase, a SAM biomarker, in a sample of predominantly White women with and without a prior diagnosis of breast cancer. The purpose was to establish an initial understanding of SAM functioning in relation to the participants’ HPA activity as measured by cortisol responses (complementary to a previously published study in the laboratory). Results of Study 1 revealed no abnormal response to stress. Virtually identical alpha-amylase slopes were observed between breast cancer survivors and control participants, except that breast cancer survivors had elevated basal levels of alpha-amylase, thus suggesting a “heightened sympathetic tone”. In Study 2 acute and diurnal cortisol profiles and their accompanying psychological stress responses were examined in a sample of Chinese and White women with and without a prior diagnosis of breast cancer. In the same participants, we also examined chronic stress levels via hair cortisol concentrations which was the subject of Study 3. In both studies 2 and 3, we assessed the potential influences of cultural orientation and ethnocultural group membership on physiological and psychological stress patterns. For the most part, Study 2 supported previous findings from the literature and our laboratory: Breast cancer survivors displayed a blunted cortisol response and their diurnal profile was comparable to that of control participants. But contrary to our hypotheses, ethnocultural membership and cultural orientation did not influence physiological patterns of stress. However, an interaction between ethnocultural group, health status, and time (i.e., from baseline to one hour after stress induction) was observed in the subjective appraisal of an acute stressor. Specifically, White breast cancer survivors reported significantly lower levels of perceived stress than the other three groups. These results suggest that health-related stress may supersede the effects of culturally related stress and indicate the potential presence of posttraumatic growth among our sample of White breast cancer survivors only. Study 3 revealed no differences in hair cortisol concentrations between breast cancer survivors and control participants, nor between Chinese and White breast cancer survivors. However, it was observed that healthy Chinese women exhibited significantly higher levels of hair cortisol concentrations than their Western counterpart. Further analyses revealed that health status and cultural orientation did not significantly predict the observed patterns of physiological or psychological stress. Although non-significant, Study 3 results offered preliminary evidence that higher orientation towards both the dominant and non-dominant cultures is associated with higher levels of chronic physiological and psychological stress. Collectively, our studies may provide support for the long-term recovery of the HPA axis via the examination of acute, diurnal, and chronic patterns of cortisol, but further research will be required. The dissertation also highlights several important key points regarding culturally related factors, health, and stress: (1) The perception of stress is influenced by one’s degree of cultural orientation and (2) effects of health-related stress may supersede those of culturally related stress, but (3) among healthy women, ethnocultural minorities may have more stressful encounters than their Western counterpart, thus having important clinical implications for ethnocultural minorities who are newly diagnosed with a chronic condition. Together, results of the studies highlight the importance of further investigating the enduring and acute implications of psychosocial variables – particularly the influence of cultural orientation – on the experience and perception of stress.

Não consta resumo na publicação. / An &#945;-amylase inhibitor purified from black beans (Phaseolus vulgaris L.) prevents the utilization of starch either in normal or diabetic rats during short-time starch loading tests. It causes hypoglycaemia and alters accordingly the serum insulin and non esterified fatty acids levels. These effects are dose dependent and doses higher than 31 mg of the inhibitor can promote the complete smoothing on serum glucose level. The existence of starch in the presence of the inhibitor in the intestine doesn\'t alter the secretion or production of &#945;-amylase by the pancreas.

Streptococcus equi is the causative agent of strangles, a prevalent and highly contagious disease of horses. Despite the animal suffering and economic burden associated with strangles, little is known about the molecular basis of S. equi virulence. Here we have investigated the contributions of a specific lipoprotein and the general lipoprotein processing pathway to the abilities of S. equi to colonize equine epithelial tissues in vitro and to cause disease in both a mouse model and the natural host in vivo. Colonization of air interface organ cultures after they were inoculated with a mutant strain deficient in the maturase lipoprotein (prtM138-213, with a deletion of nucleotides 138 to 213) was significantly less than that for cultures infected with wild-type S. equi strain 4047 or a mutant strain that was unable to lipidate preprolipoproteins (lgt190-685). Moreover, mucus production was significantly greater in both wild-type-infected and lgt190-685-infected organ cultures. Both mutants were significantly attenuated compared with the wild-type strain in a mouse model of strangles, although 2 of 30 mice infected with the lgt190-685 mutant did still exhibit signs of disease. In contrast, only the prtM138-213 mutant was significantly attenuated in a pony infection study, with 0 of 5 infected ponies exhibiting pathological signs of strangles compared with 4 of 4 infected with the wild-type and 3 of 5 infected with the lgt190-685 mutant. We believe that this is the first study to evaluate the contribution of lipoproteins to the virulence of a gram-positive pathogen in its natural host. These data suggest that the PrtM lipoprotein is a potential vaccine candidate, and further investigation of its activity and its substrate(s) are warranted.

Proteins are important biological products with unique functions, annually produced at the hundreds of millions of dollars value on a worldwide basis. The application of crystallization for these materials primarily was led by structural biologists and crystallographers who are keen on obtaining large and well-ordered crystals for protein structure determination via X-ray diffraction. Usually for this, crystallization is done on a small scale by vapor diffusion using a supersaturated solution of the material. In the past decades, production crystallization has slowly received increasing attention for the large-scale recovery of proteins. Among the numerous products, an industrial enzyme (alpha-amylase) that is extensively involved in food processing and laundry products was chosen for examination due to the lack of relevant data in the literature and the potential industrial interest in crystallizing this material. The chosen alpha-amylase is a product of Genencor International (the Danisco division) and is derived from a genetically modified Bacillus licheniformis. In parallel to the underlying principles that govern the bulk crystallization of small molecules, the broad topics of investigation for this macromolecular material included determination of solubility, studies of nucleation thresholds, and investigation of crystal growth kinetics and special phenomena accompanying the crystallization process. All these studies were preceded by a series of characterization tests conducted for the material. On the whole, this study aimed to extend the existing fundamental knowledge of bulk crystallization for biological macromolecules. Specifically, it intended to enrich the solubility and crystallization kinetic data for the alpha-amylase. The experimental data of this study were all obtained at conditions in line with industrial practice, which included the use of moderate temperatures, mild pH conditions and simple inorganic salts ((NH4)2SO4, Na2SO4 and NaCl) in order for the findings to be transferred to the industry directly. In a 20 mM sodium phosphate buffer (with no added salts), alpha-amylase solubility increased with solvent temperature and had a minimum at pH between 6.4 and 7.1. A generalized equation (as a function of pH and temperature) was obtained to correlate the data. The three inorganic salts examined affected the alpha-amylase solubility in a different manner, both qualitatively and quantitatively. Evidently, the interaction effect of a salt varied with solution pH. This confirms the importance of studying solubility with the two or more condition parameters at the same time. With relevance to crystal growth, the metastable region of the material was relatively wide at (NH4)2SO4 and Na2SO4 concentrations corresponding to maximum solubility. For example, σSNT was 1.2  0.2 in solutions with 5 wt% ammonium sulfate at pH 7.0 and 25oC. A wide metastability range is useful for the practical operation of batch crystallizers as nucleation can be minimized. This range, however, diminished as the salt concentration increased beyond the maximum solubility points, imposing a limit on the range of salt concentration favorable for growth processes. In systems with no added salts at pH 7.0, the solution metastability was slightly higher at 10oC than at 40oC. This would suggest a future further examination of the salt system at a lower temperature, say of 10oC. To develop a batch crystallizer, the growth kinetic data of the material have to be known. Throughout the growth studies, the alpha-amylase crystals obtained were lozenge-shaped thin plates. Apparently, habit was not influenced by the crystallization conditions chosen. Similar to other proteins crystallized in bulk, the growth rate of alpha-amylase demonstrated a second-order dependence upon supersaturation. Importantly, the alpha-amylase demonstrated crystal growth rate dispersion (GRD) under all the conditions tested. To simplify the analysis of growth kinetic results, the seed crystals used were common history (CH) seeds whose growth rates are proportional to their sizes. The spread of growth rates (CV) was 0.54 for the unsieved CH seeds used. Due to GRD, growth rate coefficient data varied with crystal size. For instance, in solutions containing 5 wt% ammonium sulfate at pH 7.0 and 25oC, the growth rate coefficient for seed crystals initially at 20 m was 2.47 m/hr. This order of magnitude was equivalent to that of many other proteins. Although being small, industrial crystallization was feasible with these kinetics, as demonstrated by the sample design calculations included. To improve the design, it is recommended to further examine the solubility, metastability and growth kinetics of the above system at other temperatures to obtain a wider growth rate range. As the important phenomenon of growth rate dispersion has seldom been examined for protein and enzyme materials in the crystallization literature, this study is a significant contribution to this area.

Made available in DSpace on 2014-08-20T13:25:37Z (GMT). No. of bitstreams: 1 dissertacao_rafael_nornberg.pdf: 1775290 bytes, checksum: 1aed782bae5be322b9bc4cec8e20ade0 (MD5) Previous issue date: 2012-03-13 / The climate adversities, such as high temperatures and rain before and during the harvesting period lead to a phenomenon called Pre-harvest sprouting (PHS) of wheat grain. This germination decreases the wheat flour quality through the activation of enzymes responsible for germination which degrade starch. Therefore, this study had as objective to analyse the character pre-harvest sprouting in hexaploid wheat genotypes (Triticum aestivum L.), in the Southern region of Brazil, based on distinct evaluation years. Also, the evaluations aim to indicate the most adapted cultivars for Southern Brazil and to select parental genotypes for breeding programs of wheat. The experiment was conducted in the years 2010 and 2011, in the Centro de Genômica e Fitomelhoramento, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas (CGF/FAEM/UFPel), located at Capão do Leão County/RS, Brazil. The design used was random blocks with three replications. A total of 33 genotypes were phenotyped regarding agronomic and pre-harvest sprouting characters in wheat. Simultaneously, in 2010, 56 F1 and F2 hybrid populations obtained from artificial hybridizations between eight wheat cultivars, with different tolerance to sprouting according to a complete diallel design, with reciprocals. The populations and their parental genotypes were evaluated in the agricultural season of 2011. In this experiment, the following characters were evaluated: percentage of germinated grains (GG) in the fixed generations F1 and parents, falling number index (FNI) in the fixed generations F1 and parents, and segregant populations (F2). In the analysis of genotype x year, it was detected an interference of the environment on the results of some genotypes. The genotypes: BIO 08228, RAÍZES, HORIZONTE and BRS 194 with the lowest GG values, and the cultivar GUAMIRIM with high FNI, for both evaluation years, showing the presence of PHS tolerance alleles. Based on the genetic dissimilarity, was obtained from the prediction of important parents. Thus elite genotypes such as TBIO ITAIPÚ, BIO 08228, RAÍZES, HORIZONTE, BRS 194, TBIO PIONEIRO, BIO 07367, RAÍZES, GUAMIRIM and FRONTANA were obtained to form the crossing blocks, aiming to complement the distinct alleles for PHS. The majority of genotypes presented reduced alpha-amylase activity, which causes starch degradation. The genotypes TBIO PIONEIRO, MARFIM and TBIO TIBAGI, on the other hand, presented the lowest alpha-amylase activity. There was a high heritability for both characters in both years except for GG of susceptible genotypes in 2011. The PHS tolerance is controlled by additive effects of genes evidenced by high general combining ability and by non-additive effects of genes evidenced by the specific combining ability, but it was evident that the additive effects were more important. The cultivars QUARTZO and FRONTANA presented the best potential for the use in crossing blocks aiming to increase PHS tolerance and to reduce alpha-amylase activity. Transgressive segregation was observed with high heterosis estimates for both traits FNI and GG. This, added to the loss of vigor by inbreeding for FNI, this indicates that these combinations are potential sources of tolerance to PHS. There was a high reciprocal effect that should be taken into account when producing hybrid cultivars with tolerance to pre-harvest germination and high FNI. / As adversidades climáticas como temperaturas elevadas e precipitação antes e durante o período de colheita ocasionam o fenômeno da germinação na pré-colheita (Pre-harvest sprouting PHS) dos grãos de trigo. Tal condução deprecia a qualidade da farinha por meio da atividade de enzimas responsáveis pela germinação, que degradam o amido. Desta forma, o estudo teve o objetivo de analisar o comportamento do caráter germinação na pré-colheita em genótipos de trigo hexaplóide (Triticum aestivum L.), com base em distintos anos de avaliação, gerando informação para a melhor indicação de cultivares a região sul do Brasil e na reposição de combinações promissoras em blocos de cruzamentos em programas de melhoramento genético do trigo. O experimento foi conduzido nos anos de 2010 e 2011, no Centro de Genômica e Fitomelhoramento, Faculdade de Agronomia Eliseu Maciel, Universidade Federal de Pelotas (CGF/FAEM/UFPel), situado no município do Capão do Leão/RS, Brasil. O delineamento empregado foi de blocos ao acaso com três repetições, foram analisados 33 genótipos de trigo a partir de caracteres agronômicos e relacionados a germinação na pré-colheita do trigo. Simultaneamente, no ano de 2010 foram obtidas 56 populações híbridas F1 e F2 a partir de hibridações artificiais entre oito cultivares de trigo, com distinta tolerância a germinação na espiga, seguindo o delineamento dialélico completo, com os recíprocos. As populações e os respectivos genitores foram avaliados na safra agrícola de 2011 em campo experimental, no município de Capão do Leão, adotando o delineamento em blocos ao acaso, com três repetições. Neste experimento foram avaliados os caracteres: percentual de grãos germinados (GG) nas gerações fixas F1 e genitores, e índice de queda úmido (IQU), nas gerações fixas (F1 e genitores) e populações segregantes (F2). Na análise de interação genótipo x ano foi detectada interferência do ambiente sobre alguns genótipos. Os genótipos BIO 08228, RAÍZES, HORIZONTE e BRS 194 foram aqueles de menores valores de GG, e a cultivar GUAMIRIM com elevado IQ, para os dois anos de avaliação, demonstrando a presença de genes de tolerância a PHS. Com base nos resultados da dissimilaridade genética, foi obtida a predição de importantes genitores. Assim destacam-se TBIO ITAIPÚ, BIO 08228, RAÍZES, HORIZONTE, BRS 194, TBIO PIONEIRO, BIO 07367, RAÍZES, GUAMIRIM e FRONTANA para compor os blocos de cruzamento, visando à melhor complementaridade de alelos distintos de tolerância a PHS em trigo. No desempenho per se, a maioria dos genótipos apresentaram reduzida atividade de alfa-amilase que degradam o amido. Os genótipos TBIO PIONEIRO, MARFIM e TBIO TIBAGI, expressaram reduzida atividade da enzima alfa-amilase. Houve pronunciada herdabilidades para os caracteres em ambos os anos exceto para GG nos genótipos sensíveis em 2011. A tolerância a PHS é controlada por genes de efeitos aditivos evidenciados por elevadas capacidade geral de combinação (CGC) e por genes de efeitos não aditivos evidenciado pela capacidade especifica de combinação (CEC).Se destaca que os efeitos aditivos foram mais importantes sobre a variância genética total. Neste experimento, as cultivares QUARTZO e FRONTANA apresentam o melhor potencial para o uso em blocos de cruzamentos para incremento da tolerância a PHS e redução da atividade da enzima alfa-amilase. Foram evidenciadas constituições genéticas transgressivas em F2 associadas com elevadas estimativas de heterose em F1 para ambas as características avaliadas no IQU e GG. Isto, conciliado a baixa perda de vigor por endogamia para IQU, evidenciando que as populações destas combinações são potenciais fontes de recuperação de indivíduos superiores. Existe pronunciado efeito recíproco para o caráter tolerância a germinação na pré-colheita e aumento do índice de queda.