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No. of bitstreams: 1 PDF - Géssica Laize Berto Gomes.pdf: 960450 bytes, checksum: 01f35541007decb425986338cf77d27a (MD5) Previous issue date: 2014-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Brazilian production of grains has grown every year, transforming Brazil in one of the main cereals and oilseeds producers. However, the post-harvest losses are huge, causing production loss by the inadequacy of storage, especially in order of pest occurrence. In the case of sesame, there has been a marked occurrence of P. interpuctella, which attack stored grains accompanied by other pests causing economic damage to the crop. With this, the Breeding Program Sesame Embrapa Cotton seeks alternatives to control these pests by genotypes that express high levels of protein inhibitors (PIs) with ability to inhibit proteolytic digestive enzymes of insects. The objective of this study was to detect trypsin inhibitors in genotypes of sesame (S. indicum L.) with potential use in biotechnological applications and programs to improve the species, aiming selection of genotypes resistant to stored grain pests, so we proceeded to study PIs in 10 sesame genotypes belonging to the Active Germplasm Bank at Embrapa Cotton. The total protein extracts of accessions were extracted and tested in vitro with bovine pancreatic trypsin (BPT) and intestinal homogenate of larvae of P. interpuctella. After selection of the most promising genotypes, we proceeded to a protein fractionation with ammonium sulfate. The protein fractions obtained were analyzed for inhibitory activity with BPT and intestinal homogenate of larvae of P. interpuctella, and yet, as the thermal stability and hydrogen ion and bioassays with P. interpuctella. Data were analyzed by Tukey test at 5% of probability. The results showed the presence of trypsin inhibitors in the total protein extracts of 10 sesame genotypes, ranging from 51% to 90% inhibition, among which, the BRS-SEDA, CNPA G3 and CNPA G4 stood out with 87%, 88% and 90% inhibition, respectively. These genotypes inhibitory activity have been demonstrated for the intestinal homogenate of larvae of P. interpuctella, with inhibition between 60% and 76%. The fractions F2 of the three genotypes were relatively stable to changes in temperature and pH. In the bioassay, the larvae of P. interpuctella. were susceptible to protein extracts of the three genotypes of sesame, with a maximum of 83% mortality of larvae in different strengths with total protein extracts and 58% in fractions F2, however, did not observe statistically a significant difference between genotypes. In this regard, BRS-SEDA, CNPA G3 and CNPA G4 have protein inhibitors that can prevent the development of P. interpuctella, suggesting candidates with potent insecticidal properties to control stored grain pests. / A produção brasileira de grãos tem crescido a cada ano levando o Brasil a um dos principais produtores de cereais e oleaginosas. No entanto, as perdas na pós-colheita são enormes provocando uma redução da produção em função do controle inadequado no armazenamento, especialmente em função da ocorrência de pragas. No caso do gergelim, tem havido uma ocorrência acentuada de P. interpuctella, que ataca os grãos armazenados acompanhado de outras pragas, causando danos econômicos à cultura. Com isto, o Programa de Melhoramento Genético de Gergelim da Embrapa Algodão busca alternativas para controlar essas pragas, por meio de genótipos que expressem altos níveis de inibidores proteicos (IPs) com capacidade de inibir enzimas proteolíticas digestivas de insetos. Objetivou-se com este trabalho detectar inibidores de tripsina em genótipos de gergelim (Sesamum indicum L.) com potencial uso na aplicação biotecnológica e no programa de melhoramento da espécie, visando seleção de genótipos resistentes a pragas de grãos armazenados. Assim, procedeu-se o estudo de IPs em 10 genótipos de gergelim, pertencentes ao Banco Ativo de Germoplasma da Embrapa Algodão. Os extratos proteicos totais dos acessos foram extraídos de sementes de gergelim e testados in vitro com tripsina pancreática bovina (TPB) e homogenato intestinal de larvas de P. interpuctella. Após seleção dos genótipos mais promissores, procedeu-se um fracionamento proteico com sulfato de amônia. As frações proteicas obtidas foram analisadas quanto a atividade inibitória com TPB e homogenato intestinal de larvas de P. interpuctella, e ainda, quanto a estabilidade térmica e hidrogeniônica e em bioensaios com P. interpuctella. Os dados foram analisados pelo teste de Tukey a 5% de probalibilidade. Os resultados obtidos mostraram a presença de inibidores de tripsina nos extratos proteicos totais dos 10 genótipos de gergelim, com variação de 51% a 90% de inibição, dentre os quais, o BRS-SEDA, CNPA G3 e CNPA G4 se destacaram com 87%, 88% e 90% de inibição, respectivamente. Nesses genótipos foram evidenciadas atividade inibitória para o homogenato intestinal de larvas de P. interpuctella, com inibição entre 60% e 76%. As frações F2 dos três genótipos foram relativamente estáveis às variações de temperatura e pH. No ensaio biológico, as larvas de P interpuctella mostraram-se suscetíveis aos extratos proteicos dos três genótipos de gergelim, com mortalidade máxima de 83% das larvas em diferentes dosagens com extratos proteicos totais e 58% nas frações F2, contudo, estatisticamente não observou-se diferença significativa entre os genótipos. Neste aspecto, os genótipos BRS-SEDA, CNPA G3 e CNPA G4 possuem inibidores proteicos capazes de impedir o desenvolvimento P. interpuctella, o que sugere potentes candidatos com propriedades inseticidas no controle de pragas de grãos armazenados.

No description available.

Soybean production and consumption has increased in recent decades. However, trypsin inhibitor activity and “beany” flavour are two drawbacks limiting the utilization of soybean. In the present study, micronization, ethanol washing, and enzymatic hydrolysis (alone or in combination) were used to treat soybean. Micronization at 100 °C and 135 °C decreased the activity of both trypsin inhibitors (53% and 80% respectively), and lipoxygenase (51% and 99%, respectively). Ethanol increased the trypsin inhibitor activity while alcalase hydrolysis decreased its activity. Different treatment combinations affected trypsin inhibitor activity, with micronization having a major influence. “Beany” flavour related volatiles (hexanal, (E)- 2-hexenal, 1-hexanol, heptanal, (E)-2-octenal, (E)-2-nonenal, (E,E)-2,4-nonadienal, 2,4-decadienal, (E,E)-2,4-decadienal, 1-octen-3-ol, 2-pentylfuran and 3-octen-2-one) were significantly decreased with micronization. Ethanol effects varied with different volatiles. Soybean micronized at 135°C and washed with 65% ethanol was recommended for soybean processing due to its low trypsin inhibitor activity and low “beany” related volatile content.

<p>For this dissertation, four experiments were conducted to evaluate the effect of dietary addition of exogenous protease and amylase enzymes on growth performance and nutrient utilization in broiler chickens. An additional fifth experiment was designed to determine the role of central and peripheral appetite regulators in birds fed diets deficient in dietary phosphorus (P). This arose from consistent reports in literature of a direct effect of dietary P concentration on feeding response in broiler chickens. </p><p>Experiment 1 examined the growth performance and protein utilization responses of broiler chickens to purified trypsin inhibitors (TI) and exogenous protease additions. Experimental diets were arranged as a 2 × 2 factorial with factors being dietary TI (1,033 or 10,033 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). Protease supplementation improved BW gain (<i>P < </i>0.01) and gain to feed ratio (<i>P < </i>0.05) of birds. The relative weight of pancreas increased (<i>P < </i>0.05) with added TI on d 14 and 21 but was reduced (<i>P < </i>0.001) with protease supplementation. Apparent ileal digestibility (AID) of all amino acids (AA), except methionine, decreased (<i>P < </i>0.001) with added TI, but increased (<i>P < </i>0.05) with protease supplementation. Duodenal trypsin and chymotrypsin activities were reduced (<i>P < </i>0.05) with added TI but increased <i>(P < </i>0.01) with protease supplementation. It was concluded that dietary addition of purified TI negatively affects nutrient utilization by broiler chickens and that the efficacy of the exogenous protease might be independent of dietary TI concentration. A follow-up experiment was conducted (Experiment 2) to evaluate the impact of TI and exogenous protease supplementation on endogenous AA loss in broiler chickens. Four diets were arranged as a 2 × 2 factorial with factors being dietary TI (0 or 8,000 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). There was no effect of TI, exogenous protease, or their interaction on growth performance of birds. Endogenous nitrogen (N) loss and all AA (except Cys) increased (P < 0.05) due to added dietary TI. Exogenous protease had no effect on endogenous loss of N and all AA. The AID of Ca, Fe, Mg, Mn, and Cu was reduced (P < 0.05) by added dietary TI. Protease supplementation improved the AID of Cu (P < 0.01) and K (P < 0.05). Secretion of crude mucin and sialic acid (g/kg DM intake) increased (P < 0.05) with increased dietary TI and was not recovered by protease supplementation. It was concluded from this study that TI increases the endogenous loss of AA, reduces the digestibility of minerals in broiler chickens, and that exogenous protease had no effect on endogenous AA flow, irrespective of added dietary TI. </p><p>In Experiment 3, the responses of broiler chickens fed corn-soybean meal-based diets to dietary α-amylase supplementation during 4 growth phases were evaluated. Birds were assigned to 8 treatment diet in a 2 × 4 factorial arrangement of 2 dietary levels of α-amylase supplementation (0 or 80 kilo-Novo alpha amylase units (KNU) per kg diet) and 4 post hatching growth phases (d 0 to 11, d 11 to 21, d 21 to 42, or d 42 to 56). Body weight gain and feed efficiency of birds improved (<i>P</i> < 0.01) with α-amylase supplementation. There were main effects of α-amylase, growth phase and interaction (<i>P</i> < 0.01) on AID of starch. The total tract retention (TTR) of starch increased (<i>P</i> < 0.05) with amylase supplementation but was not different across growth phases. Amylase supplementation improved (<i>P</i> < 0.05) gross energy utilization in birds, and specifically, during d 11 to 21 post hatching, the viscosity of jejunal digesta and pancreatic amylase activity increased (<i>P</i> < 0.01) with amylase supplementation. The conclusion from the study was that the growth phase of birds may affect the response to exogenous amylase. Following the result of this study, Experiment 4 was conducted to evaluate the effect of amylase supplementation on starch and energy digestibility at various intestinal sites in broiler chickens. Experimental diets comprised 3 concentrations of α-amylase supplementation (0, 80, or 160 KNU/kg diet) and sampling was done on 4 intestinal sites: anterior jejunum (AJ), posterior jejunum (PJ), anterior ileum (AI) and posterior ileum (PI). There were linear and quadratic (<i>P</i> < 0.01) responses of increasing α- amylase supplementation on starch and energy digestibility at the PJ and AI, with only linear effects on TTR of starch (<i>P</i> < 0.05). A linear increase in starch disappearance and digestible energy (kcal/kg) was observed (<i>P</i> < 0.01) with digesta flow from AJ to PJ with increasing amylase supplementation, which may be related to the observed decrease in the viscosity of the jejunal digesta (<i>P</i> < 0.05). Results from this experiment demonstrate the efficacy of exogenous amylase to improve starch, and energy digestibility in broiler chickens, with the highest impact observed in the posterior jejunum.</p><p>A final study (Experiment 5) was conducted to evaluate the impact of dietary phosphorus (P) concentration on hypothalamic molecular regulation of appetite by broiler chickens. Birds were randomly assigned to 3 experimental diets which contained 1.2 (P-deficient), 2.8 (P-marginal) or 4.4 (P-adequate) g/kg non-phytate P (nPP). A decrease in feed intake and BW gain was observed (P < 0.001) in birds fed the P-deficient diet. There was upregulation (P < 0.05) in the mRNA expression of Sodium-phosphate cotransporter (NaPi-IIb), anorexia-related hypothalamic cholecystokinin receptor (CCKAR) and melanocortin receptors (MC3R and MC4R) in birds fed P-deficient diets, whereas cholecystokinin (CCK) mRNA was downregulated (P < 0.01). It may be concluded that a deficiency in dietary P decreases feed intake in broiler chickens by altering the expression of anorexigenic genes in the gut and hypothalamus.</p>

Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-10T13:05:43Z No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) / Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-04-10T13:06:06Z (GMT) No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) / Made available in DSpace on 2018-04-10T13:06:06Z (GMT). No. of bitstreams: 1 FabioCorreiaCarneiroDissertacao2018.pdf: 2306587 bytes, checksum: ed2572dbb41a30ee1c54682a411c833d (MD5) Previous issue date: 2018-02-20 / Protease inhibitors have a broad biotechnological application, which goes since the development of several drugs to your utilization as a bioinsecticide, antifungal and as an antibacterial agent. However, those are found in small quantities in their natural sources, which unfeasible it utilization in industrial scale. Therefore, the heterologous production ends up as a method that allows the increase of scale production of those proteins. The Inga laurina Trypsin Inhibitor (ILTI) previously characterized showed an inhibitory effect in proteases extracted from the midgut of insects, besides reducing its larval developments by up to 84%, therefore, boing a candidate to be used as a potential bioinsecticide. Thus, the present work aimed at the heterologous production of ILTI in Komagataella phaffii (Pichia pastoris), followed by the optimization of the culture modes in a bioreactor with the objective of maximizing the production of the recombinant protein. For this, the gene that codifies ILTI were cloned in the expression vector pPIC9K, followed by your insertion on the strain GS115 by electroporation. PCR analysis showed that the recombinant vector was integrated into the genome of the yeast, and all the clones obtained had MutS genotype. The expression was performed for 96 hours by adding 0.5% methanol. An analysis of the proteins on the supernatant of the recombinant strain culture, by SDS-PAGE, confirmed the production of a protein with a size close to 20 KDa. Data from MALDI-TOF confirmed that the obtained protein is, in fact, the recombinant ILTI. Furthermore, inhibitory assays showed that the produced protein had activity against trypsin. Thus, culture in bioreactors was performed to optimize the production of this heterologous protein. In order to increase its expression, fed-batch was performed where on the batch phase the biomass production was favored, and the feeding phase was programmed to continuously supply methanol, based on the methanol specific consumption and its specific growth velocity, using methanol as carbon source. During the fermentations, 351.27 UIT were obtained in the extract of crude fermentation broth, and a specific activity of 2.07 UIT/mg protein. Although widely used as a host for the production of heterologous proteins, studies of the production of protease inhibitors in K. phaffii are still very limited. Until the moment, there is no report in the optimization of the production of serine protease inhibitors in K. phaffii, making this study pioneering and essential for the beginning of scaling up the process of this technology. / Os inibidores de protease possuem uma ampla aplica????o biotecnol??gica que vai desde o desenvolvimento de diversos f??rmacos at?? sua utiliza????o como bioinseticidas, antif??ngicos e como agentes antibacterianos. Por??m, estes s??o encontrados em pequenas quantidades nas suas fontes naturais, o que inviabiliza sua utiliza????o em escala industrial. Sendo assim, a produ????o heter??loga acaba sendo um m??todo que permite o aumento da escala de produ????o dessas prote??nas. O inibidor de tripsina de Inga laurina (ILTI) foi caracterizado previamente e mostrou possuir efeito inibit??rio em proteases extra??das do trato digestivo de insetos, al??m de diminuir em at?? 84% seu desenvolvimento larval, sendo, portanto, um candidato a ser utilizado como um poss??vel bioinseticida. Dessa forma, o presente trabalho teve como objetivo a produ????o heter??loga do inibidor ILTI, em Komagataella phaffii (Pichia pastoris), seguido da otimiza????o dos modos de cultivo em biorreator com o objetivo de maximizar a produ????o da prote??na recombinante. Para isso, o gene que codifica o ILTI foi clonado no vetor de express??o pPIC9K seguindo de sua inser????o na cepa GS115 por eletropora????o. An??lises de PCR mostraram que o vetor recombinante foi integrado ao genoma da levedura e que todos os clones obtidos possu??am gen??tipo MutS. A indu????o da express??o foi realizada durante 96 horas por meio da adi????o de metanol ?? 0,5%. A an??lise de prote??nas presentes no sobrenadante da cultura da cepa recombinante, por meio de SDS-PAGE, confirmou a produ????o de uma prote??na com tamanho pr??ximo a 20 KDa. Dados obtidos em MALDI-TOF confirmaram que a prote??na obtida ?? de fato ILTI recombinante. Al??m disso, ensaios inibit??rios mostraram que a prote??na produzida possui atividade contra tripsina. Dessa forma, cultivo em biorreatores foram realizados com a finalidade de otimizar a produ????o dessa prote??na heter??loga. A fim de aumentar sua express??o foram realizadas bateladas alimentadas, onde durante a fase de batelada foi favorecida a produ????o de biomassa, e a fase de alimenta????o programada para fornecer metanol de forma cont??nua, com base nos dados de velocidade espec??fica de consumo de metanol e velocidade de crescimento espec??fica nesta fonte de carbono. Ao longo das fermenta????es realizadas foi obtido 351,27 UIT no extrato bruto do caldo fermentado e uma atividade espec??fica de 2,07 UIT/mg de prote??na. Apesar de ser amplamente utilizada como hospedeira para a produ????o de prote??nas heter??logas, estudos da produ????o de inibidores em K. phaffii ainda s??o muito limitados. At?? o momento n??o existem relatos de otimiza????o da produ????o de inibidores de serinoproteases em K. phaffii, sendo esse estudo pioneiro e essencial para o in??cio do processo de escalonamento dessa tecnologia.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Cinco inibidores de tripsina da famÃlia Kunitz (JB1, JB2, JB3-1, JB3-2 e JB4) foram purificados de sementes de Pithecellobium dumosum, uma Ãrvore da subfamÃlia Mimosoideae, por precipitaÃÃo com Ãcido tricloroÃcetico (TCA), cromatografia de afinidade sobre tripsina imobilizada em Sepharose e coluna de fase reversa em sistema de CLAE. Os cinco inibidores possuem massa molecular entre 18 e 20 kDa formados por uma cadeia polipeptÃdica como determinado por SDS-PAGE na presenÃa ou ausÃncia de &#61538;-mercaptoetanol. JB1, JB3-1 e JB3-2 tÃm massas moleculares de 19,70, 19,69 e 19,69 kDa, respectivamente, por MALDI-TOF. JB2 e JB4 tÃm massa molecular de 18,08 e 20,85 kDa, respectivamente. A seqÃÃncia N-terminal de JB1, JB3-1 e JB3-2 mostrou identidade com outros inibidores da famÃlia Kunitz. Os cinco inibidores foram estÃveis Ãs variaÃÃes de temperatura e pH. A inibiÃÃo da tripsina por JB1, JB2 e JB4 foi do tipo competitivo. JB1, JB2, JB3-1 e JB3-2 tiveram Kis de 3,56 x 10-8 M, 1,65 x 10-8 M, 4,20 x10-8 M, 2,88 x 10-8 M, respectivamente para a tripsina bovina. Em comparaÃÃo com os outros inibidores JB4, com Ki de 5,70 x 10-10 M, apresentou maior afinidade para tripsina. Entre os inibidores purificados apenas JB4 inibiu moderadamente a atividade da quimotripsina. A atividade da elastase e bromelaÃna nÃo foi inibida efetivamente por esses inibidores. A inibiÃÃo de JB1, JB2, JB3-1 e JB3-2 sobre a papaÃna variaram entre 32,93 a 48,82% e foi indicativo de sua bifuncionalidade, com exceÃÃo de JB4 que inibiu fracamente essa atividade (9,93% de inibiÃÃo). A inibiÃÃo da papaÃna por JB1 e JB2 foi do tipo nÃo competitiva e os valores de Ki foram de 7,6 x 10-7 e 5,1 x 10-7 M, respectivamente. Ensaios in vitro sobre as proteinases digestÃrias de Lepidoptera, Diptera e Coleoptera foram feitos. Esses inibidores foram efetivos para enzimas digestÃrias semelhantes à tripsina desses insetos em diferentes graus. As enzimas digestivas de Zabrotes subfasciatus e Ceratitis capitata foram inibidas por JB1 em 68,87 e 65,53%, respectivamente, e as enzimas de Callosobruchus maculatus, Alabama argillaceae e Plodia interpunctella foram inibidas entre 29,18 e 44,35%. Enzimas digestÃrias de Z. subfasciatus, C. maculatus e C. capitata foram inibidas por JB2 entre 70,04 e 74,54% e as enzimas de larvas de A argillaceae e P. interpunctella foram suprimidas em 13,58 e 48,67%, respectivamente. A atividade semelhante à tripsina de larvas de Z. subfasciatus foi suprimida em 67,33 e 56,93% por JB3-1 e JB3-2, respectivamente, e a atividade de C. maculatus, A argillaceae, P. interpunctella e C. capitata foram suprimidas por esses inibidores entre 5,17 e 49,00%. JB4 inibiu entre 54,53 a 66,15 % as enzimas digestivas de C. maculatus, Z. subfasciatus e A argillaceae e inibiu as enzimas digestivas de larvas de P. intepunctella e C. capitata em 8,97 e 37,47%, respectivamente. A inibiÃÃo de proteinases semelhantes à tripsina e à papaÃna presentes no intestino de vÃrios insetos sugere que esses inibidores possam afetar o crescimento e sobrevivÃncia desses insetos pragas quando incorporados em sementes artificiais e esta bifuncionalidade à indicativo de que estes inibidores possam ser fortes candidatos para os programas de melhoramento de plantas via transgenia. / Five Kunitz-type trypsin inhibitors (JB1, JB2, JB3-1, JB3-2 and JB4) were purified from Pithecellobium dumosum seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose and reverse phase HPLC using Vydac C-18 column. The five inhibitors had Mr between 18 and 20 kDa with a single polypeptide chain as determined by SDS-PAGE with and without reduction. JB1, JB3-1 and JB3-2 had Mr of 19.70, 19.69 and 19.69 kDa, respectively, by MALDI-TOF. JB2 and JB4 had Mr of 18.08 and 20.85, respectively, by SDS-PAGE. The N-terminal sequences of JB1, JB3-1 and JB3-2 showed identity with others Kunitz-type inhibitors. The five inhibitors were stable over a wide range of temperature and pH. The inhibition of trypsin by JB1, JB2 and JB4 was competitive. JB1, JB2, JB3-1 and JB3-2 showed Ki values of 3.56 x 10-8 M, 1.65 x 10-8 M, 4.20 x 10-8 M, 2.88 x 10-8 M, respectively, against bovine trypsin. In comparison with others inhibitors JB4, with Ki of 5.70 x 10-10 M, showed a high affinity toward trypsin. Among the inhibitors purified only JB4 inhibited chymotrypsin activity. The activities of elastase and bromelain were not inhibited for these inhibitors. The inhibition of JB1, JB2, JB3-1 and JB3-2 on papain varied between 32.93 to 48.82% of inhibition and was indicative of its bifunctionality with exception of JB4 that inhibited this activity in 9.9%. The papain inhibition by JB1 and JB2 were noncompetitive type and the Kivalues were 7.6 x 10-7 and 5.1 x 10-7 M, respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. These inhibitors were effective towards trypsin-like digestive enzymes of the insect in different degrees. The digestive enzymes from Zabrotes subfasciatus and Ceratitis capitata were inhibited by JB1 in 68.87 and 65.53% respectively, and Callosobruchus maculatus, Alabama argillaceae and Plodia intepunctella enzymes were inhibited in the range of 29.18 to 44.35%. Digestive enzymes from Z. subfasciatus, C. maculatus and C. capitata were inhibited by JB2 in the range of 70.04 to 74.54%, and the enzymes of A. argillaceae and P. intepunctella were suppressed in 13.58 and 48.67%, respectively. Trypsin-like activities of larval from Z. subfasciatus were suppressed in 67.33 and 56.93% by JB3-1 and JB3-2, respectively, and the activities of C. maculatus, A. argillaceae, P. intepunctella and C. capitata were inhibited by these inhibitors in the range of 5.17-49.00%. JB4 inhibited around 54.53 to 66.15% the digestive enzymes of C. maculatus, Z. subfasciatus and A. argillaceae and the digestive enzymes from P. intepunctella and C. capitata larvae in 8.97% and 37.47%, respectively. The inhibition of trypsin-like and papain-like proteinases of several insects suggested that these inhibitors may affect the growth and survival of these insect pests when incorporated into artificial diet and their bifunctionality are indicative that these inhibitors could be strong candidates to plant management programs cross transgenia.

In the cell, as in in vitro, the final conformation of a protein is determined by it's amino acid sequence (1). Some isolated proteins can be denatured and refolded in vitro in absence of extrinsic factors. However, in order to fold in the cell, the newly synthesized polypeptide chain has to negotiate an environment far more complex than that faced by the unfolded chain in vitro. Cells have evolved proteins called “chaperones” to assist folding and assembly of polypeptides (2). Thus, the linear sequence of a protein not only contains information that specifies the final three-dimensional functional form, but also recognition motifs, which can be recognized by the cellular folding machinery. The work reported in this thesis is aimed at understanding some aspects of recognition of target substrates by the cytosolic chaperone, SecB, which forms part of the protein translocation machinery in E. coli. The sec pathway is involved in both translocation of precursor proteins across and the insertion of integral membrane proteins into the cytoplasmic membrane (3). Chapter one discusses some general aspects of protein folding and briefly describes chaperone systems, which have been extensively characterized in literature. Chapter two discusses the effect of chaperone SecB on the refolding pathway of a model substrate protein barstar, whose folding pathway has been extensively characterized (4,5). The effect of SecB on the refolding kinetics of the small protein barstar (wild type) and fluorescein labeled C82A (single Cys mutant) in 1 M guanidine hydrochloride at pH 7.0 at 25 °C has been investigated using fluorescence spectroscopy. We show that SecB does not bind either the native or the unfolded states of barstar but binds to late near-native intermediate (s) along the folding pathway. ESR studies and fluorescence anisotropy measurements show that SecB forms stable complexes with the near-native intermediate (s). For barstar, polypeptide collapse and formation of a hydrophobic surface are required for binding to SecB. Steady state polarization measurements indicated the presence of stable complexes of barstar bound to SecB. Studies on the spin labeled C82A show an immobilization of the spin label adduct at the 40th position of barstar, suggesting that the binding of SecB to barstar occurs in that region. SecB does not change the apparent rate constant of barstar refolding. The kinetic data for SecB binding to barstar are not consistent with simple kinetic partitioning models (6). Chapter three discusses the energetics of substrate:SecB interactions using the following model protein substrates: unfolded RNase A, BPTI, partially folded disulfide intermediates of alpha-lactalbumin,. The thermodynamics of binding of unfolded polypeptides to the chaperone SecB were investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of alpha-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29 and -0.41 kcal mol-1 K-1 respectively and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft (7). Chapter four discusses the thermodynamics of unfolding to gain insights into the mechanism of assembly and stability of the tetrameric structure. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. The value of ACP obtained was 10.7 ± 0.7 kcal mol-1 K-1, which is amongst the highest measured for a multimeric protein. At 298 K, pH 7.4. the AG°U for the SecB tetramer is 27.9 ± 2 kcal mol-1. Denaturant mediated unfolding of SecB was found to be irreversible. The reactivity of the 4 solvent exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four Cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well folded and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity (8). Chapter five discusses the bound state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI), as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58 residue protein and contains 3 disulfide groups between residues 5 and 55, 14 and 38, and 30 and 51. Single disulfide mutants of BPTI were reduced and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four, solvent accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The ESR data suggest that these cysteine residues are in close proximity when no substrate protein is bound, but move away from each other when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein. Chapter six discusses the mechanism of dissaggregation of a model peptide aggregate by chaperone SecB. The Hspl04, Hsp70 and Hsp40 chaperone system are capable of dissociating aggregated state(s) of substrate proteins, though little is known of the mechanism of the process. The interaction of the B chain of insulin with chaperone SecB was investigated using light scattering, pyrene excimer fluorescence and electron spin resonance spectroscopy. We show that SecB prevents aggregation of the B chain of insulin. We show that SecB is capable of dissociating soluble B chain aggregate as monitored by pyrene fluorescence spectroscopy. The kinetics of dissociation of the B chain aggregate by SecB has also been investigated to understand the mechanism of dissociation. The data suggests that SecB does not act as a catalyst in dissociation of the aggregate to individual B chains, rather it binds the small population of free B chains with high affinity, thereby shifting the equilibrium from the ensemble of the aggregate towards the individual B chains. Thus SecB can rescue aggregated, partially folded /misfolded states of target proteins by a thermodynamic coupling mechanism when the free energy of binding to SecB is greater than the stability of the aggregate. Pyrene excimer fluorescence and ESR methods have been used to gain insights on the bound state conformation of the B chain to chaperone SecB. The data suggests that the B chain is bound to SecB in a flexible extended state in a hydrophobic cleft on SecB and that the binding site accommodates approximately 10 residues of substrate (9).

This study has assessed the effects of pulse type and processing (soaking and cooking) on antinutritional factors (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins, phytic acid and oxalates) in a wide range of Canadian pulses, using soybean as a control. The contents of these antinutrients in Canadian pulses varied widely, but the levels were generally lower than those found in soybean. Analysis of variance indicated that both pulse type and processing had significant effects (P < 0.0001) on the investigated seeds. Soaking markedly decreased the contents of α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor, lectins and oxalates, but had no impact on phytic acid. Cooking of presoaked seeds appeared to be more effective; all proteinaceous antinutrients (α-amylase inhibitor, trypsin inhibitor, chymotrypsin inhibitor and lectins) were decreased by 80 – 100%, and significant reductions in phytic acid content (11 – 39%) were observed for all pulses, except common beans and soybean. / February 2016

Ziel der vorliegenden Arbeit war es, den Einfluss von unterschiedlich behandelten Sojabohnen in einer Broilerration (Mais und behandeltes Soja) auf die ernährungsphysiologischen Parameter beim wachsenden Küken zu prüfen. Unter Berücksichtigung der Einflussgrößen (z. B. Zeit, mechanischer Energieeinsatz und Futterstruktur), die die Effektivität der Futterbearbeitungsverfahren beeinflussen, wurde zunächst die Behandlung einer einheitlichen Charge von Vollfettsojabohnen unter folgenden technische Bedingungen vorgenommen: A: Walzenstuhl (WS) + Konditionierung (40 min./100 °C, Standardverfahren); B: WS + Konditionierung (40 min./100 °C) + Flockierung; C: WS + Konditionierung (10 min./100 °C); D: WS + Konditionierung (10 min. /100 °C) + Expandieren (20 kWh/t); E: Hammermühle (HM) + Konditionierung (10 min./100 °C) + Extrusion (15 kWh/t); F: HM + Konditionierung (10 min./100 °C), G: HM + Konditionierung (10 min./100 °C) + Expandieren (20 kWh/t); H: HM + Expandieren (20 kWh/t); I: HM + Expandieren (20 kWh/t) mit Dampfzufuhr; K: HM + Expandieren (40 kWh/t). Mit 6 ´ 10 männlichen Broilern der Herkunft Cobb wurde im Zeitraum vom 7. - 28. Lebenstag (LT) je Behandlung ein Wachstumsversuch durchgeführt. Während des Wachstumsversuchs wurden Daten zum Futterverzehr, Lebendmassezuwachs und Futteraufwand sowie Energie- und Proteinansatz über Körperanalyse erfaßt. Die ileale Aminosäureverdaulichkeit der einzelnen Mischungen wurde in jeweils 4 gepoolten Chymusproben (9 Tiere / gepoolte Probe) mit Hilfe eines Indikators (HCl-unlösliche Rohasche, Zusatz von 1 % Celite) nach Verfütterung vom 21. - 28. LT bestimmt. Parallel zum Wachstumsversuch wurde ein Stoffwechselversuch (6 männliche Küken der Herkunft Cobb je Behandlungsstufe) im Zeitraum vom 15. - 21. LT bei Fütterung der Versuchsmischung durchgeführt. Dabei erfolgte 3-mal täglich eine quantitative Exkrementsammlung. Neben der N- Bestimmung in den Exkrementen der Basis für die N-Bilanzmessung wurde der Bruttoenergiegehalt erfaßt und auf dieser Grundlage der Gehalt an N-korrigierter umsetzbarer Energie (MEn) in den Futtermischungen bestimmt. Weiterhin wurde aus den Daten der Bilanzmessungen der physiologische Nutzwert (PNu) als Kriterium der Proteinqualitätsbeurteilung sowie die Lysinwirksamkeit abgeleitet. Am Ende des Stoffwechselversuchs wurden die Tiere geschlachtet und die Trypsinaktivität im Chymus des Jejunums bestimmt. Die Ergebnisse lassen sich folgendermaßen zusammenfassen: 1. In Abhängigkeit von den gegebenen Bedingungen der Futterbehandlungsverfahren konnten unterschiedliche Trypsininhibitoraktivitäten (TIA) in den behandelten Sojabohnen erzielt werden. Eine starke Reduzierung des TIA war insbesondere bei den behandelten Sojabohnen A bis G erkennbar. Die relative Restaktivität lag bei diesen Gruppen (im Vergleich zur rohen Sojabohne) bei 16-27 %. Im Gegensatz dazu wurde eine höhere relative Restaktivität in den behandelten Sojabohnen H, I und K in Höhe von 75 %, 32 % und 48 % festgestellt . 2. Die Futteraufnahme, Lebendmassezunahme und Futterverwertung waren bei der Gruppe H infolge der höchsten Restaktivität des Trypsininhibitors signifikant niedriger als bei der Kontrolle A und anderen Gruppen (B, C, D, E, F, G, I und K). Analog dazu führte die höchste TIA in den Sojabohnen H ebenfalls zu einem signifikant verminderten Nährstoffansatz im Ganzkörper. Der Einfluss der höchsten Restaktivität des Trypsininhibitors auf den PNu war bei Gruppe H wenig ausgeprägt. 3. Die N-korrigierte umsetzbare Energie in den Futtermischungen B (14,87 MJ /kg T) und F (14,70 MJ /kg T) war signifikant höher als bei der Kontrolle A (13,96 MJ /kg). Der Grund für die Steigerung der N-korrigierten umsetzbaren Energie bei B und F kann dabei in der möglichen Verbesserung der Energieverfügbarkeit durch die Behandlungsverfahren Flockierung und Expander liegen. 4. Aufgrund der höchsten Restaktivität des Trypsininhibitors wurde bei der Gruppe H eine verminderte ileale Aminosäure- und Stickstoffverdaulichkeit im Vergleich zu Kontrolle A und den anderen Gruppen (B, C, D un E) festgestellt. Durch zusätzliche Dampfzufuhr und Erhöhung des Energieeintrages in den Gruppen I bzw. K konnten die antinutritiven Faktoren in den Sojabohnen reduziert werden und demzufolge die ileale Aminosäure- und Stickstoffverdaulichkeit bei den Gruppen I und K gegenüber der Gruppe H signifikant verbessert werden. Obwohl bei der Gruppe H die ileale Lysinverdaulichkeit gegenüber der Kontrolle A signifikant niedriger lag, wurde in Bezug auf die Lysinwirksamkeit kein signifikanter Unterschied zwischen den Gruppen H und der Kontrolle A festgestellt. 5. Die Trypsinaktivität im Chymus wurde auch in Abhängigkeit von der Restaktivität des Trypsininhibitors in den Sojabohnen unterschiedlich beeinflusst. Eine signifikant verminderte Trypsinaktivität im Chymus war insbesondere bei den Gruppen H sowie K feststellbar.