Th.--Sci. phys.--Nancy--I.N.P.L., 1985. / Extr. en partie de Enzyme and microbial technology, 7, 1985, 66-72 et de Biotechnology letters, 6, 1984, 12, 771-776.

Th.--Méd.--Montpellier, 1912-1913. / Montpellier, 1912-1913, n ° 73.

Orientador: Valéria Marta Gomes do Nascimento / Banca: Pedro de Oliva Neto / Banca: Cintia Regina Rodrigues Carignatto / Banca: Aneli de Melo Barbosa / Banca: Jonas Contiero / Resumo: Com o desenvolvimento de uma metodologia de purificação não convencional de passo único ("MPU") obteve-se um fator de purificação de 46,5 vezes e recuperação de 53%, observando-se banda única de 32 kDa na eletroforese SDS-PAGE. A enzima purificada apresentou atividade frente a ácidos graxos com cadeias entre 8 e 20 carbonos, destacando-se a seletividade para os ácidos palmítico (16:0) e oleico (18:1). A lipase apresentou ligeira regiosseletividade para as posições externas sn-1 e sn-3 (ésteres primários) do triacilglicerol. A cinética da reação demonstrou que a enzima segue uma cinética de Michaelis-Menten, com valor de Km de 22 mmol e Vmax de 12,7 mmol/min/mg. O valor de kcat foi de 225 s-1 e a eficiência catalítica de 104 mol-1.s-1. A temperatura para atividade máxima foi de 55 °C, sendo razoavelmente estável a 60 °C, mantendo 60% da atividade após 1 h. A atividade máxima foi obtida na faixa de pH entre 4 e 9, mantendo 100% da atividade inicial após incubação por 1 h na faixa de pH entre 2,2 e 10,0. A estabilidade em solventes orgânicos foi estudada por incubação da enzima em solventes miscíveis e imiscíveis em água. A enzima manteve 100% da atividade inicial em tolueno, n-hexano e n-heptano e apresentou estabilidade de 78%, 76% e 90% quando incubada com 50% (v/v) de metanol, etanol e isopropanol. Com relação às aplicações em reações de interesse industrial, a lipase de B. lata apresentou comportamento predominantemente de hidrolase (90%) em detrimento a ação de aciltransferase. Para a síntese de ésteres de aroma produz o éster caprilato de isoamila com 45% de rendimento (24 h e 50 °C em n-hexano). Para a síntese de um éster de cera (oleil oleato, C36), apresentou 87% de rendimento (30 h, 55 °C). A obtenção de ácidos graxos epoxidados alcançou rendimento de 44,4% em reação quimio-enzimática de epoxidação do oleato de metila ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: With the development of a non-conventional one-step purification methodology, was obtained a purification factor of 46.5 and recovery of 53%, noting single band of 32 kDa in SDS-PAGE. The purified enzyme showed activity against fatty acids with chains from 8 to 20 carbons, highlighting the preference for palmitic (16:0) and oleic acids (18:1). The lipase showed slight regioselectivity for external positions sn-1 and sn-3 (primary esters). The reaction kinetics showed that the enzyme follows a Michaelis-Menten model with Km value of 22 mmol and Vmax of 12.7 mmol/min/mg. The kcat value was 225 s-1 and the catalytic efficiency was 104 mol-1.s-1. The temperature for maximum activity was 55 °C, and reasonably stable at 60 °C, keeping 60% of activity after 1 h. The maximum activity was obtained at pH values between 4 and 9, maintaining 100% of the initial activity after 1 h incubation in a pH range from 2.2 to 10.0. Stability in organic solvents was studied by incubating the enzyme in miscible and immiscible solvents. The enzyme retained 100% activity in toluene, n-hexane and n-heptane and was 78%, 76% and 90% stable when incubated with 50% (v/v) methanol, ethanol and isopropanol, respectively. Regarding the applications in industrial interest reactions, B. lata lipase presented predominantly hydrolase behavior (90%) over acyltransferase action. For the flavor esters synthesis, the isoamyl caprylate ester is produced with 45% yield (24 h and 50 °C). For a wax ester synthesis (oleyl oleate, C36) showed 87% yield (30 h, 55 °C). The epoxidized fatty acids obtainment reached 44.4% yield in methyl oleate chemo-enzymatic epoxidation (48h, 40 °C and 10% (v/v) H2O2). The activity against spinach galactolipids in situ reached 2700 U/mg. In simulated gastric environment showed high activity and stability in the presence of proteases (pepsin and trypsin) in low pH conditions, besides demonstrating ... (Complete abstract click electronic access below) / Doutor

Orientador: Jonas Contiero / Banca: Valéria Marta Gomes de Lima / Banca: Ranulfo Monte Alegre / Resumo: Um estudo comparativo de onze métodos para determinação da atividade da dextranasacarase e de enzimas relacionadas foi realizado. Atualmente existem dois métodos usualmente empregados na determinação da atividade da dextranasacarase. Um método relativamente difundido para a reação da enzima com a sacarose consiste na medição dos valores redutores da D-frutose pelo reagente 3,5-dinitrosalicilato (DNS). Outro método é a reação da enzima com 14Csacarose, pela adição de uma alíquota da reação em papel de filtro Whatman 3MM, que posteriormente é lavado com metanol para a remoção da 14C-D-frutose e da 14C-sacarose não reagida, seguido da quantificação de 14C-dextrana por contador de cintilação líquida (CCL). Foi mostrado, pelo estudo realizado, que ambos os métodos geram dados errados. O método por quantificação do valor redutor proporciona resultados extremamente altos devido à oxidação tanto da D-frutose quanto da dextrana, e o método do 14C-papel gera resultados significativamente baixos devido à remoção de parte da 14C-dextrana do papel, pela lavagem em metanol. Neste trabalho, foram testados onze métodos, dentre os quais, dois apresentaram resultados idênticos, sendo considerados métodos confiáveis de medição da atividade da dextranasacarase. Este trabalho também reporta a caracterização físico-química de uma nova dextrana produzida por Leuconostoc mesenteroides FT045B. A estrutura química do polímero foi caracterizada por Espectroscopia de Infravermelho com Transformada de Fourier e por Espectroscopia de 1H Ressonância Magnética Nuclear. A dextrana produzida foi hidrolisada por endodextranase e seus produtos da reação do aceptor com maltose foram analisados por cromatografia em camada delgada e comparados com a dextrana comercial B512F obtida da Sigma Company. A massa molecular media e o grau de polimerização da dextrana produzida... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: A comparative study of nine assay methods for dextransucrase and related enzymes has been made. A relatively widespread method for the reaction of the enzyme with sucrose, is the measurement of the reducing value of D-fructose by alkaline 3,5- dinitrosalicylate (DNS). Another method is the reaction with 14C-sucrose, with the addition of an aliquot to Whatman 3MM paper squares that are washed 3-times with methanol to remove 14C-D-fructose and unreacted 14C-sucrose, followed by counting of 14C-dextran on the paper by liquid scintillation counting (LSC). It is shown that both methods give erroneous results. The reducing value method gives extremely high values due to over-oxidation of both D-fructose and dextran, and the 14C-paper square method gives significantly low values due to the removal of some of the 14Cdextran from the paper, by the methanol washes. In the present study, we have examined nine methods and find two that give values that are identical and are an accurate measurement of the dextransucrase reaction. This work also reports the physical and chemical characterization of a new dextran produced by Leuconostoc mesenteroides FT045B. The chemical structure of the polymer was characterized through Fourier transform infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. The dextran produced was hydrolyzed by endodextranase and its products from the acceptor reaction with maltose were analyzed using thin layer chromatography and compared with those of a commercial native dextran B512-F obtained from the Sigma Company. The average molecular weight and degree of polymerization of the dextran produced were determined through the measurement of the reducing value using the copper bicinchoninate method and the measurement of the total carbohydrates using the phenol-sulfuric acid method. All data revealed a very similar structure to that of the commercial dextran B512F from... (Complete abstract click electronic access below) / Mestre

Citrate synthase (CS) is a key mitochondrial enzyme in the tricarboxylic acid cycle (TCA). TCA provides NADH and FADH for the ETC to generate ATP through oxidative phosphorylation in muscle cells. The aim of this PhD project is to investigate the role of CS in skeletal muscle metabolism. The aim of the first study was to investigate the effects of a high fat diet (HFD, 45 % kcal fat) for 12 weeks on CS activity in the heart and gastrocnemius muscle of C57BL/6J (B6) mice and congenic (B6.A) characterised by 39% reduced CS activity. Spectrophotometric analysis of CS activity in the heart and gastrocnemius muscle revealed that HFD led to an increase in CS activity in gastrocnemius muscle but a decrease in the heart in both strains of mice. The aim of the second study was to investigate the effects of low CS activity on substrate metabolism in primary muscle cells established from B6 and B6.A mice. Primary muscle cells from both strains were incubated in radiolabelled glucose or palmitate to assess their oxidation in the mitochondria. The reduction of CS activity in B6.A muscle cells did not affect glucose and palmitate oxidation. The aim of the third study was to investigate the effects of D- and L-serine on CS activity in B6 muscle homogenates, primary muscle cells and purified CS from porcine heart. The muscle samples were incubated in D - or L-serine at 0.1 mM or 5 mM concentration and CS activity levels were assessed by spectrophotometer. D- or L-serine did not have any effect on CS activity in muscle samples. The aim of the fourth study was to investigate the effects of low CS activity on substrate metabolism in C2C12 muscle cells. Lentiviral transduction of C2C12 muscle cells with shRNA resulted in a reduction of CS activity and the metabolic pathways were assessed using XF24 Analyser, western blotting, Immunofluorescence and qRTPCR. Low CS activity was associated with a reduction in substrate oxidation by the mitochondria, an increase in glycolysis and ceramide accumulation in C2C12 muscle cells. The results highlight the significance of CS activity as a modulator of muscle metabolism.

No description available.

The object of this study was to isolate and characterise barley mutants that lacked a functional nitrite reductase activity. This work should complement previous studies on nitrate reductase to develop a fuller understanding of nitrate assimilation in barley. 30 nitrite reduction-deficient M2 barley plants (azide-treated in the M1) were identified as nitrite accumulators after treatment with nitrate. Biochemical analysis M2 selections revealed that leaf tissue from 9 selected plants lacked detectable nitrite reductase protein. Progeny from 4 of the selected plants (Golden Promise 2406, Tweed 3999, Klaxon 1010 and Klaxon 2760) inherited the phenotype of a lack of leaf nitrite reductase protein. These plants also lacked significant in vitro nitrite reductase activity. However, in vitro nitrate reductase and nitrate accumulation were comparable with wild type controls. Root tissue from nitrate-treated progeny of Golden Promise 2406, Tweed 3999, Klaxon 1010 and Klaxon 2760 also lacked nitrite reductase protein. Loss of nitrite reductase protein in selection Tweed 3999 was caused by a single recessive nuclear gene mutation. Thus, these plants are defective in nitrite reduction due to the inherited loss of nitrite reductase protein molecules in both leaf and root after treatment with nitrate in the light. This defect is caused by a mutation within a single nuclear gene in selection Tweed 3999. Analysis of wild type barley cv. Golden Promise revealed that increases in nitrite reductase activity in response to treatment with nitrate and light in leaf tissue and with nitrate in root tissue are due to de novo synthesis of enzyme molecules. In situ immunogold labelling of barley leaf sections with nitrite reductase antiserum demonstrates that the majority of labelling occurs within the chloroplasts.

The object of this research was to isolate nitrate reductase-minus cell-lines of a higher plant species in order to gain information on the genetic control of nitrate reductase production in higher plants. This demanded a culture system consisting of isolated cells or protoplasts, ideally carrying single-copy genetic information. To this end, haploid Nicotiana plants were raised by another culture and abortive attempts were made to culture haploid sylvestris protoplasts. Callus and cell suspension cultures of haploid N. sylvestris also proved unsuitable but eventually dihaploid M. tabacum suspension cultures were obtained which, it was decided, would be suitable for this work. After treatment of these cultures with EMS, 59 cell-lines were selected for resistance to chlorate, four of these were unable to grow on nitrate medium and were later shown to possess an assembled but Inactive nitrate reductase. None of the foul possessed xanthine dehydrogenase activity indicating that they were molybdenum co-factor (Mo-co) defective lines (cnx variants). These cnx lines are different from other Mo-co defective tabacum lines thus far described and are therefore new types, providing evidence that more than one gene locus is involved in the formation of functional Mo-co in N. tabacum nitrate reductase.

The secreted acid phosphatase (SAcP) of Leishmania donovani is a secreted glycoprotein modified with unique glycoforms which share a structural heterogeneity and immunological similarity with the dominant, cell surface phosphoglycolipid produced by all species of leishmania parasites. The post-translational modifications of this enzyme are structurally diverse and include standard high-mannose type N-linked glycosylations as well as a novel O-linked phosphoglycan. This dissertation encompasses the analysis of these structures including assessment of their size, hexose composition and sites of attachment to the protein. These analyses have employed both carbohydrate and protein chemistry techniques, as well as physical methods such as mass spectrometry. The N-linked glycosylations have been compared with those previously characterized on other Leishmania proteins and show substantial structural similarity. The O-linked phosphoglycans are unique to L. donovani, and are composed of phosphodisaccharides with the structure 4-O-(beta-D-galactopyranosyl)-alpha-D-mannopyranosyl-1-phosphate. These phosphodisaccharides are arranged in linear polymers by way of a phosphodiester linkage between the C1 hydroxyl of mannose and the C6 hydroxyl of galactose. Linkage of this structure to the protein is novel and proceeds via a phosphodiester to selected serine residues that are contained within a consensus protein sequence. This sequence occurs in excess of 20 times within the SAcP, which results in abundant glycan modification and contributes to the heterogeneity displayed by this enzyme. The biosynthetic machinery used to produce these structures was also investigated. The addition of phosphoglycan to the SAcP is initiated by the transfer of alpha-D-mannopyranosyl-1-phosphate from GDP-Man to the protein catalysed by a mannosyl phosphate transferase (MPT). An assay for this enzyme is described using a synthetic peptide substrate to which radiolabeled mannose can be transferred from GDP-[14C] Man. This assay has been used to partially characteriz the MPT and has assisted in the isolation of the enzyme using an affinity chromatography approach. Sequence analysis and amino acid analysis of the enzyme isolated in this way has shown that the MPT is a novel molecule that does not presently exist in the public database. / Graduate

No description available.