Adsorption and ion exchange processes for Cephamycin C purification

Cephamycin C (cepC) is a beta-lactam antibiotic produced by the actinomycetes Streptomyces clavuligerus and Nocardia lactamdurans. It is an important compound for the pharmaceutical industry because it is a raw material for some commercial antibiotics, named as cefoxitin and cefotetan. There are few studies in the literature about purification processes of this antibiotic, and most of the information available is described in patents. It has already been demonstrated that the use of the anionic resin Q Sepharose XL (QXL) in a fixed-bed column was effective in extracting cepC from the broth during column loading. However, the study did not show the separation of any contaminants during elution step, as separated peaks were not observed. Therefore, the method of column operation should be optimized and a deeper study should be carried out. In this study, three operations in sequence for cepC purification were evaluated, including a different operational method of the fixed-bed column using the resin QXL. The operations consisted of adsorption onto the neutral resin Amberlite XAD4 in a stirred reactor; fixed-bed column process using the resin QXL; and adsorption onto a C18 SPE cartridge. Ultrafiltered broth obtained after fermentation with S. clavuligerus was used. Contaminants were monitored during all steps, by different techniques: absorbance measurements between 310 to 400 nm, which are wavelengths that cepC does not absorb; biological assays using Escherichia coli ESS, that is a bacteria sensitive to cepC; mass spectrometry analyses. During adsorption in the stirred reactor with XAD4 resin, 27% of cepC and 44% of contaminants were adsorbed onto the resin. The purification factor obtained was of 1.5.The resulting broth of this step (clarified broth) was used in the fixed-bed column process. Breakthrough curves of the column process showed that cepC and contaminants competed for the biding sites on the resin. Two separated peaks with antibacterial activity were obtained during bed elution, by using 0.1 and 0.5% NaCl solution. CepC was present only in the first fraction. The compounds present in the second peak were not known. Fractions from the center of the peak containing cepC were collected and used in the next step, adsorption in the SPE cartridge. CepC completely adsorbed onto the SPE cartridge, and was eluted using 50% methanol solution. Mass spectrometry analyses of a sample collected in the elution fraction and of the clarified broth showed a reduction in the peaks corresponding to the m/z ratios of contaminants that are difficult to separate, because of the similarity between their molecular structures and that of cepC. The contaminants monitored were penicillin N, deacetylcephalosporin C, deacetoxycephalosporin C and lysine. At this final operation, a partially purified fraction of cepC was obtained. This result is of great relevance since the establishment of a suitable process for cepC purification from fermentation broth has been strongly desired

Fixed-bed column process as a strategy for separation and purification of Cephamycin C from fermented broth

Fixed-bed column processes using the anionic resin Q Sepharose XL were evaluated for Cephamycin C (CepC) purification from fermentation broth. Breakthrough and desorption curves were obtained for different flow rates (2.5, 5.0 and 7.5 mL/min). The elution method consisted of a stepwise gradient using NaCl solutions (0.1, 0.3 and 0.5%), which resulted in the separation of CepC from others antibiotics. The flow rate did not interfere on adsorption during loading of the column, but band broadening was observed during elution as the flow rate increased. After the ion exchange process, the fractions containing CepC were submitted to solid phase extraction using a C18 cartridge to remove salts. Analyses of the broth used to feed the column and of the salt-free fractions by LC-MS showed a reduction in the concentration of some contaminants (possibly penicillin N, deacetylcephalosporin C, and deacetoxycephalosporin C) compared to the concentration of CepC. In conclusion, ion exchange process followed by adsorption on a C18 adsorbent was demonstrated to be a selective and efficient procedure to purify CepC from fermentation broth.The authors acknowledge CAPES, CNPq, and FAPESP for the financial support

Clavulanic acid and cephamicin c: a perspective of the biosynthesis, isolation and action mechanism

The present article reviews different aspects of the chemistry of two widely used β-lactam antibiotics Clavulanic Acid and Cephamycin C. The article discusses important details of the biosynthesis of these compounds, their action mechanism and, principally, the methods employed in their isolation and purification, in accordance with the available literature. Despite the large quantity of available articles and patents concerning β-lactam antibiotics, those which describe the isolation and purification of Clavulanic Acid and Cephamycin C are rare. Overall, the intention of this article is to discuss the up-to-date scientific research related to the compounds under review.FAPESPCNPqCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Use of volatile fatty acids salts in the production of xanthan gum

Background: The aim of this study was the production of xanthan gum from salts of volatile fatty acids, which can be generated in anaerobic processes for the production of hydrogen from organic wastewaters. Xanthan gum was produced with three different acid salts used to replace the traditional citrate, which is normally used in the culture for the production of this biopolymer. The volatile fatty acids (VFA) salts used were sodium acetate 0.0328 M, sodium propionate 0.0219 M, and sodium butyrate 0.0164 M. Results: The values of biomass yield, (Yp/x) obtained were 9.2 g/g for acetate, 11.78 g/g for citrate, 11.80 g/g for butyrate and 14.59 g/g for propionate, while the values of the product yield (Yp/s), were 0.92; 0.59; 0.71 and 0.72 for acetate, citrate, butyrate and propionate. As for the rheological characterization, the gums produced showed a consistency index (K) and flow index (n) of 9.8 dina.s-n.cm-2 and 0.34 for acetate; 6.3 dina.s-n.cm-2 and 0.39 for citrate, 5.8 dina.s-n.cm-2 and 0.45 for butyrate, 39.2 dina.s-n.cm-2 and 0.24 for propionate, that characterize the gums with good consistency and fluidity. Conclusions: It is possible to produce xanthan gum from short-chain volatile acids in replacement by the citrate that is usually used in medium composition for the gum production. These results contribute to the feasibility studies for implementation of processes for treating wastewater generating products such as volatile acids, hydrogen and consequent use of these acids for the production of xanthan gum

Estudo cinetico de adsorção modelagem dinamica e otimização de processo continuo de purificação de cefalosporina C

Orientador: Francisco Maugeri FilhoTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de AlimentosResumo: As cefalosporinas assim como as penicilinas fazem parte de um grupo de antibióticos ß-lactâmicos produzidos por microrganismos. A cefalosporina C é produzida por mutantes do fungo Cephalosporium acremonium, e tem como característica determinante para seleção de processos de purificação, sua natureza hidrofílica, que dificulta sua extração por intermédio de compostos orgânicos. Desta forma, uma das técnicas mais utilizadas para purificação de cefalosporina C é adsorção cromatográfica. Através de simulações em computador, foi avaliado neste trabalho, um processo não convencional para purificação de cefalosporina C. O processo consiste basicamente em dois tanques agitados interligados por um reciclo, onde num primeiro estágio ocorre a adsorção e no segundo estágio a eluição do produto. Para realização deste objetivo, foi necessário estudar experimentalmente o comportamento cinético de adsorção e dessorção de cefalosporina C em resina polimérica, Amberlite XAD-2. O efeito da temperatura e do eluente (etanol) em solução foi verificado, sendo que o abaixamento da temperatura favorece a adsorção. Foram classificadas as isotermas e a cinética de adsorção na presença de etanol nas temperaturas de 10°C, 15°C e 25°C. Ensaios de adsorção em tanque agitado, permitiram determinar as constantes cinéticas, a difusividade efetiva da cefalosporina C no interior da resina e coeficiente de resistência à transferência de massa externo. Com o modelo matemático do processo contínuo e os dados cinéticos obtidos, foram realizadas simulações a respeito da dinâmica de operação, definindo-se as variáveis mais influentes. O processo foi avaliado, com base nas respostas fornecidas, sendo elas: fator de concentração (FC); fator de purificação (FP) e porcentagem de recuperação (%RC). A otimização foi feita pelo método de análise de superfície de resposta, fornecendo modelos empíricos, para os cálculos de %RC, FP e FC, dentro das faixas de operação estudas. O processo apresentou recuperação em tomo de 80%, um produto 10 vezes purificado, saindo no 2° estágio 1,6 vezes mais concentrado. Estes valores indicam, o potencial de aplicação do processo para purificação de cefalosporina C.Abstract: The cephalosporins and penicilins are constitued of the ß-lactam antibiotics group produced by microorganism. The cephalosporin C is produced by Cephalosporillm acremollillm a mutant strain, and its hidrophilic nature make the extraction process by organic compounds more difficult, so the chromatographic adsorption is a good technique to be applyied in cephalosporin C purification process with a large potential of success. Cephalosporin C purification by non-conventional process: was evaluated using computer simulation. The process is composed for two stirred tank reactors with recycle system, the adsorption occurs in the first stage and the elution of the product takes place in the second stage. The cephalosproin C adsorption and desorption kinetics in non-polar polystyrene macroporous resin were studied to achieve this aim. The temperature and eluate (ethanol) effects in solution were verified and it was noted the adsorption process is more efficient decreasing the temperature. The isotherms and kinetic adsorption in the presence of ethanol was obtained at 10°C, 15°C and 25°C. Bath adsorption experiments allows to determine the kinetic constants, the effective pore diffusion coefficient of the cephalosporin C in the particle as well as the mass transfer coefficient. The most influent parameters were defined through dynamic simulation performed by a deterministic mathematic model of the continuous process and the kinetics data. Based in the concentration factor (CF), purification factor (PF) and recovery yield (%RC) the process performance was evaluated. The surface response analyse was used for the otimization and generates the supplying %RC, PF and CF model which are valid in the considered ranges. The process shown more than 80% for RC, 10 for PF and 1,6 for FC. These values shows a promising potential of the proposed technique to cephalosporin C purification.DoutoradoDoutor em Engenharia de Alimento

Studies on the Adsorption of Inulinase from Kluyveromyces marxianus ATCC 16045 onto an Ion Exchange Resin

The adsorption of an extracellular inulinase directly onto an ion exchange resin from a clarified crude broth was investigated in this work. The enzyme inulinase was obtained from Kluyveromyces marxianus ATCC 16045 by fermentation in shaken flasks in a medium containing peptone, sucrose, yeast extract and K2HPO4 at pH 3.5, 30°C and 150 rpm for 48 hours. The final enzymatic activity was about 72 U mL–1. The crude broth filtrate was used for kinetic studies and for the adsorption isotherm of the extracellular inulinase from Kluyveromyces bulgaricus ATCC 16045 onto an ion exchange resin. The trials were carried out at pH 3.5 and 25°C in stirred tank reactors containing STREAMLINETM SP, developed for expanded bed adsorption. It was observed that the adsorption was well described by the Langmuir isotherm, and the values determined for Qm and Kd were 1,254 U mL–1 and 0.325 UmL–1, respectively. The kinetic parameters k1 (6.52 . 10–3 mL U–1 min) and k2 (2.09. 10–3 min–1) as well as the average values of 1.71 . 10–2 cm s–1 for the film coefficient (Ks) and 6.96 . 10–7 cm2 s–1 for the effective diffusion coefficient (Def), were determined using the experimental results and mathematical modelling.A adsorção de uma inulinase extracelular em resina de troca iônica diretamente a partir de um caldo filtrado foi investigada neste trabalho. A enzima inulinase foi obtida a partir de fermentação por Kluyveromyces marxianus ATCC 16045 em frascos erlenmeyers com meio contendo peptona, sacarose, extrato de levedura e K2HPO4, a pH 3,5, 30°C, 150 rpm durante 48 horas. A atividade enzimática final foi ao redor de 72 U mL–1. O caldo filtrado foi utilizado para os estudos cinéticos e de obtenção da isoterma de adsorção da inulinase extracelular de Kluyveromyces bulgaricus ATCC 16045 usando resina de troca iônica. Os ensaios foram realizados a pH 3,5 e 25°C em reatores agitados encamisados contendo resina STREAMLINETM SP, material desenvolvido para utilização em colunas de leito expandido. Observou-se que a adsorção pode ser bem descrita pela isoterma de Langmuir, sendo determinado o Qm e o Kd, 1254 U mL–1 e 0,325 U mL–1, respectivamente. Os parâmetros cinéticos k1 (6,52 . 10–3 mL U–1 min) e k2 (2,09. 10–3 min–1) bem como os valores médios 1,71 . 10–2 cm s–1 para o coeficiente de transferência. de massa na película (Ks) e 6,96 . 10–7 cm2 s–1 para o coeficiente de difusão efetiva (Def) foram determinados utilizando os resultados experimentais e modelagem matemática

Estudos de adsorção de inulinase a partir de kluyveromyces marxianus ATCC 16045 em resina de troca Iônica

Submitted by Taís Renata Pereira Amorim ([email protected]) on 2014-11-20T02:21:56Z No. of bitstreams: 1 55- Estudos de Adsorção de Inulinase a.pdf: 196303 bytes, checksum: ca2c4c775158535dc1b20204933b79e6 (MD5)Approved for entry into archive by Paula Gautério ([email protected]) on 2014-12-04T23:24:06Z (GMT) No. of bitstreams: 1 55- Estudos de Adsorção de Inulinase a.pdf: 196303 bytes, checksum: ca2c4c775158535dc1b20204933b79e6 (MD5)Made available in DSpace on 2014-12-04T23:24:06Z (GMT). No. of bitstreams: 1 55- Estudos de Adsorção de Inulinase a.pdf: 196303 bytes, checksum: ca2c4c775158535dc1b20204933b79e6 (MD5) Previous issue date: 2006The adsorption of an extracellular inulinase directly onto an ion exchange resin from a clarified crude broth was investigated in this work. The enzyme inulinase was obtained from Kluyveromyces marxianus ATCC 16045 by fermentation in shaken flasks in a medium containing peptone, sucrose, yeast extract and K2HPO4 at pH 3.5, 30°C and 150 rpm for 48 hours. The final enzymatic activity was about 72 U mL–1. The crude broth filtrate was used for kinetic studies and for the adsorption isotherm of the extracellular inulinase from Kluyveromyces bulgaricus ATCC 16045 onto an ion exchange resin. The trials were carried out at pH 3.5 and 25°C in stirred tank reactors containing STREAMLINETM SP, developed for expanded bed adsorption. It was observed that the adsorption was well described by the Langmuir isotherm, and the values determined for Qm and Kd were 1,254 U mL–1 and 0.325 UmL–1, respectively. The kinetic parameters k1 (6.52 . 10–3 mL U–1 min) and k2 (2.09. 10–3 min–1) as well as the average values of 1.71 . 10–2 cm s–1 for the film coefficient (Ks) and 6.96 . 10–7 cm2 s–1 for the effective diffusion coefficient (Def), were determined using the experimental results and mathematical modelling.A adsorção de uma inulinase extracelular em resina de troca iônica diretamente a partir de um caldo filtrado foi investigada neste trabalho. A enzima inulinase foi obtida a partir de fermentação por Kluyveromyces marxianus ATCC 16045 em frascos erlenmeyers com meio contendo peptona, sacarose, extrato de levedura e K2HPO4, a pH 3,5, 30°C, 150 rpm durante 48 horas. A atividade enzimática final foi ao redor de 72 U mL–1. O caldo filtrado foi utilizado para os estudos cinéticos e de obtenção da isoterma de adsorção da inulinase extracelular de Kluyveromyces bulgaricus ATCC 16045 usando resina de troca iônica. Os ensaios foram realizados a pH 3,5 e 25°C em reatores agitados encamisados contendo resina STREAMLINETM SP, material desenvolvido para utilização em colunas de leito expandido. Observou-se que a adsorção pode ser bem descrita pela isoterma de Langmuir, sendo determinado o Qm e o Kd, 1254 U mL–1 e 0,325 U mL–1, respectivamente. Os parâmetros cinéticos k1 (6,52 . 10–3 mL U–1 min) e k2 (2,09. 10–3 min–1) bem como os valores médios 1,71 . 10–2 cm s–1 para o coeficiente de transferência. de massa na película (Ks) e 6,96 . 10–7 cm2 s–1 para o coeficiente de difusão efetiva (Def) foram determinados utilizando os resultados experimentais e modelagem matemática

Use of volatile fatty acids salts in the production of xanthan gum

Background: The aim of this study was the production of xanthan gum from salts of volatile fatty acids, which can be generated in anaerobic processes for the production of hydrogen from organic wastewaters. Xanthan gum was produced with three different acid salts used to replace the traditional citrate, which is normally used in the culture for the production of this biopolymer. The volatile fatty acids (VFA) salts used were sodium acetate 0.0328 M, sodium propionate 0.0219 M, and sodium butyrate 0.0164 M. Results: The values of biomass yield, (Yp/x) obtained were 9.2 g/g for acetate, 11.78 g/g for citrate, 11.80 g/g for butyrate and 14.59 g/g for propionate, while the values of the product yield (Yp/s), were 0.92; 0.59; 0.71 and 0.72 for acetate, citrate, butyrate and propionate. As for the rheological characterization, the gums produced showed a consistency index (K) and flow index (n) of 9.8 dina.s-n.cm-2 and 0.34 for acetate; 6.3 dina.s-n.cm-2 and 0.39 for citrate, 5.8 dina.s-n.cm-2 and 0.45 for butyrate, 39.2 dina.s-n.cm-2 and 0.24 for propionate, that characterize the gums with good consistency and fluidity. Conclusions: It is possible to produce xanthan gum from short-chain volatile acids in replacement by the citrate that is usually used in medium composition for the gum production. These results contribute to the feasibility studies for implementation of processes for treating wastewater generating products such as volatile acids, hydrogen and consequent use of these acids for the production of xanthan gum