Streptomyces clavuligerus is a Gram-positive, filamentous bacterium which produces several important secondary metabolites, including isopenicillin N, cephamycin C and the &beta / -lactamase inhibitor clavulanic acid. Among these compounds, clavulanic acid is being used in combination with commonly used &beta / -lactam antibiotics in order to fight against bacterial infections that are resistant to such antibiotics. Among these combinations, Augmentin, composed of amoxicillin and clavulanic acid, is the most widely prescribed drug and has a market value of more than one billion dollars per year. There are two genes that act in regulation of clavulanic acid biosynthesis: ccaR located in cephamycin C gene cluster and claR located in clavulanic acid gene cluster. The goal of this study is to improve clavulanic acid production capacities of both wild type and industrial S. clavuligerus strains by integrating extra copies of claR gene into S.clavuligerus genome and its overexpression via a multicopy plasmid. Although previously has shown to be quite effective on wild type S. clavuligerus strains, claR overexpression in the industrial strain used in this study yielded only 1.4-fold increase in volumetric and 1.7-fold increase in specific CA production by the recombinant strains MA28 and MA16, respectively.

Streptomyces clavuligerus is a gram-positive, filamentous bacterium which has a great ability to produce secondary metabolites including isopenicillin N, cephamycin C and a beta-lactamase inhibitor clavulanic acid. Clavulanic acid (CA) which is a bicyclic beta-lactam, inhibits most of class A beta-lactamases by binding irreversibly to the serine hydroxyl group at the active center of beta-lactamases and resulting in the stable acyl-enzyme complexes. Clavaminate synthase (CAS) is one of the best characterized enzymes in the CA biosynthesis pathway regarding its mechanism, activity and structure. It exists as two isoenzymes, CAS1 and CAS2. cas1 gene is located in clavam biosynthetic gene cluster and its expression is nutritionally regulated. cas2 gene encodes for the rate limiting CAS2 isoenzyme which catalyzes the three distinct oxidative transformations. Conversion of deoxyguanidinoproclavaminic acid into guadinoproclavaminic acid takes part in early steps of CA biosynthesis and is catalysed by CAS2. Similarly, proclaviminic acid is converted into claviminic acid by a two-step reaction and also catalysed by CAS2. Integration of an extra copy of the cas2 gene into the chromosome of a clavulanic acid over-producer industrial strain of Streptomyces clavuligerus by means of an integration vector to improve CA production capacity of the industrial strain is the main goal of this study. Via comperative CA analysis based on HPLC, it was estimated that the recombinant strains produced higher amount of CA in comparison to the parental industrial S. clavuligerus strain. The highest CA production achieved by the recombinant strain, namely S. clavuligerus GV61 (1583.3 &mu / g/g) corresponded to more than 2 fold increase in the maximal CA titer of the parental strain.

El objetivo de esta tesis ha sido estudiar y desarrollar metodologias analíticasusando un sistema de inyección secuencial (SIA) con un espectrofotómetro de diodos enfila para obtener datos de segundo orden. Para tratar estos datos, las herramientasquimiométricas usadas han sido; resolución de curvas multivariante mediante mínimoscuadrados alternados (MCR-ALS) y otras técnicas relacionadas a ésta como el análisisde componentes principales (PCA) y SIMPLISMA. Además se han aplicado estrategiasde diseño de experimentos para obtener las condiciones experimentales óptimas. Estametodología se aplicó a la determinación de amoxicilina y ácido clavulánico enmedicamentos.El primer capítulo de la tesis contiene una descripción de la amoxicilina y del ácidoclavulánico, una explicación de los fundamentos teóricos tanto del sistema instrumentalcomo de las herramientas quimiométricas usadas y por último, se describen los diseñosde experimentos usados y la función de deseabilidad.En los dos siguientes capítulos, se muestran en forma de artículos científicos lostrabajos experimentales realizados. En un primer artículo, se realizó una clasificación delos medicamentos dependiendo si se tenían interferentes o no, para posteriormenteproponer el tipo de calibrado. Un paso previo a la diferenciación de los medicamentos,fue buscar una secuencia analítica que permitiera obtener un sistema en evolución. Estaetapa se llevó a cabo mediante un diseño de experimentos.En el segundo artículo, se determinó la cantidad de amoxicilina en losmedicamentos que tenían interferentes y además no tenían zonas selectivas. Para llevara cabo de forma correcta la etapa de calibración se realizó un estudio de una serie deparámetros asociados a MCR-ALS. En un tercer artículo se realizó la determinaciónsimultánea del ácido clavulánico y de la amoxicilina que poseían unas característicasácido-base y una sensibilidad espectral similar. Por tal de determinar simultáneamenteambos analitos se rediseñó todo el experimental. En el cuarto artículo se hizo unarevisión bibliográfica de ambas técnicas a partir del año 2004 y se discutió el potencial deusar un sistema de inyección secuencial para generar datos de segundo orden.Con la experimentación realizada se comprobó que el paso clave en estasmetodologias era obtener una buen sistema en evolución, es decir diseñar una buenasecuencia analítica. Por lo que se profundizó en estrategias basadas en diseños deexperimentos. En el quinto artículo, se estudiaron qué factores podían afectar a lasecuencia analítica. También se propusieron respuestas que representaran de una formacuantitativa una buena resolución. Se realizó un diseño Plackett-Burman con el objetivode eliminar los factores no relevantes, para posteriormente modelar una superficie derespuesta a partir de los factores relevantes que permite visualizar las condicionesóptimas de la secuencia analítica.El inconveniente de utilizar la metodología de superficie de respuesta es que enlos casos donde el número de factores relevantes sea superior a 3 o 4, el número deexperiencias aumenta considerablemente. En estos casos, una técnica alternativa essimplex que dio lugar a un sexto artículo.El último capítulo de la tesis contiene las conclusiones. Como una conclusióngeneral, se puede decir que la combinación de un sistema de inyección secuencial (SIA)y una herramienta quimiométrica como la resolución de curvas multivariante mediantemínimos cuadrados alternados (MCR-ALS) puede ser usado tanto para realizar unanálisis cualitativo y cuantitativo ya que proporciona información de los perfiles deconcentración y perfiles espectrales de las diferentes especies a estudio. / The objective of this thesis is to study and develop analytical methods to determineamoxicillin and clavulanic acid in pharmaceuticals using sequential injection analysis (SIA)with a diode-array spectrophotometric detector to obtain second-order data. To treat thesedata, the chemometric tool used was; multivariate curve resolution with alternating leastsquares (MCR-ALS) and the techniques involved in the resolution process are: principalanalysis components (PCA) and simple-to-use interactive self-modelling mixture analysis(SIMPLISMA).The first chapter contains a brief description of the theoretical backgrounds thathave been used during this thesis. We explain the characteristics and properties ofamoxicillin and clavulanic acid, we describes the instrumental and the chemometric toolsused and at the end, we introduce the experimental designs used and the desirabilityfunction.In the next two chapters contain the bulk of the work carried out for this thesis andincorporate papers published in journals. In the first paper, the pharmaceuticals wereclassified according to their selective zones in order to propose the type of calibration. In aprevious step, the experimental work was conducted to find an analytical sequence thatallows us to obtain an evolving system. This step was carried out using experimentaldesign. In the second paper, the quantity of amoxicillin in the pharmaceuticals withinterferents or without selective zones was determined. To carry out correctly thecalibration step, we studied different conditions related to the MCR-ALS process.In the third paper, we propose the simultaneous determination of amoxicillin andclavulanic acid which they have the acid-base characteristics and spectral profile similar.To determine both analytes, a new analytical sequence was redesigned. In the fourthpaper, we describe the state of the art of sequential injection analysis (SIA) andmultivariate curve resolution with alternating least squares (MCR-ALS) by reviewing thebibliography since 2004. We discuss the potential of SIA for generating second-orderdata.In previous papers, we found that the most critical step in the development ofanalytical methods based on SIA and MCR-ALS was to obtain an analytical sequence thatprovides an evolving system. To resolve so, we developed the method of experimentaldesign to obtain the optimal analytical sequence.In the forth paper, we studied all the factors and analysed how they affect to theanalytical sequence. We also proposed responses to quantitatively represent a goodresolution. Once these factors and responses were proposed, we used a Plackett-Burmandesign to remove the non-relevant factors and then modelled a response surface. In themaximum of response surface, the optimum conditions for the analytical sequence couldbe visualised. To transform several responses into a single response, we used the overalldesirability function. In the sixth paper, we applied an alternative optimisation methodknows as the simplex approach. We aimed to determine amoxicillin and clavulanic acidsimultaneously when the number of factors and responses was higher than in theprevious paper.The last chapter contains the conclusions of the thesis. In general, we concludethat a combined sequential injection analysis (SIA) with a multivariate detector (i.e. diodearray spectrophotometer) and multivariate curve resolution with alternating least squares(MCR-ALS) can be used for both qualitative and quantitative analyses since, it providesconcentration and spectra profiles for the different species of the sample.

No description available.

Orientadores: Maria Isabel Rodrigues, Francisco Maugeri Filho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-10T13:37:00Z (GMT). No. of bitstreams: 1 Forte_MarcusBrunoSoares_M.pdf: 5091221 bytes, checksum: f695e6ea266b0c682b2b8ce3dcc509ae (MD5) Previous issue date: 2008 / Resumo: O presente trabalho teve como objetivo principal a separação e purificação de acido clavulanico a partir de caldo fermentado utilizando zeolitas. Foram utilizadas zeolita natural (ZN) e sintetica faujasita (13X), ambas modificadas por troca ionica com diferentes cations de compensação (Na+1, K+1, Ca+2, Ba+2, Mg+2, Sr+2). Atraves de estudos cineticos de adsorcao de AC, usando as diferentes zeolitas nas respectivas formas cationicas, selecionou-se a zeolita 13X-Na como a mais promissora na adsorcao do referido composto. No equilibrio, houve retencao de 17,4% do AC inicial (CAC*/CAC0 = 0,826) e a quantidade de AC adsorvida, em relacao a quantidade de zeolita (q*) foi 0,4927 mg/g. O diametro médio da molecula de AC (DAC) foi estimado em 9,6 A. A zeolita 13X-Na foi caracterizada em termos de composicao (Si/Al = 1,5), densidade (dz = 2,248 g/cm3), área superficial (SBET = 444,860 m2/g), volume total de poros (Vporos = 0,308 cm3/g), volume de microporos (Vmicro = 0,203 cm3/g) e diâmetro médio de poros (Dporos = 28 A). Através desses resultados, a porosidade da particula calculada foi ep = 0,69. O leito de particulas de 13X-Na apresentou porosidade eb = 0,85. Soluções de AC puro foram obtidas através de HPLC em escala semi-preparativa. Isotermas de adsorção nas temperaturas 10, 15 e 200C, usando 13X-Na e soluções de AC puro, foram determinadas e constatou-se que, nos intervalos de temperaturas e concentrações estudados, o aumento da temperatura desfavoreceu tal processo. A partir de injeções de caldo fermentativo em coluna de leito fixo de zeolitas 13X-Na, foram observados melhores resultados na separação de acido clavulanico e proteínas usando tampão fosfato (0,2 M e pH 6,2) como efluente e frações de zeolitas na granulometria 0,053 - 0,062 mm. A eficiência de separação (ES), fator concentração (FC), fator de purificação referente as proteínas (FPP) e fator de purificação referente aos contaminantes totais (FPC) apreciados nessas condições foram, respectivamente: ES = 0,38, FC = 0,40, FPP = 1,25 e FPC = 1,28 / Abstract: In this research work, a method for separation and purification of clavulanic acid from a fermented medium was studied using natural and synthetic zeolites. The natural zeolite (ZN) and synthetic faujasite (13X) were modified by ionic exchange with different compensation cations (Na+1, K+1, Ca+2, Ba+2, Mg+2, Sr+2). The kinetic analysis by adsorption of clavulanic acid indicated that the zeolite 13X-Na was the most promising for the adsorption of the related compound. At the equilibrium conditions, the retention was about 17.4% (CAC*/CAC0 = 0.826), where the amount adsorbed clavulanic acid relative to the amount of zeolite (q*) was 0.4927 mg/g. The average diameter of a clavulanic acid molecule (DAC) was estimated as 9.6 A. The composition of the zeolite 13X-Na was characterized in terms of the ratio Si/Al (1.5), density (dz = 2.248 g/cm3), superficial area (SBET = 444.86 m2/g), total pore volume (Vpores = 0.308 cm3/g), micropore volume (Vmicro = 0.203 cm3/g) and average pore diameter (Dpores = 28 A). Accordingly, particle and bed porosity (ep and eb) were calculated as 0.69 and 0.85 respectively. Solutions of pure clavulanic acid were obtained in semi-preparative scale HPLC. The adsorption isotherm for 13X-Na and solutions of pure clavulanic acid were determined for the temperatures of 10, 15 and 200C, showing at increasing temperature, the performance of the process decreases. Addition of fermented medium in the fixed bed column of Zeolites 13X-Na improved the separation process between clavulanic acid and contaminant proteins, using as effluent phosphate buffer (0.2 M and pH 6.2) and zeolites with particle diameters in the range of 0.053 to 0.062 mm. The separation efficiency (ES), factor concentration (FC), purification factor, referred to proteins (FPP), and purification factor referred to the contaminants (FPC) were determined, as ES = 0.38, FC = 0.40, FPP = 1.25 and FPC = 1.28 respectively / Mestrado / Mestre em Engenharia de Alimentos

Ácido clavulânico (AC) é um potente inibidor de β-lactamases, produzido por Streptomyces clavuligerus, usado clinicamente em combinação com antibióticos β-lactâmicos para tratar infecções bacterianas resistentes. Apesar da produção industrial de AC já ser bem estabelecida muitos aspectos importantes relacionados com sua biossíntese permanecem carentes de estudo. Sabe-se que a via de síntese do AC envolve no mínimo 8 passos enzimáticos sendo os primeiros passos mais abordados. Por exemplo, as enzimas N2-(2-carboxietil) arginina sintase (CEAS), β-lactama sintase (BLS) e proclavaminato amidino hidrolase (PAH) são as responsáveis pela primeira, segunda e quarta reações enzimáticas respectivamente. Estudos mutagênicos recentes em S.clavuligerus relacionaram cópias extras dos genes ceas, bls e pah (ceas1, bls1 e pah1) com essa via porém nenhum ensaio enzimático foi relatado. Embora os passos finais da via ainda não estejam completamente estabelecidos, a ação de algumas enzimas putativas, como a codificada por orf12, mostraram ser essenciais a produção do AC. Assim, com o objetivo de aumentar a informação disponível sobre a biossíntese do AC estudamos quatro de seus membros: CEAS1, BLS1, PAH1 e a proteína putativa codificada pela orf12. Os genes foram isolados a partir do DNA genômico de S. clavuligerus por PCR e clonados em vetores para produção das proteínas recombinantes em E.coli. Os protocolos de expressão foram estabelecidos para CEAS1, PAH1 e ORF12 e as proteínas recombinantes foram purificadas por cromatografia de afinidade por metal. BLS foi obtida de forma isolúvel. As proteínas solúveis foram caracterizadas por meio de técnicas bioquímicas e estruturais. As análises de CEAS1 e PAH1 foram comparadas com informações já obtidas para as isozimas CEAS2 e PAH2, respectivamente. Assim, as análises de oligomerização das proteínas resultaram em uma mistura de oligômeros (monômero, dímero e tetrâmero) para CEAS1, na forma hexamérica para PAH1 e na forma dimérica para ORF12, estando de acordo com as formas solúvel e cristalográfica de CEAS2 (dímero e tetrâmero) e PAH2 (hexâmero). Espectros de dicroísmo circular mostraram que CEAS1 e PAH1 possuem um enovelamento do tipo α/β sendo estáveis até 35ºC e numa ampla faixa de pH. Os parâmetros termodinâmicos da interação entre CEAS1 e o cofator Mg+2 foram determinados mostrando que é entropicamente dirigida, com uma estequiometria de ligação de 4 : 1, com uma constante de afinidade na ordem de micromolar (KD = 1,76 ± 0.23 µM). Análises realizadas com as técnicas de reação acoplada, de Cromatografia Líquida de Alta Pressão acoplada a Espectrometria de Massas (LC-MS) e de Calorimetria de Titulação Isotérmica mostraram que CEAS1, assim como CEAS2, apresenta atividade sob o substrato gliceraldeído-3-fosfato, porém sem a formação do produto final N2-(2-carboxietil)arginina. Por outro lado, a proteína recombinante PAH1 mostrou ser inativa sobre o substrato análogo, N-α-acetil-L-arginina. Assim, apesar das isozimas manterem um padrão estrutural, podem ter mecanismos de ação distintos. Em relação a ORF12 esta proteína foi classificada com uma β-lactamase com atividade esterase de acordo com nossos estudos realizados com os substratos cefalosporina C e p-nitrofenil acetato. / Clavulanic acid (CA) is a potent inhibitor of β-lactamases, produced by Streptomyces clavuligerus, clinically used in combination with β-lactam antibiotics to treat resistant bacterial infections. Although CA industrial production is well-established, many important aspects related to its biosynthesis remains under study. It is known that CA pathway involves at least 8 enzymatic steps, being the earliest stages more addressed. For instance, N2-(2-carboxyethyl) arginine synthase (CEAS), β-lactam synthase (BLS) and proclavaminate amidinohydrolase (PAH) are responsible for the first, second and fourth enzymatic reaction, respectively. Recent mutagenic studies in S.clavuligerus have related extra copies of ceas, bls and pah genes ((ceas1, bls1 e pah1) to this pathway but none enzymatic assay was further reported. Although later stages the pathway remain unclear, the action of some putative enzymes like the codified by orf12 showed essential to CA production. Thus, aiming to increase the information available about CA biosynthesis we studied four of its members: CEAS1, BLS1, PAH1, and the putative protein codified by orf12. The genes were isolated from S.clavuligerus genomic DNA by PCR and further cloned into expression vectors in order to produce recombinant proteins in E.coli. Protocols of protein expression were established to CEAS1, PAH1 and ORF12 and recombinant proteins were purified by metal affinity chromatography. BLS was obtained as an insoluble form. Soluble proteins were characterized by means of biochemical and structural approaches. Analyses of CEAS1 and PAH1 were compared with information ever conducted to the isozymes CEAS2 and PAH2, respectively. Thus, oligomerization analysis of proteins resulted respectively in a mix of oligomers forms (monomer, dimer, tetramer) to CEAS1, hexameric form to PAH1 and dimeric form to ORF12, according to the soluble and crystallographic form of CEAS2 (dimer and tetramer) and PAH2 (hexamer). Circular Dichroism spectra showed that CEAS1 and PAH1 have an α-β conformation and were stable up to 35ºC over a wide pH range. Thermodynamic parameters of CEAS1 cofactor (Mg+2) binding were determined showing that is entropic driven, with a 4:1 binding stoichiometry, with a micro-molar affinity (KD = 1.76 ± 0.23 µM). Analyses by coupled assay, High Pressure Liquid Chromatography coupled to Mass Spectroscopy (LC-MS) and Isothermal Titration Calorimetry showed that CEAS1, as well as the CEAS2, presents activity at the substrate glyceraldehydes-3-phosphate, however without formation of final product, N2-(2-carboxyethyl)arginine. Meanwhile recombinant PAH1 showed none activity at analogous substrate, N-α-acetil-L-arginine. Thus despite isozymes maintain a structural pattern, they may have distinct action mechanism. Regards to ORF12, this protein was classified as a β-lactamase with an esterase activity according to our studies performed with the substrates cephalosporin C and p-nitrophenyl acetate.

Orientadores: Francisco Maugeri Filho, Maria Isabel Rodrigues / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-21T20:28:07Z (GMT). No. of bitstreams: 1 Forte_MarcusBrunoSoares_D.pdf: 4684118 bytes, checksum: 7ad3d65f4a3a6f6e525b286c594a4a22 (MD5) Previous issue date: 2013 / Resumo: A proposta desta tese foi o estudo da adsorção do ácido clavulânico (CA) em hidróxidos duplos lamelares (LDH) e o uso desses adsorventes em colunas de leito fixo na separação entre o CA e aminoácidos. A tese foi desenvolvida em três etapas: na primeira, utilizou-se LDH do tipo hidrotalcitas. Os adsorventes foram avaliados quanto à cinética de adsorção em banho finito. O melhor sistema foi a hidrotalcita com 70 % de magnésio na composição (HT70c) com fração de sólidos (em relação ao volume de líquido) no reator est = 15,0 mg/mL usando água como solvente. O modelo de equilíbrio melhor ajustado foi o linear com os seguintes coeficientes de adsorção: k = 0,404; 0,602; 0,849 e 1,083 L/g para 16,5; 19,0; 21,5 e 24,0°C, respectivamente, evidenciando um aumento da capacidade de adsorção com aumento da temperatura. O sistema foi avaliado termodinamicamente quanto à energia livre de Gibbs (?G°), entalpia (?H°) e entropia (?S°), sendo que os valores calculados foram: ?G° = 2,100; 1,301; 0,503 e -0,295 kJ/mol para 16,5; 19,0; 21,5 e 24,0°C, respectivamente, ?H° = 94,602 kJ/mol e ?S° = 0,319 kJ/mol.K. A segunda etapa foi desenvolvida no Laboratório de Materiais Inorgânicos da Université Blaise Pascal na França, durante Estágio de Doutorado-Sanduíche. LDH com outras composições foram sintetizados, caracterizados e avaliados quanto à adsorção de CA. A difração de raios X indicou a formação de uma estrutura cristalina com uma distância entre as lamelas de aproximadamente 22 Å. Os espectros na região do infravermelho apresentaram sinais característicos da molécula de CA: ligações C=C (720 ¿ 840, 1640 e 1657 cm-1), C-O-C e C-N (1036, 1060 e 1292 cm-1), C-N (1354 cm-1), C-OH (1392 cm-1) e COOH (1551, 1581 cm-1). A análise termogravimétrica indicou a presença de uma molécula interlamelar com peso molecular de aproximadamente 194 g/mol. Essas análises confirmam a presença do CA no espaço interlamelar e, desta forma, a afinidade da molécula de CA pelos sítios dos LDH. A adsorção de CA em diferentes fases de LDH (Zn2Cr-NO3, Zn2Al-NO3 e Mg2Al-NO3) foi avaliada por meio de isotermas de adsorção a 20,0; 30,0 e 35,0°C. O sistema escolhido como mais conveniente foi o Zn2Cr-NO3, cujos parâmetros ajustados para o modelo de Freundlich foram kF = 73,63; 128,76 e 229,62 mg1-nF.LnF/g e nF = 0,35; 0,38 e 0,33 para 20,0; 30,0 e 35,0°C. Na terceira etapa,partículas contendo cristais de Zn2Cr-NO3 encapsulados em microgéis de alginato (LDHME) foram obtidas através de gelificação iônica, a fim de possibilitar o uso desses adsorventes em colunas cromatográficas. Uma coluna foi empacotada com as partículas de LDHME a fim de avaliar a separação de CA em relação aos aminoácidos tirosina (TYR) e prolina (PRO), já que são os principais contaminantes presentes no meio fermentativo, pelo método de pulso cromatográfico. O uso das partículas de LDHME na separação de CA foi considerado satisfatório levando a fatores de purificação de 2,32 vezes o inicial e a um grau de pureza das soluções de 93 %. Um modelo matemático foi desenvolvido para adsorção de ácido clavulânico (CA) em coluna de leito fixo, empacotada com partículas LDHME. Curvas de ruptura foram obtidas experimentalmente e o sistema foi avaliado em relação aos tempos de operação assim como eficiências e produtividades em função de variações simultâneas das condições experimentais, vazão (Q) e altura de leito (L), através de um delineamento composto central rotacional (DCCR). A vazão foi avaliada no intervalo de 0,19 a 0,82 mL/min e a altura do leito de 3,0 a 5,0 cm. O algoritmo particle swarm optimization (PSO) se mostrou eficaz na estimação dos parâmetros do modelo proposto. Depois de validado experimentalmente em condições distintas das usadas na estimação, o modelo foi usado como ferramenta na otimização da adsorção de CA através de um DCCR, cujos resultados foram obtidos com simulações usando níveis superiores para as variáveis de entrada (Q de 0,80 a 1,50 mL/min e L de 7,0 a 10,0 cm) considerando somo respostas o tempo de ruptura (tb), tempo de exaustão (te), eficiência de recuperação (frec), eficiência de utilização da coluna (fcol) e a produtividade (P). Esse procedimento foi considerado satisfatório já que houve diminuições consideráveis nos tempos de operação e aumentos nas respectivas eficiências e produtividades. As melhores condições de operação do sistema estudado para adsorção de CA foram: Q = 1,15 mL/min e L = 10,0 cm, cujas respostas foram: tb = 24 min, te = 110 min, frec = 96 %, fcol = 37 % e P = 117 kg/h / Abstract: The main focus of this thesis was to study the adsorption of clavulanic acid (CA) in layered double hydroxides (LDH) and to use these adsorbents in fixed bed columns for separation of CA from amino acids. The thesis was developed in three steps: first, it was used LDH-type hydrotalcites. Adsorption studies were performed in stirred batch glass reactors. It was shown that distilled water and a solid/liquid ratio of 15.0 mg/L generated the most favourable conditions for adsorption. A calcined hydrotalcite containing 70 % of MgO was selected for further study since it presented the best adsorption performance. Adsorption equilibrium was evaluated from adsorption isotherms determined at different temperatures. The experimental isotherm data were well fitted by a linear equilibrium model with the corresponding adsorption constants being klin = 0.404, 0.602, 0.849 and 1.083 L/g at 16.5, 19.0, 21.5 and 24.0°C, respectively, showing an increased adsorption capacity at higher temperatures. Thermodynamic evaluation of the process allowed the Gibbs¿ free energy (?G°), the standard enthalpy change (?H°) and the standard entropy change (?S°) to be estimated as follows: ?G° = 2.100, 1.301, 0.503 and ¿0.295 kJ/mol at 16.5, 19.0, 21.5 and 24.0°C, respectively; ?H° = 94.602 kJ/mol; and ?S° = 0.319 kJ/(mol.K), respectively. The second step was developed at the Laboratory of Inorganic Materials of the Blaise Pascal University France during a doctoral internship in France. Different LDH compositions have been investigated: Zn2Cr-Cl, Zn2Cr-NO3, Zn2Al-NO3 and Mg2Al-NO3. Zn2Cr-CA hybrid LDH assemblies were prepared through coprecipitation method to evaluate the affinity between the CA molecule and the interlayer sites. Experiments were carried out using two different sources of CA, i.e. the pharmaceutical product Clavulin® and Clavulanate: Avicel (1:1). The resulting inorganic-organic "sandwich" structure was characterized by a combination of techniques showing an expansion of the layered structure from 0.78 nm to values between 2.30 and 2.60 nm upon CA intercalation; a molecular sieve role of LDH host is also pointed out. Isotherms studies were carried out to evaluate the adsorption capacity of LDH towards CA in presence of contaminants such as amino acids. The Freundlich adsorption model was found to fit the data well and indicating relatively large adsorption capacity, as high as 73.63, 128.76 and 229.62 mg1-nFLnFg-1 at 20, 30 and 35°C, respectively. Besides, the adsorption of CA is classified as an endothermic and spontaneous process with the following calculated thermodynamic parameters: ?H° = 20.516 kJ/mol, ?S° = 0.081 kJ/mol.K and ?G° = -3.271, -4.082, -4.488 kJ/mol for 20, 30 and 35°C, respectively. Particularly, the adsorption of CA onto Zn2Cr-NO3 is a favorable process thus allowing us to envisage the use of LDH in CA biomolecule separation. In the third step, microparticles containing crystals of Zn2Cr-NO3 microencapsulated in alginate (LDHME) were obtained by ionic gelation in order to allow the use of these adsorbents in chromatography columns. A column was packed with particles of LDHME to evaluate the separation of CA from mixtures with amino acids such as tyrosine (TYR) and proline (PRO) using the chromatographic pulse methodology. The use of these LDHME microparticles in the CA separation was satisfactory leading to purification factors of 2.32 times the original and a purity of 93% of the solutions. A mathematical model has been developed for adsorption of CA in a fixed bed column packed with LDHME particles. Breakthrough curves were obtained and the system was experimentally assessed in relation to the time of operation as well as efficiencies and yields due to simultaneous variations of experimental conditions, flow rate (Q) and bed height (L) using a central composite rotatable design (CCRD), considering as responses the breakthrough time (tb), the exhaustion time (te), the recovery efficience (frec), the column utilization efficience (fcol) and the process productivity (P). The flow rate was assessed in the range from 0.19 to 0.82 mL/min and the bed height from 3.0 to 5.0 cm. The particle swarm optimization algorithm (PSO) was shown to be effective in the estimation of the parameters of the proposed model. Once validated experimentally under different conditions from those used in the estimation, the model has been used as a tool in optimizing the adsorption of CA by a CCRD, whose results were obtained using simulations with higher levels for the input variables (0.80 < Q < 1.50 mL/min and 7.0 < L < 10.0 cm). This procedure was considered satisfactory since there were significant reductions in operating times and increases in their productivity and efficiencies. The best operating condition of the system studied for CA adsorption were: Q = 1.15 mL/min and L = 10.0 cm, the answers were: tb = 24 min, trec = 96 %, frec = 110 min, frec = 37% and P = 117 kg/h / Doutorado / Engenharia de Alimentos / Doutor em Engenharia de Alimentos

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Professional nurses and their patients are directly influenced by insufficient medication, causing a decrease in the quality of care, delays in hospitalisation and it might lead to resistance. In some cases professional nurses have to leave the unit in search of medicine. Amoxicillin-clavulanic acid 1.2 gram for intravenous administration is prescribed to the majority of patients in the medical units in public South African hospitals. Yet there are intermitted insufficient stock levels and challenged inventory systems for amoxicillin-clavulanic acid 1.2 gram in some public hospitals. This fact is positioned against the background of a South African health system that has undergone major changes since the fall of Apartheid in 1994 and amidst major positive changes, is still challenged by overburdened hospital admissions and a quadruple disease burden. The aim of this research was to enhance optimal levels of amoxicillin-clavulanic acid 1.2 gram in medical units in public hospitals to ensure sufficient stock levels and timeous administration. The aim was achieved by identifying and describing the current supply chain of intravenous amoxicillin-clavulanic acid 1.2 gram in two medical units in a district (level 2) public hospital in the North West Province (from here referred only as North West) by identifying inefficiencies in the current supply chain and to formulate recommendations for management to enhance the supply chain of intravenous amoxicillin-clavulanic acid 1.2 gram to medical units in public hospitals. An exploratory case study approach was followed to explain the supply chain of amoxicillin-clavulanic acid 1.2 gram by utilising a qualitative, descriptive, explorative and contextual design. A case study approach was chosen as it examined single units within the context of real life as environment, which in this case were medical units in a level two public hospital, North West. The case selection was motivated and described, followed by case records of policies and standard operational procedures. Field participants included all levels of nurses (professional, enrolled and auxiliary) in medical male and female units on day and night duty, and the head of pharmacy [n=8]. Non-probable, purposive sampling was conducted according to inclusion criteria after all levels of ethical clearance and consent were granted. Three semi-structured individual interviews followed, after which two focus groups were conducted. Thematic analysis of transcriptions was done, followed by an analysis of case records regarding where after all results were integrated. Results indicated complex organisational, unit-specific and behavioural challenges that impact on the supply chain management of amoxicillin-clavulanic acid 1.2 gram and insufficient stock levels are predominantly positioned within retailer and customer aspects of the supply chain. Despite well-formulated standard operational procedures, the realisation thereof lacks, implicating a greater need for managerial control. Recommendations were formulated for management to enhance the supply chain of amoxicillin-clavulanic acid 1.2 gram in medical units in public South African hospitals integrated with good pharmacy practices. The close collaboration, mutual respect and effective communication between health professionals in the multi-professional team are reiterated. / MCur, North-West University, Potchefstroom Campus, 2015

Professional nurses and their patients are directly influenced by insufficient medication, causing a decrease in the quality of care, delays in hospitalisation and it might lead to resistance. In some cases professional nurses have to leave the unit in search of medicine. Amoxicillin-clavulanic acid 1.2 gram for intravenous administration is prescribed to the majority of patients in the medical units in public South African hospitals. Yet there are intermitted insufficient stock levels and challenged inventory systems for amoxicillin-clavulanic acid 1.2 gram in some public hospitals. This fact is positioned against the background of a South African health system that has undergone major changes since the fall of Apartheid in 1994 and amidst major positive changes, is still challenged by overburdened hospital admissions and a quadruple disease burden. The aim of this research was to enhance optimal levels of amoxicillin-clavulanic acid 1.2 gram in medical units in public hospitals to ensure sufficient stock levels and timeous administration. The aim was achieved by identifying and describing the current supply chain of intravenous amoxicillin-clavulanic acid 1.2 gram in two medical units in a district (level 2) public hospital in the North West Province (from here referred only as North West) by identifying inefficiencies in the current supply chain and to formulate recommendations for management to enhance the supply chain of intravenous amoxicillin-clavulanic acid 1.2 gram to medical units in public hospitals. An exploratory case study approach was followed to explain the supply chain of amoxicillin-clavulanic acid 1.2 gram by utilising a qualitative, descriptive, explorative and contextual design. A case study approach was chosen as it examined single units within the context of real life as environment, which in this case were medical units in a level two public hospital, North West. The case selection was motivated and described, followed by case records of policies and standard operational procedures. Field participants included all levels of nurses (professional, enrolled and auxiliary) in medical male and female units on day and night duty, and the head of pharmacy [n=8]. Non-probable, purposive sampling was conducted according to inclusion criteria after all levels of ethical clearance and consent were granted. Three semi-structured individual interviews followed, after which two focus groups were conducted. Thematic analysis of transcriptions was done, followed by an analysis of case records regarding where after all results were integrated. Results indicated complex organisational, unit-specific and behavioural challenges that impact on the supply chain management of amoxicillin-clavulanic acid 1.2 gram and insufficient stock levels are predominantly positioned within retailer and customer aspects of the supply chain. Despite well-formulated standard operational procedures, the realisation thereof lacks, implicating a greater need for managerial control. Recommendations were formulated for management to enhance the supply chain of amoxicillin-clavulanic acid 1.2 gram in medical units in public South African hospitals integrated with good pharmacy practices. The close collaboration, mutual respect and effective communication between health professionals in the multi-professional team are reiterated. / MCur, North-West University, Potchefstroom Campus, 2015