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The research project, The Enzymatic Synthesis of CoA Derivatives Using a New ATP Regeneration System, describes the multiple lab trials conducted to develop an ATP regeneration system using various concentrations of specific substrates and the two enzymes MatB and PrpE. Reactions were combined using different concentrations of ADP, the addition or removal of ADK (adenylate kinase), and the substitution of either MatB or PrpE. Further reactions were combined using trans-crotonyl, trifluoroacetic acid, butyric acid, acetic acid, and creatine phosphokinase. This report also includes the methods used and analysis of the different chromatographs of each sample tested. / text

Orientador: Francisco Maugeri Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-07T18:40:17Z (GMT). No. of bitstreams: 1 Hernalsteens_Saartje_D.pdf: 2071351 bytes, checksum: 1b18d6d6f4088be5bdd8d6168a5c2d5b (MD5) Previous issue date: 2006 / Resumo: Devido ao aumento da demanda por alimentos saudáveis e ao aumento da aplicação, de oligossacarídeos prebióticos, na indústria cosmética, agro-química, farmacêutica e na indústria de alimentos, a pesquisa visando a utilização de diferentes enzimas na produção dos oligossacarídeos se tornou algo necessário. O objetivo deste trabalho foi obter linhagens de leveduras, produtoras de frutooligossacarídeos a partir da sacarose. As linhagens foram isoladas de flores e frutos de diversas eco-regiões do Brasil. A partir da gama de microrganismos isolados foram selecionadas 4 cepas potencialmente aplicáveis na produção de frutooligossacarídeos: Candida sp. LEB-I3; Rhodotorula sp. LEB-U5; Cryptococcus sp. LEB-V2 e Rhodotorula sp. LEB-V10. Os quatro microrganismos estudados produzem enzimas semelhantes em relação a algumas características bioquímicas. As condições ótimas para a atividade de transferência de frutose foram pH entre 4,0 e 5,0 e temperaturas entre 65 e 70ºC, enquanto que para a atividade de frutofuranosidase o pH ótimo foi de 3,0 a 4,0 e a temperatura ótima entre 55 e 75ºC. A cinética enzimática (em relação à atividade de transferência de frutose) da enzima I3 seguiu o modelo de Michaelis-Menten, enquanto que U5 e V2 seguiram o modelo de inibição pelo substrato e a enzima V10 apresentou uma cinética de ¿cooperatividade¿. O estudo da síntese de FOS mostrou que o microrganismo Rhodotorula sp. LEB-V10 foi o único cuja enzima promoveu uma síntese constante de FOS, não apresentando nenhum indício de hidrólise dos frutooligossacarídeos, sendo devido a esta característica que tanto a produção da enzima quanto a síntese de frutooligossacarídeos foram otimizadas. Neste caso a produção de enzima foi máxima nas seguintes condições: 9% ± 1% de AMM e 7,5% ± 0,7% de açúcares redutores totais (melaço), com uma agitação de 250 rpm, a 30-35ºC, pH inicial do meio de 4,5. A produção de frutooligossacarídeos por esta enzima também foi otimizada chegando-se a 55-65% de rendimento nas seguintes condições: 50% de sacarose (P.A, comercial ou melaço), 6,5 (± 0,5) UTF.ml-1, temperatura entre 50 ºC (± 1ºC) e pH 5,0 (± 0,5). Desta forma foi alcançado o rendimento dos processos comerciais, com a vantagem de estarmos trabalhando com enzimas e não células. Além disso, a produção da enzima utilizando meios industriais, e o uso de açúcar cristal e mesmo melaço na síntese enzimática resultam em uma diminuição dos custos de produção. Desta forma há uma chance de que a continuação destes estudos resulte em um processo economicamente viável / Abstract: In response to the increasing demand for healthier foods and as a result of the expanding applications of oligosaccharides in the cosmetic, agrochemical, pharmaceutical and food industries, the search for ¿new¿ enzymes concerning the oligosaccharides production, became necessary. The present study reports on the screening for high transfructosylating enzymes in yeasts strains isolated from fruits and flowers obtained from tropical Brazilian biomass. The efforts made to screen for high extra-cellular transfructosylating enzyme producing yeasts provided very promising results. Although the enzymes from the strains Candida sp. LEB-I3, Rhodotorula sp. LEB-U5 and Cryptococcus sp. LEB-V2 showed high hydrolytic activity, the production of fructooligosaccharides (FOS) by the Rhodotorula sp. LEB-V10 enzyme was successful, showing a continuous increase in FOS concentration up to the end of the synthesis reaction. The best operational conditions for these enzymes, considering the transfructosylating activities, were determined to be in the pH range from 4.0 to 5.0 and temperatures from 65 to 70°C. While the fructofuranosidase activities had shown the optimum activity on pH values from 3.0 to 4.0 and temperatures between 55 and 75°C. The enzymatic kinetic (fructosyl transferase activity) of the Candida sp. LEB-I3 showed a Michaellis-Mentem behavior, while the Rhodotorula sp. LEB-U5 and Cryptococcus sp. LEB-V2 showed a substrate inhibitory kinetic and the Rhodotorula sp. LEB-V10 showed a sigmoid shape, similar to that of allosteric enzymes. Considering that the Rhodotorula sp. LEB-V10 process was the only one that may be regarded as economically possible, the response surface methodology was employed to study the fermentation and the synthesis condition aiming the process optimization. On basis of the experimental results, the optimum conditions to obtain high fructosyl transferase activity were: 250 rpm, 30-35°C, 9% ± 1% (w/v) corn steep liquor and 7.5% ± 0.7% (w/v) of total reducing sugar from sugar cane molasses. The synthesis of FOS was also optimized (55 to 65% of yield), being the optimum conditions: 50% sucrose (P.A., commercial or from sugar cane molasses), 50°C (± 1°C), pH 5.0 (± 0.5) and 6.5 FTA.ml-1 (± 0.5). This data is very similar to those from the commercial process, and the use of commercial sucrose and sugar cane molasses led to a reduction on the production cost, consequently, further studies on the enzyme and fructooligosaccharides production conditions may show its potential for commercial application / Doutorado / Doutor em Engenharia de Alimentos

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13178号 / 論農博第2857号 / 新制||農||1061(附属図書館) / 学位論文||H30||N5100(農学部図書室) / (主査)教授 小川 順, 教授 栗原 達夫, 教授 三上 文三 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2008. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-09-24T17:54:51Z No. of bitstreams: 1 dissertao de mestrado cristiane.pdf: 830083 bytes, checksum: 8a0fe102af2eaf27474ab995424a5309 (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-12-01T16:25:51Z (GMT) No. of bitstreams: 1 dissertao de mestrado cristiane.pdf: 830083 bytes, checksum: 8a0fe102af2eaf27474ab995424a5309 (MD5) / Made available in DSpace on 2012-12-01T16:25:51Z (GMT). No. of bitstreams: 1 dissertao de mestrado cristiane.pdf: 830083 bytes, checksum: 8a0fe102af2eaf27474ab995424a5309 (MD5) Previous issue date: 2008 / Os galactooligossacarídeos (GOS) vêm sendo considerados como novos ingredientes funcionais dos alimentos, apresentando um grande potencial para melhorar a qualidade dos mesmos, sendo denominados como prebióticos, devido às suas características fisiológicas e tecnológicas. Podem ser produzidos a partir de lactose, principal açúcar presente no soro de leite, através de uma reação enzimática com o uso da enzima β-galactosidase, que possui atividade de transgalactosilação. Este trabalho tem como objetivo principal estudar a obtenção de galactooligosacarídeos por via enzimática. Foi utilizada b-galactosidase de Kluyveromyces lactis Lactozym® 3000L (Novozymes) e, como substratos, a lactose e o soro de leite. Um planejamento experimental 23 totalizando 11 ensaios foi proposto para cada um dos substratos, verificando-se a influência da temperatura (30 a 40°C), concentração de lactose (20 a 40%) e concentração da enzima (5 a 10 U.mL-1), obtendo-se como respostas a concentração de GOS máxima, o rendimento de GOS máximo, a produtividade de GOS máxima e conversão de lactose máxima. Os ensaios foram conduzidos a 180 rpm de agitação, em meio aquoso e pH 7,0 (tampão fosfato de sódio 0,1M), retirandose alíquotas ao longo do tempo, sendo os açúcares presentes em cada amostra quantificados através de um sistema de cromatografia líquida de alta eficiência HPLCPAD (Dionex). A enzima Lactozym® 3000L apresentou atividade de transgalactosilação na presença dos dois substratos. Utilizando como substrato lactose o rendimento atingiu 43,8% e a concentração de GOS foi de 175,3 g/L no sistema reacional composto por 40% de lactose e 10 U.mL-1 de enzima a 30°C. No entanto a produtividade máxima alcançada, de 41,1 g.L-1.h-1, bem como a máxima conversão de lactose (80,5%), foram obtidas em condições de síntese distintas. Utilizando como substrato soro de leite, os valores máximos obtidos para as respostas concentração de GOS (119,8 g.L-1) e rendimento (29,95%) em 4 h de processo foram obtidos no sistema reacional composto por 40% de lactose e 10 U.mL-1 de enzima a 40°C. Nestas condições a produtividade máxima alcançada foi de 64,4 g.L-1.h-1. O máximo valor alcançado para conversão de lactose (87,8%) foi obtido nas mesmas condições de temperatura e concentração de enzima, mas com 20% de lactose. / Galactooligosaccharides (GOS) have been considered as new functional food ingredients, presenting a great potential to improve its quality. They are denominated as prebiotics, due to their physiological and technological characteristics. They can be produced from lactose, the main sugar of the whey, through an enzymatic reaction with the use of the enzyme β-galactosidase, with transgalactosylation activity. The main goal of this work was to study the enzymatic production of galactooligosaccharides. It was used β-galactosidase from Kluyveromyces lactis Lactozym® 3000L (Novozymes) and, as substrates, lactose and whey. A 23 experimental design (11 assays) was proposed for each substrate, verifying the effect of the temperature (30 to 40°C), lactose concentration (20 to 40%) and concentration of the enzyme (5 to 10 U.mL-1). The responses were maximum GOS concentration, maximum GOS yield, maximum productivity and maximum lactose conversion. Assays were carried out at 180 rpm in aqueous medium and pH 7.0 (0.1 M sodium phosphate buffer). Samples were collected and sugars were quantified by high performance liquid chromatography (HPLC-PAD system, Dionex). For lactose the yield reached 43.8% and GOS concentration was 175.3 g.L-1, in the system composed by 40% of lactose and 10 U.mL-1 of enzyme at 30°C. However, maximum productivity (41.1 g.L-1.h-1) as well as maximum lactose conversion (80.5%) were obtained in different conditions. For whey, the maximum values for the responses GOS concentration (119.8 g.L-1) and yield (29.95%) in 4 h of process were obtained in the system composed by 40% of lactose and 10 U.mL-1 of enzyme at 40°C. In these conditions, maximum productivity was 64.4 g.L-1.h-1. The maximum value for lactose conversion (87.8%) was obtained in the same temperature and enzyme concentration, but with 20% of lactose.

La bioraffinerie, avec l’utilisation de la biomasse végétale comme matière première renouvelable, est un moyen alternatif aux ressources fossiles qui s’amenuisent pour l’obtention de molécules d’intérêt, de biomatériaux ou de biocarburant nécessaires à notre vie quotidienne. Les hémicelluloses constituent 20 à 45% de la biomasse végétale et sont riches en pentoses (D-xylose en particulier) dans le cas des xylanes. Dans le cadre du projet multi-partenaires XYLOCOS, financé par la Région Champagne-Ardenne et le FEDER, l’objectif de cette thèse consiste en l’utilisation d’une xylanase pour mettre au point la préparation par voie enzymatique ou chimio-enzymatique de nouveaux β-xylosides et β-xylobiosides présentant des aglycones de différentes natures et d’évaluer leurs propriétés biologiques. L’étude d’une xylanase GH11 a d’abord été réalisée pour améliorer son activité de transglycosylation en présence de divers accepteurs. Une étude de modélisation in silico a été effectuée et a conduit à cibler la mutation du résidu W126. Les paramètres cinétiques et les propriétés de transglycosylation de la xylanase sauvage et du mutant ont été comparés et ont montré que le mutant a une capacité de transglycosylation améliorée. La synthèse de séries de xylosides et xylobiosides comportant une partie aglycone aromatique par voie enzymatique ou présentant des hétérocycles triazoles diversement fonctionnalisés par voie chimio-enzymatique a été effectuée. L’influence de la nature de la partie aglycone mais également du degré de polymérisation des différents xylosides sur leur capacité à amorcer la biosynthèse de glycosaminoglycanes a été étudiée en présence d’un modèle cellulaire. L’ensemble des xylosides et xylobiosides synthétisés amorce la biosynthèse des GAGs. Les résultats obtenus montrent que les xylosides sont plus efficaces que leurs analogues xylobiosides. Certains xylosides à aglycones hydrophobes représentent de bons candidats pour des applications cosmétiques. / Biorefinery, with the use of plant biomass as a renewable raw material, is an alternative means to fossil resources for obtaining molecules of interest, biomaterials or biofuel. Hemicelluloses represent about 20 to 45% of the plant biomass and are rich in pentoses (D-xylose in particular) in the case of xylans. XYLOCOS is a multi-partner project funded by the Champagne-Ardenne Region and the FEDER. In this context the objective of this work consists in the use of a xylanase to develop the enzymatic or chemo-enzymatic synthesis of new β-xylosides and β-xylobiosides featuring different kinds of aglycone moieties and to assess their biological properties.First, the study of a GH11 xylanase was carried out to improve its transglycosylation activity in the presence of various acceptors. A in silico study was performed and led to target the mutation of W126 residue. Kinetic parameters and transglycosylation properties of the wild and mutant xylanases were compared and showed that the mutant has an improved capacity for transglycosylation.The synthesis of xylosides and xylobiosides bearing an aromatic aglycone part by an enzymatic transformation or functionalized triazole heterocycles by a chemo-enzymatic pathway was performed. The influence of the nature of the aglycone part but also the degree of polymerization of different xylosides was studied through their ability to initiate the biosynthesis of glycosaminoglycans in the presence of a cell model. All the xylosides and xylobiosides prepared act as GAGs primers. The obtained results show that xylosides are more efficient primers than the corresponding xylobiosides. Xylosides carrying hydrophobic aglycones represent good candidates for cosmetic applications.

Natural products continue to be a major chemical lead matter for drug discovery due to their diverse chemical structures and bioactivities. Clinically significant natural products include anti-cancer and anti-infective compounds and while many more of these compounds show promising bioactivity, their clinical relevance is often limited by toxicity or poor solubility. Combinatorial biosynthesis can be employed to modify existing chemical scaffolds towards reducing these limitations. To fully take advantage of these biochemical tools, it is important to understand the biosynthesis and mechanism of action of the molecules. Saccharides in glycosylated natural products provide specific interactions with cellular targets and are often crucial for a compound’s bioactivity. Genetic engineering of sugar pathways can modify glycosylation patterns leading to the diversification of natural products. Saquayamycins (SQN) H and I are cytotoxic angucycline antibiotics containing five deoxyhexoses including the rare amino sugar rednose. Elucidating the biosynthetic pathway of rednose could add to the arsenal of combinatorial biosynthesis tools for drug development. Our research goal of investigating the rednose biosynthetic pathway was pursued through two specific aims: the identification of the Streptomyces sp. KY 40-1 gene cluster involved in the biosynthesis of SQN H and I (sqn) (specific aim 1), and the validation of the proposed L-rednose biosynthetic pathway up to the glycosyl transfer through enzymatic synthesis of NDP-3,6-dideoxy-L-idosamine (specific aim 2). The sqn gene cluster revealed deoxysugar biosynthetic genes that could be used to alter glycosylation patterns to generate novel compounds while the enzymatic synthesis afforded novel genetic engineering tools to generate novel TDP-deoxysugars that could be used to diversify compounds such as aminoglycosides to circumvent resistance mechanisms. The first step to generate TDP-glucosamine enzymatically was accomplished, however later steps were unsuccessful. The aureolic acid mithramycin (MTM) was recently tested in clinical trials for Ewing sarcoma following the discovery of MTM as a potent inhibitor of the oncogenic transcription factor EWS-FLI1 present only in Ewing sarcoma cells It is understood that MTM binds the minor groove of G/C rich DNA as an Mg2+-coordinated dimer disrupting transcription of proto-oncogenes; however, the DNA recognition rules were not completely understood, making further interrogation of MTM’s DNA binding preferences necessary. This research goal of further understanding the mechanism of action for MTM was approached through two specific aims: the investigation of the dimerization of MTM (specific aim 3), and the investigation of MTM’s DNA binding preferences (specific aim 4). This work established that MTM and its biosynthetic precursor premithramycin B (PreMTM B), and several MTM analogues with modified 3-side chains: mithramycin SDK (MTM SDK), mithramycin SA tryptophan (MTM SA-Trp), and mithramycin SA alanine (MTM SA-Ala) dimerize even in the absence of DNA under physiologically relevant conditions. The study also demonstrated that modification of the 3-side chain modulates DNA binding affinity of MTM analogues, established a minimum MTM binding site on DNA, and revealed MTM DNA recognition is driven by direct (sequence) and not indirect (conformation) readout laying the foundation for subsequent research based on the interaction between MTM, DNA, and the oncogenic transcription factor EWS-FLI1 in the rational design of new MTM analogues for the treatment of Ewing sarcoma.

Mono- and bis-aspidosperma indole alkaloid natural products have been shown to be cytotoxic against various cancer cell lines and are potential leads in therapeutics against diabetes and neurodegenerative diseases. Tabersonine, a monoterpene indole alkaloid isolated from the family Apocynaceae, was known to be the bio-synthetic precursor of its mono and dimeric analogs, including vindoline, conophylline, and vinblastine. The first synthesis of bis-aspidosperma indole alkaloid (–)-melodinine K was achieved in a chemoenzymatic fashion, featuring the T16H hydroxylation and the biomimetic dimerization via the Polonovski-Potier reaction. Moreover, eight tabersonine derivatives bearing one or two hydroxy or methoxy groups at C15-C17 were synthesized up to grams scale via a halogenation/borylation/oxidation sequence. These analogs exhibited potent inhibitory activities against cancer cells with GI50 values in the single digit micromolar level. This research on aspidosperma indole alkaloids could help guide the development of future cancer therapeutics as well as elucidate their biosynthetic pathways.Histone deacetylases (HDACs), regulating gene expression and multiple cellular activities, are important biomarkers and potential therapeutic targets. In this project, a group of fluorescent S-acetamidomethyl homocysteine-containing molecules were designed and synthesized to monitor HDAC’s activity and elucidate the temporal and spatial dynamics of HDACs in live cells. The free thiol warhead, released from the probe upon deacetylation by HDACs, would trigger an intramolecular Michael Addition to the coumarin-derived fluorophore and induce a blue shift for the fluorescence, which could be applied to the quantitative ratiometric study of HDACs. It provides a modular platform for discovering and developing class-/isoform-specific HDAC probes and future therapeutic lead compounds through modifications of fluorophores, C-terminal amino acids, and the capping acyl groups. / Chemistry

O objetivo desta dissertaÃÃo foi sintetizar derivados de vitamina A por rota enzimÃtica, como alternativa à rota quÃmica, que à caracteristicamente mais agressiva ao meio ambiente. A sÃntese do palmitato de retinila resultaria em produto com melhor aceitaÃÃo de mercado, jà que à mais estÃvel do que o Ãster que foi utilizado como substrato, acetato de retinila. Jà a sÃntese de adipato de retinila, tinha como principal finalidade, disponibilizar um novo derivado de vitamina A. Para ambos, visava-se a aplicaÃÃo nas indÃstrias farmacÃutica, cosmÃtica e alimentÃcia. Nesse contexto, utilizou-se lipase de Candida antarctica tipo B imobilizada (Novozyme 435 com 571,48 UI/g  55,47) e os substratos acetato de retinila, Ãcidos palmÃtico e adÃpico. AlÃm destes, peneira molecular (PM) 3Ǻ tambÃm foi adicionada, jà que as reaÃÃes propostas liberam Ãgua para o meio reacional, desfavoredendo a sÃntese do Ãster desejado. Dois planejamentos foram realizados para se avaliar a sÃntese de palmitato de retinila, ambos em volume reacional total de 2 mL. No primeiro, trÃs variÃveis foram analisadas: proporÃÃo entre os substratos, solvente e temperatura. A quantidade de acetato de retinila foi mantida em 0,1 mmol e a do Ãcido palmÃtico variando entre 0,1 e 0,5 mmol. Levando em consideraÃÃo o coeficiente de partiÃÃo do Ãcido utilizado, foram testados tolueno e hexano. As temperaturas variaram entre 25 e 40ÂC, de acordo com o planejamento fatorial 23 blocado com ponto central. No segundo, estudou-se a influÃncia da quantidade de lipase (25, 50 e 75 mg) e PM (20, 50, 80 mg) em tolueno e hexano, conforme planejamento fatorial 32. Ensaios em maior escala foram realizados nÃo apenas para o palmitato, o qual foi submetido a teste de estabilidade tÃrmica, mas tambÃm para o adipato, que por nÃo ser comercializado precisou ser separado da reaÃÃo e identificado por ressonÃncia magnÃtica nuclear. Com uma anÃlise estatÃstica dos resultados, pÃde-se observar que os parÃmetros que tiveram efeito significativo no primeiro planejamento, foram a temperatura e a interaÃÃo desta com a razÃo molar entre os substratos. No segundo, tanto a enzima como a relaÃÃo quadrÃtica da PM foram significantes no rendimento de sÃntese com tolueno, e apenas a enzima, quando utilizado o hexano. A separaÃÃo do palmitato de retinila foi realizada em coluna de sÃlica C18, tendo sido avaliada em calorimetria exploratÃria diferencial (do inglÃs, differencial screening calorimetry - DSC) e observado eventos tÃrmicos por volta de 6,54ÂC. Quanto ao adipato de retinila, nenhum procedimento de separaÃÃo foi eficaz para a separaÃÃo da mistura formada entre o mesmo e o adipato de diretinila. / The main objective of this work was to synthesize vitamin A derivatives through an enzymatic route, as an alternative to chemistry route, more aggressive to the environment. The conversion of retinyl acetate into retinyl palmitate would result in a product with better market acceptance, since it is more stable than the ester used as substrate. Retinyl adipate synthesis, on the other hand, was studied in order to prepare a new vitamin A derivative. Both Vitamin A derivatives synthesized in this work were aiming the industrial production of cosmetics and foods. In this context, immobilized Candida antarctica type B lipase (Novozyme 435 with 571,48 UI/g  55,47) was used to catalyze the conversion of the substrates retinyl acetate, palmitic and adipic acids. In addition to these, molecular sieves was also added since the proposed reactions release water to the reaction media, which is not favorable to the desired ester synthesis. The retinyl palmitate synthesis was investigated by two factorial experimental design, using a total reactional volume of 2 mL. In the first, three variables were analyzed: rate between substrates, type of solvent and temperature. Retinyl acetate was kept in 0,1 mmol and palmitic acid varied between 0,1 and 0,5 mmol. Considering the acid partition coefficient, toluene and hexane were tested as solvents. The temperatures varied between 25 and 40ÂC, following a 23 factorial experimental design blocked with central points. In the second design, the influence of lipase (25, 50 e 75 mg) and molecular sieves (20, 50, 80 mg) amounts were studied using toluene or hexane as solvent, in accordance with a 32 factorial design. Experiments in a larger scale were performed not only to the produce retinyl palmitate, which was submitted to termic stability tests, but also to retinyl adipate, which is not commercially available and thereof it was recovered from the reactional media and identified by nuclear magnetic resonance. The statistical analysis of the results allowed the observation of significant effects. In the first planning, temperature and their interaction with the molar rate between the substrates were the significant variables. In the second, enzyme and the molecular sieves quadratic relation were significant in the yield of synthesis with toluene, but only the enzyme was significant when hexane was utilized. The retinyl palmitate separation was performed in silica C18 column and the purified sample was analyzed by differential scanning calorimetric â DS with a thermal event around 6.54ÂC. In the case of retinyl adipate, no separation procedure was effective since there is a mixture formed between retinyl and diretinyl adipates.