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Thiago Faggion De Padua - One of the best experts on this subject based on the ideXlab platform.
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Monitoramento de reator enzimático para produção de ampicilina
Programa de Pós-graduação em Engenharia Química, 2008Co-Authors: Thiago Faggion De PaduaAbstract:This work is part of a research project in DEQ-UFSCar (Department of Chemical Engineering at Federal University of São Carlos, Brazil). The focus is to develop a competitive industrial Enzymatic process to obtain semi-synthetic β- Lactam antibiotics. This process rivals with the conventional one, which uses toxic solvents and generates non-recyclable residues. Thus, the increasing rigor of environmental regulations is an important driving force for the research. The Enzymatic production of ampicillin, in aqueous medium, uses a derivate from an acyl donor (in this work, D-phenylglycine methyl ester, PGME) and 6-aminopenicillanic acid (6-APA). Penicillin G acylase (PGA) is usually the biocatalyst for this process. Unfortunately, PGA acts either as transferase, producing ampicillin (AMP), or as hydrolase, producing D-phenylglycine (PG) from PGME (and from ampicillin). This is a series-parallel reaction scheme, with the antibiotic being the desired intermediate product. This work studied the use of multivariate calibration in a spectrophotometer for on-line monitoring the Enzymatic synthesis Reactor. High Performance Liquid Chromatography (HPLC) is the usual analytical procedure, which is time demanding, expensive and generates amounts of disposals. Multivariate calibration, on its turn, only uses dilutions of Reactor samples and a spectrophotomer (with UV detector). Some multivariate calibration techniques (SPA, PCR, PLS) were tested and compared. They didn t show representative differences in cross-validations or in test data. The work also focused the automation of a Reactor and its accessories, where the Enzymatic production of ampicillin and 6-APA (after Penicillin G Enzymatic hydrolysis) takes place. The software was implemented in LabVIEW (National Instruments), which controls the Automatic Sampler (AS) for multivariate analysis, among other equipments. The development of this system provided a real-time concentration response from the Reactor, for all chemical species. Following an optimal trajectory in the Enzymatic Reactor by adding reactants is one of the most important strategies to reach a competitive process. Hence, the concentration sensor represents an important step for the feasibility of the Enzymatic process, because it could be used to monitor and control the concentrations of substrates during the industrial feed-batch. Several assays, among then batch and fed-batch runs, were done and many modifications on the experimental scheme were implemented or proposed. Some of these assays could be treated as validation of monitoring software and concentration sensor, which showed good results.Financiadora de Estudos e ProjetosEste trabalho é parte de linha de pesquisa do DEQ/UFSCar, cujo objetivo é o desenvolvimento de processo enzimático para produção de antibióticos β-lactâmicos semi-sintéticos. Este processo se propõe a substituir a rota química convencional, que possui ressalvas devido ao crescente rigor das legislações ambientais, por utilizar solventes tóxicos e gerar quantidades apreciáveis de resíduos não recicláveis. No processo enzimático, a produção de ampicilina é conduzida em meio aquoso, a partir de um derivado ativado do doador acil e do ácido 6- Aminopenicilânico (6-APA). Geralmente, utiliza-se a enzima Penicilina G Acilase (PGA) como catalisador. Neste trabalho, o Éster Metílico de Fenilglicina, EMFG (derivado da Fenilglicina, FG), foi empregado. A mesma enzima atua também nas hidrólises do derivado ativado (EMFG, produzindo FG e metanol) e do antibiótico (ampicilina, AMP, produzindo 6-APA e FG), caracterizando um sistema reacional série-paralelo, onde o produto intermediário é o desejado. Uma das contribuições deste trabalho foi estudar a substituição da análise por HPLC ( High Performance Liquid Chromatography ) pela multicalibração espectrofotométrica, que tornou possível obter as concentrações dos analitos, em solução aquosa multicomponente (AMP, FG, EMFG e 6-APA em água), através da leitura das absorbâncias correspondentes a diferentes comprimentos de onda. A substituição se justifica devido à demanda elevada do HPLC por recursos, tempo e cuidados experimentais. Além disso, viabiliza-se assim o monitoramento do reator em tempo real. Várias técnicas de multicalibração (SPA, PCR e PLS) foram utilizadas e comparadas. Todas apresentaram desempenho semelhante tanto na validação cruzada como durante a apresentação de dados de teste. O trabalho também focou a automação de um reator piloto, que tem utilidade para produção de ampicilina e também 6-APA (pela hidrólise de penicilina G microbiana, catalisada por PGA). A linguagem de programação LabVIEW (National Instruments) foi utilizada nos trabalhos de automação. O programa de monitoramento gerado inclui, entre outros, sistema de amostragem automática (SAA) com análises por multicalibração espectrofotométrica. A aplicação do SAA, integrado a multicalibração, funciona como um sensor de concentrações para produção enzimática de ampicilina e pode ser utilizado no monitoramento e controle do reator em tempo real. Assim, o desenvolvimento do sistema representa um passo marcante rumo à viabilidade técnica e econômica do processo industrial, uma vez que a alimentação de reagentes, visando a obter trajetórias ótimas no reator, é uma das estratégias mais importantes para competitividade do processo. Vários ensaios experimentais, com o reator operando em batelada e em batelada alimentada, foram realizados e motivaram propostas para modificação do aparato experimental, como a retirada de cristais durante o processo reacional. Esses ensaios também podem ser visualizados como validações para o sensor de concentrações desenvolvido e para o sistema automatizado, que mostraram comportamento adequado para o monitoramento do processo
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Monitoramento de reator enzimático para produção de ampicilina
Universidade Federal de São Carlos, 2008Co-Authors: Thiago Faggion De PaduaAbstract:Este trabalho é parte de linha de pesquisa do DEQ/UFSCar, cujo objetivo é o desenvolvimento de processo enzimático para produção de antibióticos β-lactâmicos semi-sintéticos. Este processo se propõe a substituir a rota química convencional, que possui ressalvas devido ao crescente rigor das legislações ambientais, por utilizar solventes tóxicos e gerar quantidades apreciáveis de resíduos não recicláveis. No processo enzimático, a produção de ampicilina é conduzida em meio aquoso, a partir de um derivado ativado do doador acil e do ácido 6- Aminopenicilânico (6-APA). Geralmente, utiliza-se a enzima Penicilina G Acilase (PGA) como catalisador. Neste trabalho, o Éster Metílico de Fenilglicina, EMFG (derivado da Fenilglicina, FG), foi empregado. A mesma enzima atua também nas hidrólises do derivado ativado (EMFG, produzindo FG e metanol) e do antibiótico (ampicilina, AMP, produzindo 6-APA e FG), caracterizando um sistema reacional série-paralelo, onde o produto intermediário é o desejado. Uma das contribuições deste trabalho foi estudar a substituição da análise por HPLC (High Performance Liquid Chromatography) pela multicalibração espectrofotométrica, que tornou possível obter as concentrações dos analitos, em solução aquosa multicomponente (AMP, FG, EMFG e 6-APA em água), através da leitura das absorbâncias correspondentes a diferentes comprimentos de onda. A substituição se justifica devido à demanda elevada do HPLC por recursos, tempo e cuidados experimentais. Além disso, viabiliza-se assim o monitoramento do reator em tempo real. Várias técnicas de multicalibração (SPA, PCR e PLS) foram utilizadas e comparadas. Todas apresentaram desempenho semelhante tanto na validação cruzada como durante a apresentação de dados de teste. O trabalho também focou a automação de um reator piloto, que tem utilidade para produção de ampicilina e também 6-APA (pela hidrólise de penicilina G microbiana, catalisada por PGA). A linguagem de programação LabVIEW (National Instruments) foi utilizada nos trabalhos de automação. O programa de monitoramento gerado inclui, entre outros, sistema de amostragem automática (SAA) com análises por multicalibração espectrofotométrica. A aplicação do SAA, integrado a multicalibração, funciona como um sensor de concentrações para produção enzimática de ampicilina e pode ser utilizado no monitoramento e controle do reator em tempo real. Assim, o desenvolvimento do sistema representa um passo marcante rumo à viabilidade técnica e econômica do processo industrial, uma vez que a alimentação de reagentes, visando a obter trajetórias ótimas no reator, é uma das estratégias mais importantes para competitividade do processo. Vários ensaios experimentais, com o reator operando em batelada e em batelada alimentada, foram realizados e motivaram propostas para modificação do aparato experimental, como a retirada de cristais durante o processo reacional. Esses ensaios também podem ser visualizados como validações para o sensor de concentrações desenvolvido e para o sistema automatizado, que mostraram comportamento adequado para o monitoramento do processo.This work is part of a research project in DEQ-UFSCar (Department of Chemical Engineering at Federal University of São Carlos, Brazil). The focus is to develop a competitive industrial Enzymatic process to obtain semi-synthetic β- Lactam antibiotics. This process rivals with the conventional one, which uses toxic solvents and generates non-recyclable residues. Thus, the increasing rigor of environmental regulations is an important driving force for the research. The Enzymatic production of ampicillin, in aqueous medium, uses a derivate from an acyl donor (in this work, D-phenylglycine methyl ester, PGME) and 6-aminopenicillanic acid (6-APA). Penicillin G acylase (PGA) is usually the biocatalyst for this process. Unfortunately, PGA acts either as transferase, producing ampicillin (AMP), or as hydrolase, producing D-phenylglycine (PG) from PGME (and from ampicillin). This is a series-parallel reaction scheme, with the antibiotic being the desired intermediate product. This work studied the use of multivariate calibration in a spectrophotometer for on-line monitoring the Enzymatic synthesis Reactor. High Performance Liquid Chromatography (HPLC) is the usual analytical procedure, which is time demanding, expensive and generates amounts of disposals. Multivariate calibration, on its turn, only uses dilutions of Reactor samples and a spectrophotomer (with UV detector). Some multivariate calibration techniques (SPA, PCR, PLS) were tested and compared. They didnt show representative differences in cross-validations or in test data. The work also focused the automation of a Reactor and its accessories, where the Enzymatic production of ampicillin and 6-APA (after Penicillin G Enzymatic hydrolysis) takes place. The software was implemented in LabVIEW (National Instruments), which controls the Automatic Sampler (AS) for multivariate analysis, among other equipments. The development of this system provided a real-time concentration response from the Reactor, for all chemical species. Following an optimal trajectory in the Enzymatic Reactor by adding reactants is one of the most important strategies to reach a competitive process. Hence, the concentration sensor represents an important step for the feasibility of the Enzymatic process, because it could be used to monitor and control the concentrations of substrates during the industrial feed-batch. Several assays, among then batch and fed-batch runs, were done and many modifications on the experimental scheme were implemented or proposed. Some of these assays could be treated as validation of monitoring software and concentration sensor, which showed good results
Raquel L C Giordano - One of the best experts on this subject based on the ideXlab platform.
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kinetics of β lactam antibiotics synthesis by penicillin g acylase pga from the viewpoint of the industrial Enzymatic Reactor optimization
Biotechnology Advances, 2006Co-Authors: Roberto C Giordano, Marcelo Perencin De Arruda Ribeiro, Raquel L C GiordanoAbstract:Competition with well-established, fine-tuned chemical processes is a major challenge for the industrial implementation of the Enzymatic synthesis of beta-lactam antibiotics. Enzyme-based routes are acknowledged as an environmental-friendly approach, avoiding organochloride solvents and working at room temperatures. Among different alternatives, the kinetically controlled synthesis, using immobilized penicillin G acylase (PGA) in aqueous environment, with the simultaneous crystallization of the product, is the most promising one. However, PGA may act either as a transferase or as a hydrolase, catalyzing two undesired side reactions: the hydrolysis of the acyl side-chain precursor (an ester or amide, a parallel reaction) and the hydrolysis of the antibiotic itself (a consecutive reaction). This review focuses specially on aspects of the reactions' kinetics that may affect the performance of the Enzymatic Reactor.
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kinetics of β lactam antibiotics synthesis by penicillin g acylase pga from the viewpoint of the industrial Enzymatic Reactor optimization
Biotechnology Advances, 2006Co-Authors: Roberto C Giordano, Marcelo Perencin De Arruda Ribeiro, Raquel L C GiordanoAbstract:Abstract Competition with well-established, fine-tuned chemical processes is a major challenge for the industrial implementation of the Enzymatic synthesis of β-lactam antibiotics. Enzyme-based routes are acknowledged as an environmental-friendly approach, avoiding organochloride solvents and working at room temperatures. Among different alternatives, the kinetically controlled synthesis, using immobilized penicillin G acylase (PGA) in aqueous environment, with the simultaneous crystallization of the product, is the most promising one. However, PGA may act either as a transferase or as a hydrolase, catalyzing two undesired side reactions: the hydrolysis of the acyl side-chain precursor (an ester or amide, a parallel reaction) and the hydrolysis of the antibiotic itself (a consecutive reaction). This review focuses specially on aspects of the reactions' kinetics that may affect the performance of the Enzymatic Reactor.
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analysis of a taylor poiseuille vortex flow Reactor i flow patterns and mass transfer characteristics
Chemical Engineering Science, 1998Co-Authors: Roberto C Giordano, Raquel L C Giordano, D M F Prazeres, Charles L CooneyAbstract:This paper shows the results of flow visualization and residence time distribution experiments in a Taylor-Poiseuille vortex flow apparatus. It is the first of a series that starts with the identification of flow patterns inside the device and goes up to the assessment of its performance as an Enzymatic Reactor. The approach is to study in depth one single geometric configuration (radius ratio η=0.677 and aspect ratio Γ=18.30), adequate for use as a heterogeneous Reactor and or adsorption system in bio-processes, rather than spanning a range of geometries and proposing empirical expressions for mass transport coefficients. The range of rotations and axial flow rates used in this article correspond to low moderate rotational Reynolds numbers (Re θ from 130 to 615, with 1.6
vortices is slower than the mean axial velocity. The implications of this fact on the Reactor performance are discussed.
J M Lema - One of the best experts on this subject based on the ideXlab platform.
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Sequential Reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles
2017Co-Authors: M. Gamallo, Gemma Eibes, J M Lema, G. Feijoo, Y. Moldes-diz, M. T. MoreiraAbstract:The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two Enzymatic Reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of Enzymatic Reactors operated in sequential mode: a membrane Reactor using fsMP-laccase and a Reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.
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removal of estrogenic compounds from filtered secondary wastewater effluent in a continuous Enzymatic membrane Reactor identification of biotransformation products
Environmental Science & Technology, 2013Co-Authors: L Lloret, Gemma Eibes, Gumersindo Feijoo, Teresa M Moreira, J M LemaAbstract:In the present study, a novel and efficient technology based on the use of an oxidative enzyme was developed to perform the continuous removal of estrogenic compounds from polluted wastewaters. A 2 L Enzymatic membrane Reactor (EMR) was successfully operated for 100 h with minimal requirements of laccase for the transformation of estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2)from both buffer solution and real wastewater (filtered secondary effluent). When the experiments were performed at high and low concentrations of the target compounds, 4 mg/L and 100 μg/L, not only high removal yields (80–100%) but also outstanding reduction of estrogenicity (about 84–95%) were attained. When the EMR was applied for the treatment of municipal wastewaters with real environmental concentrations of the different compounds (0.29–1.52 ng/L), excellent results were also achieved indicating the high efficiency and potential of the Enzymatic Reactor system. A second goal of this study relied on the identifi...
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Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed Reactor
Biodegradation, 2012Co-Authors: L Lloret, Gemma Eibes, F. Hollmann, G. Feijoo, M. T. Moreira, J M LemaAbstract:Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively. Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based on the ratio between the turnover number and the Michaelis constant, k_cat/K_M) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed bed Reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals: estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2 and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected. In addition, a 79% decrease in estrogenic activity was detected in the effluent of the Enzymatic Reactor while only 14% was attained by inactivated laccase.
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degradation of estrogens by laccase from myceliophthora thermophila in fed batch and Enzymatic membrane Reactors
Journal of Hazardous Materials, 2012Co-Authors: L Lloret, Gemma Eibes, Gumersindo Feijoo, Maria Teresa Moreira, J M LemaAbstract:Several studies reported that natural and synthetic estrogens are the major contributors to the estrogenic activity associated with the effluents of wastewater treatment plants. The ability of the enzyme laccase to degrade these compounds in batch experiments has been demonstrated in previous studies. Nevertheless, information is scarce regarding in vitro degradation of estrogens in continuous Enzymatic bioReactors. The present work constitutes an important step forward for the implementation of an Enzymatic Reactor for the continuous removal of estrone (E1) and estradiol (E2) by free laccase from Myceliophthora thermophila. In a first step, the effect of the main process parameters (pH, enzyme level, gas composition (air or oxygen) and estrogen feeding rate) were evaluated in fed-batch bioReactors. E1 and E2 were oxidized by 94.1 and 95.5%, respectively, under the best conditions evaluated. Thereafter, an Enzymatic membrane Reactor (EMR) was developed to perform the continuous degradation of the estrogens. The configuration consisted of a stirred tank Reactor coupled with an ultrafiltration membrane, which allowed the recovery of enzyme while both estrogens and degradation products could pass through it. The highest removal rates at steady state conditions were up to 95% for E1 and nearly complete degradation for E2. Furthermore, the residual estrogenic activity of the effluent was largely reduced up to 97%.
Andreas Seidelmorgenstern - One of the best experts on this subject based on the ideXlab platform.
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racemization of undesired enantiomers immobilization of mandelate racemase and application in a fixed bed Reactor
Biotechnology Journal, 2016Co-Authors: Katarzyna Wrzosek, Marie Anel Garcia Rivera, Katja Bettenbrock, Andreas SeidelmorgensternAbstract:Production of optically pure products can be based on simple unselective synthesis of racemic mixtures combined with a subsequent separation of the enantiomers; however, this approach suffers from a 50% yield limitation which can be overcome by racemization of the undesired enantiomer and recycling. Application of biocatalyst for the racemization steps offers an attractive option for high-yield manufacturing of commercially valuable compounds. Our work focuses on exploiting the potential of racemization with immobilized mandelate racemase. Immobilization of crude mandelate racemase via covalent attachment was optimized for two supports: Eupergit(®) CM and CNBr-activated Sepharose 4 Fast Flow. To allow coupling of Enzymatic reaction with enantioselective chromatography, a mobile phase composition compatible with both processes was used in Enzymatic Reactor. Kinetic parameters obtained analyzing experiments carried out in a batch Reactor could be successfully used to predict fixed-bed Reactor performance. The applicability of the immobilized enzyme and the determined kinetic parameters were validated in transient experiments recording responses to pulse injections of R-mandelic acid. The approach investigated can be used for futher design and optimization of high yield combined resolution processes. The characterized fixed-bed Enzymatic Reactor can be integrated e.g. with chromatographic single- or multicolumn steps in various configurations.
Tatiana Souza Porto - One of the best experts on this subject based on the ideXlab platform.
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immobilization of a commercial aspergillus aculeatus enzyme preparation with fructosyltransferase activity in chitosan beads a kinetic thermodynamic study and fructo oligosaccharides continuous production in Enzymatic Reactor
Food and Bioproducts Processing, 2020Co-Authors: Rodrigo Lira De Oliveira, Marcos Fellipe Da Silva, Suzana Pedroza Da Silva, Jorge Vinicius Fernandes Lima Cavalcanti, Attilio Converti, Tatiana Souza PortoAbstract:Abstract Pectinex Ultra SP-L, a food-grade Enzymatic preparation with high transfructosylating activity, was covalently immobilized on chitosan beads. The highest immobilization yield (95.9%) was obtained using 4.0% glutaraldehyde for 60 min and 80 rpm at 25 ± 1 °C. The immobilized biocatalyst showed good operational stability, being able to retain, after the third cycle of reuse, no less than 100.0 and 73.9% of starting hydrolytic and transfructosylating activities, respectively. Results of residual activity experiments allowed estimating, for irreversible thermal inactivation of both free and immobilized enzyme, the activation energy (E*d = 234.3 and 242.2 kJ·mol−1), enthalpy (234.4 ≤ ΔH*d ≤ 234.2 kJ·mol−1 and 239.4 ≤ ΔH*d ≤ 239.3 kJ·mol−1), entropy (381.2 ≤ ΔS*d ≤ 379.8 J·mol−1·K−1 and 390.4 ≤ ΔS*d ≤ 389.7 J·mol−1·K−1) and Gibbs free energy (109.3 ≤ ΔG*d ≤ 103.9 kJ·mol−1 and 111.3 ≤ ΔG*d ≤ 103.6 kJ·mol−1). The use of the immobilized enzyme in a packed bed Reactor allowed obtaining, after 100 min, a fructo-oligosaccharide mixture containing more than 25 g L−1 1-kestose.