No epitélio do intestino médio de S. frugiperda encontra-se 90% da atividade de trealase solúvel. A trealase solúvel foi purificada até a homogeneidade por uma série de passos cromatográficos. A enzima possui um sítio hidrofóbico adjacente ao sítio ativo. Mudanças conformacionais aparentemente ocorrem quando metil-a-glicosídeo liga-se ao sítio ativo. A trealase solúvel é inibida competitivamente por amigdalina (Ki=0,21 mM), prunasina (Ki=0,92 mM), mandelonitrila (Ki = 1,14 mM), metil-α-glicosídeo (Ki=89 mM), metil-α-manosídeo (Ki=6,2 mM )e salicina (Ki= 19 mM). Florizina é um inibidor acompetitivo hiperbólico da trealase solúvel (Ki=0,087 mM, α =β =0,35) e seu aglicone floretina é um inibidor não competitivo (Ki=0,029 mM). Tris e mandelonitrila ligam-se a regiões diferentes da enzima enquanto mandelonitrila e floretina não podem ligar-se concomitantemente à enzima. Os pKs da enzima livre (pKe) e do complexo enzima substrato (pKes) foram determinados a partir de valores de Km e Vmáx/Km obtidos em vários pHs. Os valores encontrados foram: pKe1=4,47; pKe2=8,0l ; pKes1=4,83; pKes2=7,59. A trealase solúvel não perde a atividade quando incubada com reagentes que modificam grupos sufidrila, thiol e fenol. Com modificador de grupo imidazol, a enzima perde 60% da atividade somente na presença de metil-α-glucosídeo (inibidor competitivo da trealase). Essa modificação é protegida por trealose, indicando a presença de uma Histidina não essencial para catálise. Modificadores de grupo carboxila e guanidino inativam a enzima, com pKs de, respectivamente, 4,87 e 7,84. a similaridade desses pKs com os determinados cineticamente sugere que os resíduos envolvidos em catálise são uma arginina e um asparto ou glutamato. β-glicosídeos tóxicos produzidos por plantas e seus aglicones inibem trealoses presentes em diferentes órgãos de várias ordens de insetos. Essa inibição foi menor em insetos que se alimentam somente de vegetais, provavelmente devido a uma adaptação desses organismos. / The epithelium of S. frugiperda midgut has a trehalase activity that is mainly soluble (90%). The soluble trehalase was purified by several chromatographic steps. The enzyme has an hydrophobic site near the active site. Conformational changes apparently occur in the enzyme when methyl-α-glucoside binds to the active site. Trehalase has a Km=0.37 mM and is a competitively inhibited by methyl-α-glucoside (Ki=89 mM); methyl-α-mannosideo (Ki=6.2 mM); amygdalin (Ki=0.21); prunasin (Ki=0.92 mM); mandelonitrile (Ki=l.14 mM); Tris (Ki=0.55 mM) and salicin (Ki=l9 mM). Phlorizin is an hyperbolic acompetitive inhibitor (α=β=0.35; Ki=0.0087 mM), whereas its aglycon, phloretin, is a non-competitive inhibitor (Ki=0.029 mM). Tris and mandelonitrile bind at the same region in the active site. On the other hand, mandelonitrile and phloretin cannot be bound to the enzyme at the same time. Enzyme pKs (pKe) and enzyme substrate pKs (pKes) were determined from Km and Vmax/Km values obtained at different pHs. The values are: pKe1=4.47; pKe2=8.0l ; pKes1=4.83; pKes2=7.59. Trehalase is not inactivated when incubated with compounds that react with thiol, imidazole or phenol groups. Trealase lose 60% of its activity in the presence of methyl-α-glucoside (acompetitive inhibitor) plus a compound that reacts with imidazole groups. This inactivation is decreased by trehalose, indicating that there is a non-essential histidine in the active site substances that react with carboxyl guanidine groups inactivate the enzyme. The modified groups have pH of respectively, 4.87 and 7.84. The resemblance of these pKs with the one determined from Km and Vmax values suggest that the prototropic groups of the enzyme are in residues of arginine and aspartic acid or glutamic acid. Toxic β-glucosides from plants and their aglycons inhibit trealase from different organs of insects from several orders. This inhibition is lower in herbivorous insects, possibly due to their adaptation in ingesting vegetal tissues.

Inositol dehydrogenase (IDH) catalyzes the oxidation of myo-inositol to scyllo-inosose using NAD+ as the coenzyme. IDH-related genes (Lp_iolG1 to Lp_iolG4) from Lactobacillus plantarum WCSF1 and (Lc_iolG1 and Lc_iolG2) from Lactobacillus casei BL23 were cloned into the vector pQE-80L, expressed in E. coli host cells and the proteins were purified to homogeneity. IDH activity of the purified enzymes was explored with myo-inositol and other structurally related compounds. It was found that IDH-related enzymes from L. plantarum WCSF1 did not exhibit any activity with tested substrates but, LcIDH1 and LcIDH2 from L. casei BL23 showed activity with myo-inositol and other related compounds. pH-rate profile studies have demonstrated the optimum pH for the reactions catalyzed by the active enzymes. Steady-state kinetics of the active enzymes was performed as with IDH from Bacillus subtilis (BsIDH), revealing that LcIDH1 is a myo-inositol dehydrogenase and LcIDH2 is a scyllo-inositol dehydrogenase. Both LcIDH1 and LcIDH2 are observed to be NAD+-dependent. Kinetic isotopic effect experiments for LcIDH1 have demonstrated that the chemical step in the reaction is partly rate-limiting. Substrate spectrum of LcIDH1 and LcIDH2 was explored and compared to BsIDH. Finally, a multiple sequence alignment of IDH-related enzymes was performed and the proposed consensus sequence motifs were considered to understand the activity differences between these enzymes.

Type 1 diabetes is the direct result of an autoimmune attack on the pancreatic islet cells. The islets contain b cells, which are the only type of cell capable of supplying insulin in the human body. The destruction of these cells leaves the diabetic to rely on exogenous insulin to maintain a normal blood sugar level. Insulin therapy allows the diabetic to deal with the symptoms of the disease, but does nothing for the underlying condition. In order to truly cure the disease, the strategy is to replenish the b cells in the diabetic. Islet neogenesis associated protein (INGAP) has been shown to regenerate islet cells and reverse experimentally-induced diabetes in animal models. The INGAP pentadecapeptide is a 15 amino acid peptide from INGAP with comparable activity to the full-length protein. This 15-mer is undergoing clinical trials for treating diabetes. The overall goal of the project described in this work is to determine the structure of the INGAP pentadecapeptide for use in structure-based drug design of non-peptide mimics of the 15-mer. The first set of experiments in the present work directly examined the 15-mer in solution using NMR. No stable structure of the small peptide was found. The second set of experiments involved a homolog of INGAP, called INGAP-related protein, or INGAPrP. INGAPrP was recombinantly produced in E. coli and subsequently purified and refolded. Refolding of INGAPrP was verified by a 1H-15N HSQC experiment. CD experiments supported the NMR study, indicating helical content in INGAPrP. The folded nature of the protein will allow for the three-dimensional structure of INGAPrP to be determined. The protein structure will show the fold of the 15-mer within the full-length protein. This information will be valuable for the ultimate goal of producing structural mimics of the INGAP pentadecapeptide. Non-peptide mimics should have better oral bioavailability and longer half-lives in vivo.

Plants are potential sources of valuable recombinant and native proteins that can be purified for pharmaceutical, nutraceutical, and food applications. Transgenic rice and corn germ were evaluated for the production of novel protein products. This dissertation addresses: 1) the extraction and purification of the recombinant protein, human lysozyme (HuLZ), from transgenic rice and 2) the processing of dry-milled corn germ for the production of high protein germ and corn protein concentrate (CPC). The factors affecting the extraction and purification of HuLZ from rice were evaluated. Ionic strength and pH was used to optimize HuLZ extraction and cation exchange purification. The selected conditions, pH 4.5 with 50 mM NaCl, were a compromise between HuLZ extractability and binding capacity, resulting in 90% purity. Process simulation was used to assess the HuLZ purification efficiency and showed that the processing costs were comparable to native lysozyme purification from egg-white, the current predominant lysozyme source. Higher purity HuLZ (95%) could be achieved using pH 4.5 extraction followed by pH 6 adsorption, but the binding capacity was unexpectedly reduced by 80%. The rice impurity, phytic acid, was identified as the potential cause of the unacceptably low capacity. Enzymatic (phytase) treatment prior to adsorption improved purification, implicating phytic acid as the primary culprit. Two processing methods were proposed to reduce this interference: 1) pH 10 extraction followed by pH 4.5 precipitation and pH 6 adsorption and 2) pH 4.5 extraction and pH 6 adsorption in the presence of TRIS counter-ions. Both methods improved the binding capacity from 8.6 mg/mL to >25 mg/mL and maintained HuLZ purity. Processing of dry-milled corn germ to increase protein and oil content was evaluated using germ wet milling. In this novel method, dry-milled germ is soaked and wet processed to produce higher value protein products. Lab-scale and pilot-scale experiments identified soaking conditions that reduced germ starch content, enhanced protein and oil content, and maintained germ PDI (protein dispersibility index). Soaking at neutral pH and 25 degrees C maintained germ PDI and improved CPC yield from defatted germ flour. CPC with greater than 75% protein purity was produced using protein precipitation or membrane filtration.

Ephrin type-B receptor 2 (EphB2) is a receptor tyrosine kinase which phosphorylates proteins and thereby regulates cell migration, vascular development, axon guidance synaptic plasticity, and formation of borders between tissues. It has been seen overexpressed in several cancers, which make it an interesting protein to study. In this thesis EphB2 kinase domain (KD) and juxtamembrane segment with kinase domain (JMS-KD) have been expressed, purified and studied using analytical ultracentrifugation to evaluate the oligomerisation of the KD and how the double mutation S677/680A affects this. A program for data analysis have been written and used for analysis of the acquired data. The values of the dissociation constant were 2.94±1.04 mM for KD wild type and 3.46±2.26 mM for JMS-KD wild type have been calculated. Due to varied problems with the measurements no data was acquired on the double mutant, and not enough data was gained to draw any conclusions. Additional experiments will be needed to understand the oligomerisation of this intriguing protein.

Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.

Affibody® molecules are small therapeutic proteins which mimics antibody functionality. This is a report of several methods for increasing productivity and yield in recombinant production of Affibody® molecules. This literature study shows several steps in the production line which can be optimised, several novel methods for cultivating and harvesting cells and purication of proteins. There is also a section about validation of therapeutic protein production according to The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) are presented.

Orientadores: Aline Carvalho da Costa, Sindelia Freitas Azzoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-26T08:50:46Z (GMT). No. of bitstreams: 1 Bussamra_BiancaConsorti_M.pdf: 2624016 bytes, checksum: d09caf6db1941dc3b8ff17c802bb5ddb (MD5) Previous issue date: 2014 / Resumo: O alto custo da hidrólise enzimática para a conversão de biomassa lignocelulósica é um problema recorrente que limita a produção de bioetanol a partir desta fonte renovável e altamente energética. Muitos fatores afetam a eficiência do processo de hidrólise, como a recalcitrância do substrato lignocelulósico, eficiência do pré-tratamento, proporções e cargas das enzimas, inibidores e perda de atividade devido à adsorção não específica. Somado a estes fatores, o alto custo de produção de coquetéis enzimáticos limita a carga enzimática, o que contribui para a inviabilização técnica e econômica do processo. Dentre as abordagens consideradas promissoras para otimizar a reação de hidrólise, pode-se destacar o desenvolvimento de coquetéis artificiais bem balanceados envolvendo diferentes classes de enzimas. Este projeto estuda coquetéis enzimáticos otimizados para a hidrólise do bagaço de cana-de-açúcar pré-tratado, onde as enzimas provieram tanto de fonte comercial quanto de cultivos microbianos (Trichoderma reesei e Escherichia coli geneticamente modificada), seguidas de purificação. Planejamento experimental de mistura lattice simplex, seguido de planejamento fatorial, foram utilizados para a otimização da proporção enzimática. Em ambos os experimentos, a concentração de glicose liberada (g/L) foi a resposta avaliada. A fim de se reduzir o consumo de reagente, as hidrólises foram conduzidas em escala reduzida, cuja metodologia foi validada neste trabalho. A mistura enzimática otimizada, compreendida da fração T. reesei (80%), endoglucanase (10%) e ?-glicosidase (10%), foi capaz de converter 68,36% ± 5,57 da celulose contida no bagaço hidrotérmico, enquanto que os coquetéis comerciais Cellic CTec H2 e Celluclast converteram 70,43% ± 5,03 e 49,11% ± 0,49 do mesmo material, respectivamente, sob as mesmas condições de operação. Desta forma, este trabalho contribuiu com a identificação de atividades necessárias e de suas proporções ótimas para melhorar o coquetel enzimático de T. reesei, resultando no aumento da conversão do bagaço hidrotérmico. As estratégias de purificação de proteínas e análises de planejamento experimental e de sinergismo podem ser empregadas para a obtenção de coquetéis melhorados feitos sob medida para a hidrólise de outros materiais / Abstract: The high cost of converting lignocellulose by enzymatic hydrolysis is a recurring problem that limits the production of bioethanol from a renewable and highly energetic source. Many factors affect the efficiency of the hydrolysis process, such as lignocellulosic substrates recalcitrance, pretreatment efficiency, enzyme ratios and loadings, inhibitors and non-productive adsorption. Furthermore, the high production costs of enzyme cocktails limit the enzyme loads, which contribute to technical and economic infeasibility of this process. Among the promising approaches to optimize hydrolysis reaction, there is the development of well-balanced cocktails involving different classes of enzymes. This project studies optimized enzyme cocktails for hydrolysis of pretreated sugar cane bagasse, where the enzymes were provided from both commercial source and microorganism cultivation (Trichoderma reesei and genetically modified Escherichia coli), followed by purification. Experimental simplex lattice design, followed by factorial design, were performed to optimize the enzymatic proportion. In both experiments, released glucose concentration was the result evaluated. To reduce reagent consumption, the hydrolysis was carried out in micro assays scale, which methodology was validated in this study. The optimized enzyme mixture, comprised of T. reesei fraction (80%), endoglucanase (10%) and ?-glucosydase (10%), was capable of converting 68,36% ± 5,57 of the cellulose present in hydrothermal pretreated bagasse, whereas commercial cocktails Cellic CTec H2 and Celluclast converted 70,43% ± 5,03 e 49,11% ± 0,49 of the same material, respectively, under the same conditions. Thus, the work reported here contributed to the identification of needed activities and their optimal proportions to improve T. reesei enzymatic cocktail, enhancing hydrothermal bagasse conversion. Protein purification strategies and analyses of experimental design and synergism can be used to obtain improved cocktails to hydrolyze other materials / Mestrado / Engenharia Química / Mestra em Engenharia Química

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Drug resistant strains of Clostridium difficile are a major health concern with over 3 million cases costing over 1 billion $ per year in the United-States. The diseases associated with these bacteria (CDAD) are toxin-mediated which offers a mean of treating and lessening the severity of CDAD symptoms. Toxin inactivation via antibodies therapy can drastically reduce CDAD morbidity and this project was aiming at investigating the large-scale production and recovery of a novel llama single domain antibody (pSJF2H-ToxA5.1) in recombinant Escherichia coli TG1 targeting C. difficile enterotoxin A (TcdA). In order to achieve these objectives, the project was divided into four segments: 1) ToxA5.1 being an intracellular recombinant protein, obtaining a high biomass production was the first step towards large-scale production. To achieve HCDC, effects of initial glucose concentration and pH-stat feeding strategy were studied; 2) Upon achieving HCDC, effects of parameters such as temperature, induction timing and media supplementation with complex nitrogen sources were investigated; 3) Once large-scale production of ToxA5.1 was obtained, the recombinant protein needed to be recovered and a selective cell lysis scheme where synergistic lysis effects of Triton X-100 and temperature were studied. And finally 4) Single-step purification using nickel nanoparticles (NNP) synthesized via a modified polyol method was studied. Combining the HCDC strategy with a temperature shift and yeast extract addition at the time of induction, ToxA5.1 concentration of 127 mg/L was obtained. Synergistic and selective cell lysis using Triton X-100 and temperature was achieved where 95% of the available ToxA5.1 was recovered and still functional while ToxA5.1 fraction in the resulting lysate increased to 27% in the cell lysate. Single-step purification was achieved using the synthesized NNP which proved to be highly selective and could be used up to five times. Diameter of the NNP synthesized was controlled by using various concentration of ranging from 131 ± 80 nm to 47 ± 20 nm. Using experimental data from binding isotherm, the ToxA5.1-NNP system was modeled.