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Francisco J. Fernandez - One of the best experts on this subject based on the ideXlab platform.
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DeaCetylCephalosporin C produCtion in peniCillium Chrysogenum by expression of the isopeniCillin n epimerization ring expansion and aCetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Javier Casqueiro, Sonia Campoy, Francisco J. FernandezAbstract:PeniCillium Chrysogenum npe6 laCking isopeniCillin N aCyltransferase aCtivity is an exCellent host for produCtion of different beta-laCtam antibiotiCs. We have ConstruCted P. Chrysogenum strains expressing CefD1, CefD2, CefEF, and CefG genes Cloned from ACremonium Chrysogenum. Northern analysis revealed that the four genes were expressed in P. Chrysogenum. The reCombinant strains TA64, TA71, and TA98 seCreted signifiCant amounts of DeaCetylCephalosporin C, but Cephalosporin C was not deteCted in the Culture broths. DAC-aCetyltransferase aCtivity was found in all transformants Containing the CefG gene. HPLC analysis of Cell extraCts showed that transformant TA64, TA71, and TA98 aCCumulate intraCellularly DeaCetylCephalosporin C and, in the last strain (TA98), also Cephalosporin C. Mass speCtra analysis Confirmed that transformant TA98 synthesize true DeaCetylCephalosporin C and Cephalosporin C. Even when aCCumulated intraCellularly, Cephalosporin C was not found in the Culture broth.
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DeaCetylCephalosporin C produCtion in peniCillium Chrysogenum by expression of the isopeniCillin n epimerization ring expansion and aCetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Javier Casqueiro, Sonia Campoy, Francisco J. FernandezAbstract:Summary PeniCillium Chrysogenum npe6 laCking isopeniCillin N aCyltransferase aCtivity is an exCellent host for produCtion of different β-laCtam antibiotiCs. We have ConstruCted P. Chrysogenum strains expressing CefD1 , CefD2 , CefEF , and CefG genes Cloned from ACremonium Chrysogenum . Northern analysis revealed that the four genes were expressed in P. Chrysogenum . The reCombinant strains TA64, TA71, and TA98 seCreted signifiCant amounts of DeaCetylCephalosporin C, but Cephalosporin C was not deteCted in the Culture broths. DAC-aCetyltransferase aCtivity was found in all transformants Containing the CefG gene. HPLC analysis of Cell extraCts showed that transformant TA64, TA71, and TA98 aCCumulate intraCellularly DeaCetylCephalosporin C and, in the last strain (TA98), also Cephalosporin C. Mass speCtra analysis Confirmed that transformant TA98 synthesize true DeaCetylCephalosporin C and Cephalosporin C. Even when aCCumulated intraCellularly, Cephalosporin C was not found in the Culture broth.
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The CefG Gene of Cephalosporium aCremonium Is Linked to the CefEF Gene and EnCodes a DeaCetylCephalosporin C ACetyltransferase Closely Related to
1991Co-Authors: Homoserine O-acetyltransferase, Javier Velasco, Santiago Gutierrez, Francisco J. FernandezAbstract:The gene (CefG) enCoding the aCetyl Coenzyme A.DeaCetylCephalosporin C aCetyltransferase of Cephalospo-rium aCremonium (synonym ACremonium Chrysogenum) C10 has been Cloned. It Contains two introns and enCodes a protein of 444 amino aCids with an Mr of 49,269 that Correlates well with the Mr deduCed by gel filtration. The CefG gene is linked to the CeJEF gene (enCoding the bifunCtional deaCetoxyCephalosporin C synthase/hydroxylase), but it is expressed in an orientation opposite that of the CeJEF gene. Two transCripts of 1.2 and 1.4 kb were found in C. aCremonium that Correspond to the CefEF and CefG genes, respeCtively; the degree of expression of the CeiG gene was Clearly lower than that of the CeJEF gene in 48-h Cultures. The Cloned CefG Complemented the defiCienCy of DeaCetylCephalosporin aCetyltransferase in the nonproduCer mutant C. aCremonium ATCC 20371 and restored Cephalosporin biosynthesis in this strain. Heterologous expression of the CefG genes took plaCe in PeniCilium Chrysogenum. The DeaCetylCephalosporin aCetyltransferase showed a muCh higher degree of homology with the O-aCetylhomoserine aCetyltransferases of SaCCharomyCes Cerevisiae and AsCobolus immersus than with other O-aCetyltransferases. The CefEF-CeIG Cluster of genes enCodes the enzymes that Carry out the three late steps of the Cephalosporin biosynthetiC pathway and is not linked to thepCbAB-pCbC gene Cluster that enCodes the first two steps of the pathway. Cephalosporin is a P-laCtam antibiotiC formed in Cepha
Liliane Maciel De Oliveira - One of the best experts on this subject based on the ideXlab platform.
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PurifiCação da CefamiCina C por proCesso de adsorção em Coluna de leito fixo
Programa de Pós-graduação em Engenharia Química, 2013Co-Authors: Liliane Maciel De OliveiraAbstract:Cephalosporins are β-laCtam antibiotiCs whiCh are widely used in the treatment of baCterial infeCtions. Within this group, CephamyCin C (CefC) stands out from the others Cephalosporins antibiotiCs due to its greater resistanCe to β-laCtamases enzymes and its broader speCtrum of aCtion against Gram-negative pathogens. It is produCed by the baCteria NoCardia laCtamdurans and StreptomyCes Clavuligerus, usually in submerged fermentation. In the literature, the available information about the purifiCation proCesses of this antibiotiC are restriCted to patents or papers published in the past deCades. As CefC is the raw material for the produCtion of important semi-synthetiC antibiotiCs and due to the laCk of sCientifiCally substantiated information about its separation and purifiCation proCesses, the motivation for the development of this thesis has arisen. Then, the aim of this thesis was to study the purifiCation proCess of CefC produCed by S. Clavuligerus, by using the proCesses of adsorption and ion exChange in fixed bed Columns. The teChniques of adsorption and ion exChange present themselves as good strategies for CefC purifiCation due to the amphoteriC nature of the antibiotiC moleCule, whiCh has positive or negative Charges depending on the environmental pH, and the biCyCliC nuCleus in its struCture, whiCh allows the interaCtion with hydrophobiC struCtures. As there is no ChemiCal standard of CefC available on the market, in this study we have only used Culture broth Containing this antibiotiC. The adsorbents evaluated were the neutral resins Amberlite XAD4 and Amberlite XAD16 and the anioniC resin Q Sepharose XL. At first, ion exChange Column experiments using the resin QXL were Carried out. The results showed that the variation of the flow rate in the range of 2.5 mL/min to 7.5 mL/min did not affeCt the effiCienCies of produCt reCovery nor of bed utilization, and it did not interfere with the bioaCtive Compounds elution. The isoCratiC elution modes using 0.1% or 1% NaCl (w / v) were able to separate CefC from other (s) Compound (s) with antimiCrobial aCtivity present in the broth, whiCh were not identified. But the use of 1% NaCl solution promoted the antibiotiC reCovery in a shorter proCess time and smaller volume than using 0.1 % NaCl solution. After analysis by mass speCtrometry, Carried out by ESI ionization in the positive mode, it was observed that the ion exChange proCess with QXL resin followed by adsorption on a C18 SPE Cartridge were able to separate CefC from Compound (s) whose moleCules after ionization aCquired m/z ratios Corresponding to the lysine s moleCule, and they also reduCed the ConCentration of Contaminants with m/z ratios Corresponding to the moleCules of peniCillin N, DeaCetylCephalosporin C and deaCetoxyCephalosporin C. As the ion exChange studies were done, the adsorption studies using the neutral resins were initiated. At first, adsorption isotherms of CefC on XAD4 and XAD16 resins were obtained, and the effeCts of pH and temperature in the adsorption equilibrium were evaluated. The results showed that the broth pH had strong influenCe on the adsorption and the best Condition was observed at pH 2.6 at 15° C. The kinetiC experiments have shown that equilibrium is reaChed more quiCkly on the resin XAD16. Estimation of intrinsiC kinetiC parameters and mass transfer CoeffiCients returned higher values for this resin. The Column adsorption proCesses with XAD4 resin, when the Column feed Consisted of the ultrafiltered or Clarified broth and the elution solutions were 1% or 5% (v/v) EtOH solutions, did not lead to separation of CefC from Contaminants that were monitored in the experiments. But when the feed Consisted of a preAbstraCt iv purified fraCtion, obtained in the ion exChange Column with QXL resin, added with Cephalosporin C, it was possible to separate the two antibiotiCs. Cephalosporin C was added to simulate the presenCe of some Contaminants in the Culture broth - DeaCetylCephalosporin C and deaCetoxyCephalosporin C. As these Contaminants are in very low ConCentrations and there are not CommerCial ChemiCal standards available to purChase, neither an analytiCal methodology to quantify them, it was deCided to add Cephalosporin C, whose ChemiCal struCture is very similar to the Contaminants, the CommerCial standard is CommerCially available and there is a quantifiCation method to determinate it. The Column experiments with resin XAD16 showed similar results to the experiments with resin XAD4, in whiCh it was also observed the separation of CefC from Cephalosporin C. The moment analysis of the ChromatographiC peaks showed that CefC average retention times and peak dispersions were smaller in the Column assays with XAD16 than with XAD4. Thus, a more ConCentrated fraCtion Containing CefC was obtained in the Column experiments with XAD16 resin. Therefore, it was demonstrated by the purifiCation proCesses studied that the teChniques of adsorption and ion exChange were effeCtive in the purifiCation of CefC in relation to Contaminants that are diffiCult to separateUniversidade Federal de Sao CarlosAs Cefalosporinas são antibiótiCos β-laCtâmiCos muito utilizados no tratamento de infeCções baCterianas. Dentro do grupo, a CefamiCina C (CefC) se destaCa devido a sua maior resistênCia à ação das β-laCtamases baCterianas e ao seu maior espeCtro de ação Contra patógenos Gramnegativos. Ela é produzida pelas baCtérias NoCardia laCtamdurans e StreptomyCes Clavuligerus, geralmente em Cultivos submersos. Na literatura, as informações existentes sobre os proCessos de purifiCação deste antibiótiCo estão disponíveis apenas em patentes ou em artigos publiCados em déCadas passadas. Sendo um Composto que é matéria-prima para produção de antibiótiCos semissintétiCos de grande importânCia ClíniCa e ComerCial, e devido à falta de informações fundamentadas CientifiCamente sobre os proCessos de separação e purifiCação deste antibiótiCo, surgiu a motivação para o desenvolvimento desta tese. Assim, o objetivo deste trabalho foi estudar a purifiCação da CefC produzida por S. Clavuligerus, utilizando o proCesso de adsorção e troCa iôniCa em Colunas de leito fixo. As téCniCas de adsorção e troCa iôniCa apresentam-se Como uma boa estratégia para purifiCação da CefC devido à natureza anfótera da moléCula do antibiótiCo, que possui Cargas positivas ou negativas dependendo do pH do meio, e ao núCleo biCíCliCo em sua estrutura, que permite a interação Com estruturas hidrofóbiCas. Como não há o padrão da CefC disponível no merCado, neste estudo trabalhou-se apenas Com o Caldo de Cultivo. Os adsorventes avaliados foram as resinas neutras Amberlite XAD4 e Amberlite XAD16 e a resina de troCa iôniCa Q Sepharose XL. IniCialmente, foram realizados experimentos em Coluna de troCa iôniCa Com a resina QXL. Os resultados destes ensaios mostraram que a variação da vazão no intervalo de 2,5 mL/min a 7,5 mL/min não alterou as efiCiênCias de reCuperação do produto e de utilização do leito, nem interferiram na eluição dos Compostos bioativos. A eluição no modo isoCrátiCo Com solução de NaCl 1% (m/v) possibilitou a separação da CefC de outro(s) Composto(s) Com atividade antimiCrobiana presentes no Caldo, mas não identifiCados, e promoveu uma reCuperação do antibiótiCo em um tempo menor de proCesso e em um volume menor de solução que a eluição Com NaCl 0,1%. Após análise por espeCtrometria de massas, Com ionização feita por ESI no modo positivo, observou-se que o proCesso de troCa iôniCa seguido da adsorção em CartuCho de SPE C18 foi Capaz de separar a CefC de Composto(s) Com a razão m/z Correspondente à lisina, e diminuir a ConCentração de Contaminantes presentes no Caldo Cujas moléCulas apresentaram, após ionização, razão m/z Correspondentes à das moléCulas de peniCilina N, desaCetilCefalosporina C e desaCetoxiCefalosporina C. Finalizados os experimentos de troCa iôniCa, iniCiaram-se os estudos de adsorção utilizando as resinas neutras. Os primeiros experimentos Consistiram na determinação de isotermas de adsorção da CefC nas resinas XAD4 e XAD16, avaliando a influênCia do pH e da temperatura no equilíbrio de adsorção. Os resultados mostraram que o pH do Caldo teve drástiCa influênCia na adsorção, sendo que a melhor Condição observada foi no valor de pH 2,6 à 15°C. Os ensaios CinétiCos mostraram que o equilíbrio é atingido mais rapidamente na resina XAD16. A estimativa dos parâmetros CinétiCos intrínseCos e de transferênCia de massa retornaram valores maiores para esta resina. O proCesso de adsorção em Coluna Com a resina XAD4, quando a alimentação da Coluna Consistiu do Caldo ultrafiltrado ou ClarifiCado e a eluição foi feita Com as soluções de EtOH 1% ou 5% (v/v), não levou à separação da CefC dos Contaminantes que Resumo ii foram monitorados nos ensaios. Mas quando a alimentação foi feita Com uma fração prépurifiCada, oriunda da troCa iôniCa, adiCionada de Cefalosporina C, foi possível obter a separação dos dois antibiótiCos. A Cefalosporina C foi adiCionada para simular a presença de alguns Contaminantes presentes no Caldo de Cultivo a desaCetilCefalosporina C e desaCetoxiCefalosporina C. Como estes Contaminantes estão em ConCentrações muito baixas e não existem os padrões ComerCiais disponíveis para serem adquiridos, nem metodologia analítiCa para quantifiCá-los, optou-se por adiCionar Cefalosporina C, Cuja estrutura químiCa é bem similar à dos Contaminantes, existe o padrão ComerCial para ser adquirido e possui um método de quantifiCação. Os experimentos em Coluna Com a resina XAD16 mostraram resultados similares aos experimentos Com a resina XAD4, nos quais também foi observada a separação entre a CefC e a Cefalosporina C. Após análise dos momentos dos piCos CromatográfiCos, verifiCou-se que a CefC apresentou tempos de retenção médios menores na resina XAD16 e a dispersão dos piCos foi menor. Desta forma, foi obtida uma fração mais ConCentrada em CefC nos ensaios em Coluna Com esta resina do que Com a resina XAD4. Portanto, pôde-se Constatar pelo proCesso de purifiCação estudado que as téCniCas de adsorção e troCa iôniCa mostraram-se efiCientes na purifiCação da CefC em relação a Contaminantes difíCeis de serem separados
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PurifiCação da CefamiCina C por proCesso de adsorção em Coluna de leito fixo
Universidade Federal de São Carlos, 2013Co-Authors: Liliane Maciel De OliveiraAbstract:As Cefalosporinas são antibiótiCos β-laCtâmiCos muito utilizados no tratamento de infeCções baCterianas. Dentro do grupo, a CefamiCina C (CefC) se destaCa devido a sua maior resistênCia à ação das β-laCtamases baCterianas e ao seu maior espeCtro de ação Contra patógenos Gramnegativos. Ela é produzida pelas baCtérias NoCardia laCtamdurans e StreptomyCes Clavuligerus, geralmente em Cultivos submersos. Na literatura, as informações existentes sobre os proCessos de purifiCação deste antibiótiCo estão disponíveis apenas em patentes ou em artigos publiCados em déCadas passadas. Sendo um Composto que é matéria-prima para produção de antibiótiCos semissintétiCos de grande importânCia ClíniCa e ComerCial, e devido à falta de informações fundamentadas CientifiCamente sobre os proCessos de separação e purifiCação deste antibiótiCo, surgiu a motivação para o desenvolvimento desta tese. Assim, o objetivo deste trabalho foi estudar a purifiCação da CefC produzida por S. Clavuligerus, utilizando o proCesso de adsorção e troCa iôniCa em Colunas de leito fixo. As téCniCas de adsorção e troCa iôniCa apresentam-se Como uma boa estratégia para purifiCação da CefC devido à natureza anfótera da moléCula do antibiótiCo, que possui Cargas positivas ou negativas dependendo do pH do meio, e ao núCleo biCíCliCo em sua estrutura, que permite a interação Com estruturas hidrofóbiCas. Como não há o padrão da CefC disponível no merCado, neste estudo trabalhou-se apenas Com o Caldo de Cultivo. Os adsorventes avaliados foram as resinas neutras Amberlite XAD4 e Amberlite XAD16 e a resina de troCa iôniCa Q Sepharose XL. IniCialmente, foram realizados experimentos em Coluna de troCa iôniCa Com a resina QXL. Os resultados destes ensaios mostraram que a variação da vazão no intervalo de 2,5 mL/min a 7,5 mL/min não alterou as efiCiênCias de reCuperação do produto e de utilização do leito, nem interferiram na eluição dos Compostos bioativos. A eluição no modo isoCrátiCo Com solução de NaCl 1% (m/v) possibilitou a separação da CefC de outro(s) Composto(s) Com atividade antimiCrobiana presentes no Caldo, mas não identifiCados, e promoveu uma reCuperação do antibiótiCo em um tempo menor de proCesso e em um volume menor de solução que a eluição Com NaCl 0,1%. Após análise por espeCtrometria de massas, Com ionização feita por ESI no modo positivo, observou-se que o proCesso de troCa iôniCa seguido da adsorção em CartuCho de SPE C18 foi Capaz de separar a CefC de Composto(s) Com a razão m/z Correspondente à lisina, e diminuir a ConCentração de Contaminantes presentes no Caldo Cujas moléCulas apresentaram, após ionização, razão m/z Correspondentes à das moléCulas de peniCilina N, desaCetilCefalosporina C e desaCetoxiCefalosporina C. Finalizados os experimentos de troCa iôniCa, iniCiaram-se os estudos de adsorção utilizando as resinas neutras. Os primeiros experimentos Consistiram na determinação de isotermas de adsorção da CefC nas resinas XAD4 e XAD16, avaliando a influênCia do pH e da temperatura no equilíbrio de adsorção. Os resultados mostraram que o pH do Caldo teve drástiCa influênCia na adsorção, sendo que a melhor Condição observada foi no valor de pH 2,6 à 15C. Os ensaios CinétiCos mostraram que o equilíbrio é atingido mais rapidamente na resina XAD16. A estimativa dos parâmetros CinétiCos intrínseCos e de transferênCia de massa retornaram valores maiores para esta resina. O proCesso de adsorção em Coluna Com a resina XAD4, quando a alimentação da Coluna Consistiu do Caldo ultrafiltrado ou ClarifiCado e a eluição foi feita Com as soluções de EtOH 1% ou 5% (v/v), não levou à separação da CefC dos Contaminantes que Resumo ii foram monitorados nos ensaios. Mas quando a alimentação foi feita Com uma fração prépurifiCada, oriunda da troCa iôniCa, adiCionada de Cefalosporina C, foi possível obter a separação dos dois antibiótiCos. A Cefalosporina C foi adiCionada para simular a presença de alguns Contaminantes presentes no Caldo de Cultivo a desaCetilCefalosporina C e desaCetoxiCefalosporina C. Como estes Contaminantes estão em ConCentrações muito baixas e não existem os padrões ComerCiais disponíveis para serem adquiridos, nem metodologia analítiCa para quantifiCá-los, optou-se por adiCionar Cefalosporina C, Cuja estrutura químiCa é bem similar à dos Contaminantes, existe o padrão ComerCial para ser adquirido e possui um método de quantifiCação. Os experimentos em Coluna Com a resina XAD16 mostraram resultados similares aos experimentos Com a resina XAD4, nos quais também foi observada a separação entre a CefC e a Cefalosporina C. Após análise dos momentos dos piCos CromatográfiCos, verifiCou-se que a CefC apresentou tempos de retenção médios menores na resina XAD16 e a dispersão dos piCos foi menor. Desta forma, foi obtida uma fração mais ConCentrada em CefC nos ensaios em Coluna Com esta resina do que Com a resina XAD4. Portanto, pôde-se Constatar pelo proCesso de purifiCação estudado que as téCniCas de adsorção e troCa iôniCa mostraram-se efiCientes na purifiCação da CefC em relação a Contaminantes difíCeis de serem separados.Cephalosporins are β-laCtam antibiotiCs whiCh are widely used in the treatment of baCterial infeCtions. Within this group, CephamyCin C (CefC) stands out from the others Cephalosporins antibiotiCs due to its greater resistanCe to β-laCtamases enzymes and its broader speCtrum of aCtion against Gram-negative pathogens. It is produCed by the baCteria NoCardia laCtamdurans and StreptomyCes Clavuligerus, usually in submerged fermentation. In the literature, the available information about the purifiCation proCesses of this antibiotiC are restriCted to patents or papers published in the past deCades. As CefC is the raw material for the produCtion of important semi-synthetiC antibiotiCs and due to the laCk of sCientifiCally substantiated information about its separation and purifiCation proCesses, the motivation for the development of this thesis has arisen. Then, the aim of this thesis was to study the purifiCation proCess of CefC produCed by S. Clavuligerus, by using the proCesses of adsorption and ion exChange in fixed bed Columns. The teChniques of adsorption and ion exChange present themselves as good strategies for CefC purifiCation due to the amphoteriC nature of the antibiotiC moleCule, whiCh has positive or negative Charges depending on the environmental pH, and the biCyCliC nuCleus in its struCture, whiCh allows the interaCtion with hydrophobiC struCtures. As there is no ChemiCal standard of CefC available on the market, in this study we have only used Culture broth Containing this antibiotiC. The adsorbents evaluated were the neutral resins Amberlite XAD4 and Amberlite XAD16 and the anioniC resin Q Sepharose XL. At first, ion exChange Column experiments using the resin QXL were Carried out. The results showed that the variation of the flow rate in the range of 2.5 mL/min to 7.5 mL/min did not affeCt the effiCienCies of produCt reCovery nor of bed utilization, and it did not interfere with the bioaCtive Compounds elution. The isoCratiC elution modes using 0.1% or 1% NaCl (w / v) were able to separate CefC from other (s) Compound (s) with antimiCrobial aCtivity present in the broth, whiCh were not identified. But the use of 1% NaCl solution promoted the antibiotiC reCovery in a shorter proCess time and smaller volume than using 0.1 % NaCl solution. After analysis by mass speCtrometry, Carried out by ESI ionization in the positive mode, it was observed that the ion exChange proCess with QXL resin followed by adsorption on a C18 SPE Cartridge were able to separate CefC from Compound (s) whose moleCules after ionization aCquired m/z ratios Corresponding to the lysines moleCule, and they also reduCed the ConCentration of Contaminants with m/z ratios Corresponding to the moleCules of peniCillin N, DeaCetylCephalosporin C and deaCetoxyCephalosporin C. As the ion exChange studies were done, the adsorption studies using the neutral resins were initiated. At first, adsorption isotherms of CefC on XAD4 and XAD16 resins were obtained, and the effeCts of pH and temperature in the adsorption equilibrium were evaluated. The results showed that the broth pH had strong influenCe on the adsorption and the best Condition was observed at pH 2.6 at 15 C. The kinetiC experiments have shown that equilibrium is reaChed more quiCkly on the resin XAD16. Estimation of intrinsiC kinetiC parameters and mass transfer CoeffiCients returned higher values for this resin. The Column adsorption proCesses with XAD4 resin, when the Column feed Consisted of the ultrafiltered or Clarified broth and the elution solutions were 1% or 5% (v/v) EtOH solutions, did not lead to separation of CefC from Contaminants that were monitored in the experiments. But when the feed Consisted of a preAbstraCt iv purified fraCtion, obtained in the ion exChange Column with QXL resin, added with Cephalosporin C, it was possible to separate the two antibiotiCs. Cephalosporin C was added to simulate the presenCe of some Contaminants in the Culture broth - DeaCetylCephalosporin C and deaCetoxyCephalosporin C. As these Contaminants are in very low ConCentrations and there are not CommerCial ChemiCal standards available to purChase, neither an analytiCal methodology to quantify them, it was deCided to add Cephalosporin C, whose ChemiCal struCture is very similar to the Contaminants, the CommerCial standard is CommerCially available and there is a quantifiCation method to determinate it. The Column experiments with resin XAD16 showed similar results to the experiments with resin XAD4, in whiCh it was also observed the separation of CefC from Cephalosporin C. The moment analysis of the ChromatographiC peaks showed that CefC average retention times and peak dispersions were smaller in the Column assays with XAD16 than with XAD4. Thus, a more ConCentrated fraCtion Containing CefC was obtained in the Column experiments with XAD16 resin. Therefore, it was demonstrated by the purifiCation proCesses studied that the teChniques of adsorption and ion exChange were effeCtive in the purifiCation of CefC in relation to Contaminants that are diffiCult to separat
Ricardo V. Ullán - One of the best experts on this subject based on the ideXlab platform.
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DeaCetylCephalosporin C produCtion in peniCillium Chrysogenum by expression of the isopeniCillin n epimerization ring expansion and aCetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Javier Casqueiro, Sonia Campoy, Francisco J. FernandezAbstract:Summary PeniCillium Chrysogenum npe6 laCking isopeniCillin N aCyltransferase aCtivity is an exCellent host for produCtion of different β-laCtam antibiotiCs. We have ConstruCted P. Chrysogenum strains expressing CefD1 , CefD2 , CefEF , and CefG genes Cloned from ACremonium Chrysogenum . Northern analysis revealed that the four genes were expressed in P. Chrysogenum . The reCombinant strains TA64, TA71, and TA98 seCreted signifiCant amounts of DeaCetylCephalosporin C, but Cephalosporin C was not deteCted in the Culture broths. DAC-aCetyltransferase aCtivity was found in all transformants Containing the CefG gene. HPLC analysis of Cell extraCts showed that transformant TA64, TA71, and TA98 aCCumulate intraCellularly DeaCetylCephalosporin C and, in the last strain (TA98), also Cephalosporin C. Mass speCtra analysis Confirmed that transformant TA98 synthesize true DeaCetylCephalosporin C and Cephalosporin C. Even when aCCumulated intraCellularly, Cephalosporin C was not found in the Culture broth.
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DeaCetylCephalosporin C produCtion in peniCillium Chrysogenum by expression of the isopeniCillin n epimerization ring expansion and aCetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Javier Casqueiro, Sonia Campoy, Francisco J. FernandezAbstract:PeniCillium Chrysogenum npe6 laCking isopeniCillin N aCyltransferase aCtivity is an exCellent host for produCtion of different beta-laCtam antibiotiCs. We have ConstruCted P. Chrysogenum strains expressing CefD1, CefD2, CefEF, and CefG genes Cloned from ACremonium Chrysogenum. Northern analysis revealed that the four genes were expressed in P. Chrysogenum. The reCombinant strains TA64, TA71, and TA98 seCreted signifiCant amounts of DeaCetylCephalosporin C, but Cephalosporin C was not deteCted in the Culture broths. DAC-aCetyltransferase aCtivity was found in all transformants Containing the CefG gene. HPLC analysis of Cell extraCts showed that transformant TA64, TA71, and TA98 aCCumulate intraCellularly DeaCetylCephalosporin C and, in the last strain (TA98), also Cephalosporin C. Mass speCtra analysis Confirmed that transformant TA98 synthesize true DeaCetylCephalosporin C and Cephalosporin C. Even when aCCumulated intraCellularly, Cephalosporin C was not found in the Culture broth.
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expression of Cefd2 and the Conversion of isopeniCillin n into peniCillin n by the two Component epimerase system are rate limiting steps in Cephalosporin biosynthesis
Molecular Genetics and Genomics, 2004Co-Authors: Ricardo V. Ullán, Javier Casqueiro, Leopoldo Naranjo, I Vaca, Juan F. MartínAbstract:The Conversion of isopeniCillin N into peniCillin Ni nACremonium Chrysogenum is Catalyzed by an epi- merization system that involves an isopeniCillin N-CoA synthethase and isopeniCillin N-CoA epimerase, enCoded by the genes CefD1 and CefD2. Several trans- formants Containing two to seven additional Copies of both genes were obtained. Four of these transformants (TMCD26, TMCD53, TMCD242 and TMCD474) showed two-fold higher IPN epimerase aCtivity than the untransformed A. Chrysogenum C10, and produCed 80 to 100% more Cephalosporin C and DeaCetylCephalosporin C than the parental strain. A seCond Class of transfor- mants, inCluding TMCD2, TMCD32 and TMCD39, in Contrast, showed a drastiC reduCtion in Cephalosporin biosynthesis relative to the untransformed Control. These transformants had no deteCtable IPN epimerase aCtivity and did not produCe Cephalosporin C or DeaCetylCephalosporin C. They also expressed both endogenous and exogenous CefD2 genes only after long periods (72-96 h) of inCubation, as shown by Northern analysis, and were impaired in myCelial branChing in liquid Cultures. The negative effeCt of amplifiCation of the CefD1 - CefD2 gene Cluster in this seCond Class of transformants is not Correlated with high gene dosage, but appears to be due to exogenous DNA integration into a speCifiC loCus, whiCh results in a pleiotropiC effeCt on growth and CefD2 expression.
Keqian Yang - One of the best experts on this subject based on the ideXlab platform.
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saturation mutagenesis of aCremonium Chrysogenum deaCetoxy DeaCetylCephalosporin C synthase r308 site Confirms its role in Controlling substrate speCifiCity
Biotechnology Letters, 2011Co-Authors: Xiuyun Tian, Keqiang Fan, Keqian YangAbstract:DeaCetoxy/DeaCetylCephalosporin C synthase (aCDAOC/DACS) from ACremonium Chrysogenum is a bifunCtional enzyme that Catalyzes both the ring-expansion of peniCillin N to deaCetoxyCephalosporin C and the hydroxylation of the latter to DeaCetylCephalosporin C. The R308 residue loCated in Close proximity to the C-terminus of aCDAOC/DACS was mutated to the other 19 amino aCids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed signifiCant improvement in their ability to Convert peniCillin analogs, thus Confirming the role of R308 in Controlling substrate seleCtivity, the four amino aCids all possess short aliphatiC sideChains that may improve hydrophobiC interaCtions with the substrates. The mutant R308I showed the highest reaCtivity for peniCillin G, with 3-fold inCrease in k(Cat)/K(m) ratio and 7-fold inCrease in relative aCtivity.
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Saturation mutagenesis of ACremonium Chrysogenum deaCetoxy/DeaCetylCephalosporin C synthase R308 site Confirms its role in Controlling substrate speCifiCity
2010Co-Authors: Xiuyun Tian, Keqiang Fan, Keqian YangAbstract:AbstraCt DeaCetoxy/DeaCetylCephalosporin C syn-thase (aCDAOC/DACS) from ACremonium Chrysog-enum is a bifunCtional enzyme that Catalyzes both the ring-expansion of peniCillin N to deaCetoxyCephalo-sporin C and the hydroxylation of the latter to DeaCetylCephalosporin C. The R308 residue loCated in Close proximity to the C-terminus of aCDAOC/ DACS was mutated to the other 19 amino aCids. In the resulting mutant pool, R308L, R308I, R308T and R308V showed signifiCant improvement in their ability to Convert peniCillin analogs, thus Confirming the role of R308 in Controlling substrate seleCtivity, the four amino aCids all possess short aliphatiC sideChains that may improve hydrophobiC interaC-tions with the substrates. The mutant R308I showed the highest reaCtivity for peniCillin G, with 3-fold inCrease in kCat/Km ratio and 7-fold inCrease in relative aCtivity
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C terminus mutations of aCremonium Chrysogenum deaCetoxy DeaCetylCephalosporin C synthase with improved aCtivity toward peniCillin analogs
Fems Microbiology Letters, 2005Co-Authors: Keqiang Fan, Qinhong Wang, Keqian YangAbstract:DeaCetoxy/DeaCetylCephalosporin C synthase (aCDAOC/DACS) from ACremonium Chrysogenum is a bifunCtional enzyme that Catalyzes both the ring-expansion of peniCillin N to deaCetoxyCephalosporin C (DAOC) and the hydroxylation of the latter to DeaCetylCephalosporin C (DAC). Three residues N305, R307 and R308 loCated in Close proximity to the C-terminus of aCDAOC/DACS were eaCh mutated to leuCine. The N305L and R308L mutant aCDAOC/DACSs showed signifiCant improvement in their ability to Convert peniCillin analogs. R308 was identified for the first time as a CritiCal residue for DAOC/DACS aCtivity. KinetiC analyses of purified R308L enzyme indiCated its improved CatalytiC effiCienCy is due to Combined improvements of Km and kCat. Comparative modeling of aCDAOC/DACS supports the involvement of R308 in the formation of substrate-binding poCket.
Sonia Campoy - One of the best experts on this subject based on the ideXlab platform.
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DeaCetylCephalosporin C produCtion in peniCillium Chrysogenum by expression of the isopeniCillin n epimerization ring expansion and aCetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Javier Casqueiro, Sonia Campoy, Francisco J. FernandezAbstract:Summary PeniCillium Chrysogenum npe6 laCking isopeniCillin N aCyltransferase aCtivity is an exCellent host for produCtion of different β-laCtam antibiotiCs. We have ConstruCted P. Chrysogenum strains expressing CefD1 , CefD2 , CefEF , and CefG genes Cloned from ACremonium Chrysogenum . Northern analysis revealed that the four genes were expressed in P. Chrysogenum . The reCombinant strains TA64, TA71, and TA98 seCreted signifiCant amounts of DeaCetylCephalosporin C, but Cephalosporin C was not deteCted in the Culture broths. DAC-aCetyltransferase aCtivity was found in all transformants Containing the CefG gene. HPLC analysis of Cell extraCts showed that transformant TA64, TA71, and TA98 aCCumulate intraCellularly DeaCetylCephalosporin C and, in the last strain (TA98), also Cephalosporin C. Mass speCtra analysis Confirmed that transformant TA98 synthesize true DeaCetylCephalosporin C and Cephalosporin C. Even when aCCumulated intraCellularly, Cephalosporin C was not found in the Culture broth.
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DeaCetylCephalosporin C produCtion in peniCillium Chrysogenum by expression of the isopeniCillin n epimerization ring expansion and aCetylation genes
Chemistry & Biology, 2007Co-Authors: Ricardo V. Ullán, Javier Casqueiro, Sonia Campoy, Francisco J. FernandezAbstract:PeniCillium Chrysogenum npe6 laCking isopeniCillin N aCyltransferase aCtivity is an exCellent host for produCtion of different beta-laCtam antibiotiCs. We have ConstruCted P. Chrysogenum strains expressing CefD1, CefD2, CefEF, and CefG genes Cloned from ACremonium Chrysogenum. Northern analysis revealed that the four genes were expressed in P. Chrysogenum. The reCombinant strains TA64, TA71, and TA98 seCreted signifiCant amounts of DeaCetylCephalosporin C, but Cephalosporin C was not deteCted in the Culture broths. DAC-aCetyltransferase aCtivity was found in all transformants Containing the CefG gene. HPLC analysis of Cell extraCts showed that transformant TA64, TA71, and TA98 aCCumulate intraCellularly DeaCetylCephalosporin C and, in the last strain (TA98), also Cephalosporin C. Mass speCtra analysis Confirmed that transformant TA98 synthesize true DeaCetylCephalosporin C and Cephalosporin C. Even when aCCumulated intraCellularly, Cephalosporin C was not found in the Culture broth.
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moleCular CharaCterization of the aCremonium Chrysogenum Cefg gene produCt the native DeaCetylCephalosporin C aCetyltransferase is not proCessed into subunits
Biochemical Journal, 1999Co-Authors: Javier Velasco, Santiago Gutierrez, Sonia Campoy, Juan F. MartínAbstract:ConstruCtions starting at eaCh of the three in-frame ATG Codons of the ACremonium Chrysogenum CefG gene (Met1, Met46 and Met60) were expressed in EsCheriChia Coli, obtaining proteins of 49, 44 and 43 kDa, respeCtively. All three proteins showed DeaCetylCephalosporin C (DAC) aCetyltransferase aCtivity. The native A. Chrysogenum DAC aCetyltransferase was purified to eleCtrophoretiC homogeneity by immunoaffinity Chromatography. It showed a moleCular mass of 50 kDa by filtration in Calibrated Sephadex G-75 SF or Superose 12 (FPLC) Columns. The N-terminal end of the pure DAC aCetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, whiCh matChed perfeCtly the deduCed amino aCid sequenCe starting at Met1. The putative alpha- and beta-subunits of DAC aCetyltransferase were also obtained in E. Coli but showed no enzymiC aCtivity either separately or in Combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC aCetyltransferase showed high protein levels in A. Chrysogenum Cultures at 72 and 96 h and deCreased sharply thereafter, but in all Cases no deteCtable proCessing of the enzyme into subunits was found. Three different A. Chrysogenum strains (inCluding the wild-type Brotzu strain and two high-Cephalosporin-produCing mutants) showed the same unproCessed 50 kDa DAC aCetyltransferase. The non-produCer mutant ATCC 20371 showed no DAC aCetyltransferase protein band but formed a normal transCript of 1.4 kb.