Dissertations / Theses on the topic 'Cephalosporin C'

The biosynthetic pathway which leads, in Cephalosporium acremonium, to the production of the commercially important β-lactam antibiotic Cephalosporin C (CPC) has been the subject of extensive biochemical studies and is now well characterized. In contrast, genetic analysis in this organism was limited until the application of protoplast fusion techniques facilitated parasexual analysis and allowed a genetic map to be established. (Hamlyn 1982; Hamlyn et al 1985). Subsequently, work leading to our understanding of the genetic basis of the CPC biosynthetic pathway in C. acremonium began. (Perez-Martinez 1984; Perez-Martinez and Peberdy in preparation). The studies described here were aimed at extending this understanding to a point at which individual genes implicated in the pathway could be identified and positioned on the linkage map. A programme of mutagenesis resulted in the production of a number of 'blocked' mutant strains of C. acremonium which were phenotypically particular steps of the CPC biosynthetic pathway. The segregation of several of these mutations relative to other genetic markers was examined. Crosses designed to detect complementation between mutations resulting in a 'blocked' phenotype were carried out and involved strains produced in other laboratories in addition to those characterized during this work. Complementation was shown between two mutations which apparently affected the same step in CPL biosynthesis (the conversion of penicillin N into deacetoxycephalosporin C) and evidence for the linkage of one of the mutations (cnp-6) to a mutation resulting in a requirement for inositol was obtained. During the course of the complementation studies, it was noted that the haploid and heterozygous products obtained following C. acremonium protoplast fusion crosses did not always behave in the typical manner described previously. (Hamlyn 1984). The persistent heterogeneity of these fusion products and the possible implications of this are discussed.

Kyoto University (京都大学)
0048
新制・論文博士
博士(農学)
乙第11556号
論農博第2543号
新制||農||901(附属図書館)
学位論文||H16||N3979(農学部図書室)
22816
UT51-2004-T200
(主査)教授 江﨑 信芳, 教授 清水 昌, 教授 加藤 暢夫
学位規則第4条第2項該当

Orientador: Maria Lucia Gonsales da Costa Araujo
Banca: Oswaldo Garcia Junior
Banca: Alberto Colli Baldino Junior
Resumo: Os grupos de antibióticos mais importantes clinicamente são os dos b-lactâmicos, aminoglicosídeos e tetraciclinas. Streptomyces clavuligerus produz vários compostos b- lactâmicos, com destaque para os antibióticos envolvidos na rota biossintética da cefalosporina C (penicilina N, deacetoxicefalosporina C e cefamicina C) e o ácido clavulânico (AC) que, embora não tenha atividade biológica significativa, é um potente inibidor de b-lactamases (penicilinases e cefalosporinases). A cefamicina C (CefC) é uma 7-metoxi-cefalosporina que apresenta maior atividade que a cefalosporina C (CPC), produzida somente por fungos, por ser resistente a b-lactamases. Apesar das rotas biossintéticas de AC e CefC serem completamente independentes em S. clavuligerus, são controladas pelo mesmo elemento multifuncional (ccaR), o que dificulta a indução da produção de um ou outro composto durante o processo fermentativo. No presente trabalho, procurou-se obter maiores concentrações de CefC manipulando-se componentes em meio solúvel de cultivo de S. clavuligerus, selecionados dentre compostos que, segundo a literatura, atuam como agentes reguladores da síntese daquele antibiótico. As fermentações foram realizadas em frascos agitados (28ºC, 260 rpm) para selecionar fontes de C e de N e, então, avaliar o processo no melhor meio-padrão, variando-se concentrações combinadas de L-lisina (10 a 108 mM) e -cetoglutarato (3 a 110 mM) através de metodologia de planejamento experimental. A presença de -cetoglutarato acarretou em aumento indesejável de pH, afetando negativamente o processo e os melhores resultados (entre 300 e 400 mg CPC totais/L após 72 horas de fermentação) foram obtidos no meio adotado como meio-controle, contendo amido e extrato protéico de semente de algodão como principais fontes de C e N, respectivamente, e L-lisina.
Abstract: The most important groups of antibiotics, from a clinical standpoint, are -lactams, aminoglycosides and tetracyclines. Streptomyces clavuligerus produces several -lactam compounds, primarily the antibiotics involved in the biosynthetic route of cephalosporin C (penicillin N, deacetoxycephalosporin C and cephamycin C) and clavulanic acid (CA), which, despite its slight biological activity, is a potent inhibitor of -lactamases (penicillinases and cephalosporinases). Cephamycin C (CMC) is a 7-methoxycephalosporin with higher bioactivity than cephalosporin C (CPC) because it is more resistant to -lactamases. Although the biosynthetic routes of CA and CMC are completely independent in S. clavuligerus, they are controlled by a common multi-functional element (ccaR), which hinders induction of the production of one or the other compound during the fermentation process. In this work, we sought to obtain higher concentrations of CMC by handling compounds in a soluble medium of S. clavuligerus, which were selected from compounds that, according to the literature, act as regulating agents in the synthesis of that antibiotic. Fermentation was carried out in flasks under shaking (28ºC, 260 rpm), in order to select sources of C and N. The process was then evaluated in the best standard medium, by varying combined concentrations of lysine (10 to 108 mM) and -ketoglutarate (3 to 110 mM) using an experimental planning methodology. The presence of -ketoglutarate caused an undesirable increase in pH, negatively affecting the process. The best results (between 300 and 400 mg/L of total CPC after 72 h of fermentation) were obtained in the medium used as the control, which contained starch and cottonseed protein extract as main sources, respectively, of C and N, and L-lysine.
Mestre

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Os grupos de antibióticos mais importantes clinicamente são os dos b-lactâmicos, aminoglicosídeos e tetraciclinas. Streptomyces clavuligerus produz vários compostos b- lactâmicos, com destaque para os antibióticos envolvidos na rota biossintética da cefalosporina C (penicilina N, deacetoxicefalosporina C e cefamicina C) e o ácido clavulânico (AC) que, embora não tenha atividade biológica significativa, é um potente inibidor de b-lactamases (penicilinases e cefalosporinases). A cefamicina C (CefC) é uma 7-metoxi-cefalosporina que apresenta maior atividade que a cefalosporina C (CPC), produzida somente por fungos, por ser resistente a b-lactamases. Apesar das rotas biossintéticas de AC e CefC serem completamente independentes em S. clavuligerus, são controladas pelo mesmo elemento multifuncional (ccaR), o que dificulta a indução da produção de um ou outro composto durante o processo fermentativo. No presente trabalho, procurou-se obter maiores concentrações de CefC manipulando-se componentes em meio solúvel de cultivo de S. clavuligerus, selecionados dentre compostos que, segundo a literatura, atuam como agentes reguladores da síntese daquele antibiótico. As fermentações foram realizadas em frascos agitados (28ºC, 260 rpm) para selecionar fontes de C e de N e, então, avaliar o processo no melhor meio-padrão, variando-se concentrações combinadas de L-lisina (10 a 108 mM) e -cetoglutarato (3 a 110 mM) através de metodologia de planejamento experimental. A presença de -cetoglutarato acarretou em aumento indesejável de pH, afetando negativamente o processo e os melhores resultados (entre 300 e 400 mg CPC totais/L após 72 horas de fermentação) foram obtidos no meio adotado como meio-controle, contendo amido e extrato protéico de semente de algodão como principais fontes de C e N, respectivamente, e L-lisina.
The most important groups of antibiotics, from a clinical standpoint, are -lactams, aminoglycosides and tetracyclines. Streptomyces clavuligerus produces several -lactam compounds, primarily the antibiotics involved in the biosynthetic route of cephalosporin C (penicillin N, deacetoxycephalosporin C and cephamycin C) and clavulanic acid (CA), which, despite its slight biological activity, is a potent inhibitor of -lactamases (penicillinases and cephalosporinases). Cephamycin C (CMC) is a 7-methoxycephalosporin with higher bioactivity than cephalosporin C (CPC) because it is more resistant to -lactamases. Although the biosynthetic routes of CA and CMC are completely independent in S. clavuligerus, they are controlled by a common multi-functional element (ccaR), which hinders induction of the production of one or the other compound during the fermentation process. In this work, we sought to obtain higher concentrations of CMC by handling compounds in a soluble medium of S. clavuligerus, which were selected from compounds that, according to the literature, act as regulating agents in the synthesis of that antibiotic. Fermentation was carried out in flasks under shaking (28ºC, 260 rpm), in order to select sources of C and N. The process was then evaluated in the best standard medium, by varying combined concentrations of lysine (10 to 108 mM) and -ketoglutarate (3 to 110 mM) using an experimental planning methodology. The presence of -ketoglutarate caused an undesirable increase in pH, negatively affecting the process. The best results (between 300 and 400 mg/L of total CPC after 72 h of fermentation) were obtained in the medium used as the control, which contained starch and cottonseed protein extract as main sources, respectively, of C and N, and L-lysine.

This thesis describes an investigation of the mechanism of the bifunctional, a-ketoglutarate dependent dioxygenase, deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS), which catalyses the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of this to deacetylcephalosporin C (DAC). The conversion of the unnatural substrate 3-exomethylene cephalosporin C by DAOC/DACS has been investigated in detail. A new metabolite was isolated from incubations of the deuterated [4-²H]-3-exomethylene cephalosporin C, and was identified as the 3β-spiroepoxide cepham, (2Ṟ,3Ṟ,6Ṟ,7Ṟ)-l-aza-[2-²H]-3-spiroepoxy-7-[(5Ṟ)-5-amino- 5-carboxypentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylic acid. The results obtained indicate that this metabolite is a shunt product whose formation is enhanced by the operation of a deuterium kinetic isotope effect on an enzyme-bound intermediate. It has also been found that this 3β-spiroepoxide cepham is further converted by DAOC/DACS to 3-formyl cephalosporoate products. The mechanism of oxygenation of DAOC/DACS was investigated through ¹⁸O-labelling studies. Incubations of [2-¹³C,3-²H]penicillin N and [4-²H]-3-exomethylene cephalosporin C with DAOC/DACS were carried out under ¹⁸O₂ or in H₂¹⁸O. Incorporation of ¹⁸O-label into the products [3-¹³C]DAC, [3-¹³C,4-²H]-3β-hydroxycepham and 3β-spiroepoxide cepham was observed from both sources. The results suggest that intermediates capable of oxygen-exchange are formed during the enzymatic reactions. Two substrate analogues, the 5-epipenicillin N and the 2β-difluoromethyl penicillin N, have been synthesised in order to probe the substrate specificity of DAOC/DACS with respect to the ring-expansion activity. The 5-epipenicillin N was not accepted as a substrate by DAOC/DACS, and the observations made indicate that it was unstable under the incubation conditions. No product was either observed from incubations of the 2β-difluoromethyl penicillin N with DAOC/DACS, although bioassay tests suggested a cephem product had been formed in very small amounts. Finally, the results of a substrate specificity comparison between the soluble recombinant enzymes deacetoxy/deacetylcephalosporin C synthase (DAOC/DACS) from Cephalosporium acremonium and deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus are described.

Orientador: Gil Eduardo Serra
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Este trabalho teve como objetivo estudar um meio de cultivo industrial para produção de cefalosporina C, utilizando a linhagem C-1 O de Cephalosporium acremonium (A TCC 11550) em experimentos conduzidos em frascos agitados. Foram testadas diferentes fontes de proteína padronizadas, de uso indicado para fermentações industriais, fornecidas pela QUEST INTERNACIONAL - Divisão SHEFFIELD, constituídas à base de soja (Hy-Soy), lacto albumina (Edamin'S), caseína (N-Z Amine A e N-Z Amine As), milho (Hydrolyse Com Gluten ) e algodão (Hydrolyse Cottonseed). As fontes protéicas foram adicionadas a um meio basal, em quantidade equivalente a 4g/1 de nitrogênio total. O meio de cultivo que apresentou uma maior produção de cefalosporina C ( 0,63 gll) foi formulado à base de soja, sendo o meio à base de caseína (N-Z Amine A) o que apresentou uma menor produção do antibiótico. A partir disto foi feito um estudo de cinética da produção de cefalosporina C com esses dois meios de cultivo. Os resultados confirmaram a soja como melhor substrato para produção do antibiótico e mostraram também, ser esta a matéria prima mais produtiva, uma vez que com 144 horas de fermentação houve a produção de 0,93 gll de cefalosporina C. A caseína precisou de 192 horas de fermentação para produzir apenas 0,64 gll do antibiótico. Para os dois substratos testados foi verificada ainda a degradação do antibiótico após o pico de máxima produção. Em uma segunda etapa do trabalho foi avaliada a associação de fontes protéicas na formulação do meio de cultivo. Além de Hy-Soy e Edamin'S, que foram as que apresentaram os melhores resultados na primeira fase, foi introduzido um extrato protéico padronizado, à base de soja (Samprosoy), que é produzido no Brasil pela SANTISTA ALIMENTOS divisão SAMBRA. Neste experimento foram formulados sete diferentes meios de cultivo contendo 4 g/l de N total. Os resultados mostraram um sinergismo na associação de Samprosoy, Hy-Soy e Edamin'S, com uma maior produção do antibiótico (1,43 g/l em 144 horas). Esta composição foi utilizada como ponto central (36 g/l de saca rose, 27 gI1 de glicose e 4 g/l de N total) na primeira fase da otimização do meio de cultivo utilizando planejamento fatorial completo de dois níveis e três variáveis. As variáveis estudadas foram a concentração de sacarose, glicose e nitrogênio, e a variável resposta foi a concentração de cefalosporina C no caldo fermentado. Os resultados obtidos mostraram que maiores concentrações de glicose e nitrogênio levam a um aumento na produção do antibiótico, sendo a variação da concentração de sacarose não significativa na produção da cefalosporina C. Em uma segunda fase da otimização do meio de cultivo o ponto central foi deslocado de modo que esse meio apresentasse maiores concentrações de glicose e nitrogênio (37 g/l de glicose e 6 g/l de N total) e menor quantidade de sacarose (26 g/l). Confirmou-se que altas concentrações de glicose (acima de 45 g/l) e nitrogênio (acima de 6 g/l) direcionam a um aumento na produção, e que a concentração de sacarose não influencia no aumento da produção do antibiótico. Em uma última etapa foi conduzida uma fermentação simultânea em fermentador e em frascos agitados para o estudo do efeito da aeração na produção de cefalosporina C. Parte do meio preparado e inoculado no fermentador foi transferido para frascos Erlenmayer. Os resultados mostram que no fermentador a quantidade de cefalosporina C produzida foi 2,82 vezes maior que as obtidas em frascos agitados. A diferença certamente está associada ao nível constante da concentração de oxigênio no meio de fermentação, possível de se controlar no fermentador, mas não em frascos agitados.
Abstract: The purpose of this work was to study an industrial growing medium for cephalosporin C production in a shaker, using the C-1 O strain of Cephalosporuium acremonium (A TCC 11550). Different sources of standard protein have been tested, all of which recommended for industrial fermentation, and provided by QUEST INTERNACIONAL¬SHEFFIELD Division, soy-based (Hy Soy), Lactoalbumin (Edamin'S), casein (N-Z Amine and N_Z Anime A), com (Hidrolyse Com Gluten) and cotton (Hydrolyse Cottonseed). The protein sources were added to a basic medium amounting to 4 g/l of the total nitrogen. The growing medium that reached the highest cephalosporin C production (0.63 g/l) was soy-based, and the medium that had the lowest amount of the antibiotic was the casein-based (N-Z Amine A). These two growing mediums were then used for a kinetic study of cephalosporin C production. The results have confirmed soybean as the best substratum for the antibiotic production as well as the most productive raw material, since there was a 0.93 g/l cephalosporin C production within 144 hours of fermentation. The casein took 192 hours of fermentation to produce only 0.64 g/l of the antibiotic. It has been observed for both substrata that there was a degradation of the antibiotic after the maximum production peak was reached. In the second stage of the trial, the association of protein sources in the growing medium formulation was evaluated. In the addition to Hy-Soy and Edamin'S, proteins that presented the best results in the first stage, a standard soy-based proteinic extract (Samprosoy) produced in Brazil by SANTISTA ALIMENTOS - SAMBRA division was introduced. Seven different growing mediums containing 4 g/l of total N were formulated. The results have showed a synergism in the association among Samprosoy, Hy-Soyand Edamin'S, with a higher antibiotic production (1,43 g/l within 144 hours). This composition was used as a central point (36 g/l sucrose, 27 g/l glucose and 4 g/l of total N) at the first optimization stage of growing medium a complete two-Level and three-variable factorial design. The variable studied was the sucrose, glucose and nitrogen concentration, and the response variable was the cephalosporin C concentration in the fermented broth. The results have shown that higher glucose and nitrogen concentration have led to an increase in the antibiotic production, even though the variation in sucrose concentration was insignificant for cephalosporin C production. In the second stage of growing medium optimization the central point was dislocated in order to present higher glucose and nitrogen concentrations (37 g/l of glucose and 6 g/l of N total) and lower sucrose concentration (26 g/l). It has been confirmed that high glucose (over 45 g/l) and nitrogen (over 6 g/l) concentration result in an increase in the production, and that sucrose concentration has no effect in the increase of antibiotic production. In the last stage, a simultaneous fermentation in a fermenter and in a shaker was performed for the purpose of studying the aeration effect in cephalosporin C production. Part of the medium prepared and inoculated in the fermenter was transferred to Erlenmeyer flasks. The results have shown that in the fermenter, the amount of cephalosporin C produced was 2.82 times higher than in the shaker. The difference is surely associated with the constant oxygen concentration level in the fermentation medium, which was possible to the controlled in the fermenter but not in the shaker.
Doutorado
Doutor em Tecnologia de Alimentos

The order of events in the Deacetoxycephalosporin C/Deacetylcephalosporrn C Synthetase (DAOC/DAC Synthetase) catalysed ring expansion of penicillin N to deacetoxycephalosporin C has been investigated by the use of labelled/unlabelled penicillin N mixed competitive kinetic isotope effect experiments, in which the labelled penicillin N substrates were either labelled in the pro R- and pro S-methyl groups or at C-3. In addition, to assisting in the determination of the position of the first irreversible event in this reaction, deuteration at C-3 gave rise to a bifurcation of the natural biosynthetic pathway which led to enhanced production of the shunt metabolite, (2R,3S,6R,7R)-l-aza-3- methyl-3-hydroxy-7-[(5R)-5-amino-5-carboxy-pentanamido]-8-oxo-5-thiabicyclo[4.2.0]octane-2-carboxylate. The biosynthetic precursor to the 3S-hydroxycepham shunt metabolite has been investigated and the origin of the 3S-hydroxyl oxygen atom has been determined by the use of labelling studies with ¹⁸O₂ and shown to be derived from molecular oxygen. ¹³C-labelling studies are described which indicate that the ring expansion process is stereospecific to within the limits of the detection system employed. These experiments confirm earlier investigations but, in addition to improving upon the assessment of the degree of stereospecificity, have shown that the 3S- hydroxycepham shunt metabolite is produced with the same stereospecificity as that observed for the usual biosynthetic products, DAOC and DAC. Chapter 5 describes an investigation of the anomalous C-2 deuterium exchange detected in DAOC produced by incubation of di-(²H₃-methyl)-penicillin N with DAOC/DAC synthetase. The preliminary results from this study indicate that initially exchange occurs stereospecifically with the pro R C-2 deuterium atom being replaced by a hydrogen atom. The origins of the unusual tripeptides L-α-aminoadipyl-L-serinyl-D-valine (L,L,D-ASV), α-aminoadipyl-serinyl-isodehydrovaline (ASdV) and α-aminoadipyl-cysteinyl- β-hydroxyvaline (AC-[β-OH]-V) isolated from Penicillium chrysogenum and Cephalosporium acremonium, have been examined by the use of variously ¹³C-labelled L,L,D-α-aminoadipyl-cysteinyl-valine (L,L,D-ACV) and D,L,D-α-aminoadipyl- cysteinyl-valine (D,L,D-ACV) tripeptide isotopomers. The initial results obtained from this investigation may be considered as circumstantial evidence that ASdV is formed by the action of IPNS upon L,L,D-ACV. Finally, various substrate analogues have been prepared and evaluated as substrates for the ring expansion and hydroxylation activities of the bifunctional DAOC/DAC synthetase enzyme.

Philosophiae Doctor - PhD
Background: Extended-spectrum beta-lactamase-producing (ESBL) and carbapenemaseproducing Gram-negative bacilli showing resistance to cephalosporins and carbapenems respectively, have been reported from several countries globally and recently among Libyan combatants who have been transferred to European countries for advanced medical care. However, there is a lack of data about their presence in Misrata and in Libya in general. This is the first documented study aimed at investigating the prevalence and resistance mechanisms of ESBL and carbapenemase-producing K. pneumoniae isolates from Misrata. Materials and Methods: Two hundred Gram-negative bacillus isolates were collected and identified from hospitals and pathology laboratories in Misrata. Following antimicrobial susceptibility screening, those showing resistance to cephalosporin and carbapenem were tested for ESBL activity using the Modified double disc synergy test, Sensititer ESBL confirmatory MIC plates and MAST AmpC detection sets D52C and D68C. Carbapenemase activity was detected using RAPIDEC CARBA NP test, Modified Hodge test (MHT), carbapenem inactivation methods (CIM), carbapenem combined test (CCT), and by MAST carba puls set. ESBL and carbapenemases genes were detected using multiplex PCR. Results: K. pneumoniae was the predominant species (85/200) of the 14 species identified, with 56 (65.8%) showing carbapenem resistance, 16 (18.8%) were cephalosporin-resistant carbapenem-susceptible and 13 (15.2%) were susceptible to all antibiotics except ampicillin. OXA-48 was the only carbapenemase detected, with SHV, TEM and CTX-M group 1 found in almost all carbapenem and cephalosporin resistant K. pneumoniae. Rep-PCR analysis revealed multiple clones and some K. pneumoniae strains were genetically related or indistinguishable despite differences in ESBL genes or carbapenemase activity. Conclusion: The findings of this study show that carbapenemase- and ESBL-producing K. pneumoniae are prevalent in Misrata and emphasize the urgent need for optimized infection control and antibiotic stewardship programmes in the Libyan hospitals to prevent further spread of these organisms.

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The scope of the current work was to investigate the effect of glucose, amonium acetate, DL-methionine, phosphorus, oleic acid and salt solution on the maximum specific growth rate (µmax), as well as the effect of sucrose and DL-methionine on the specific cefalosporin C production rate (µp), by Cephalosporium acremonium ATCC 48272, using factorial design and response surface techniques. The initial medium of fermentation was used according to factorial design and the experiments were carried out in shaker flasks at controlled temperature at 28 °C, initial pH of 7,0+0,1 and agitation speed 250 rpm. For both specific rate the variables involved were correlated by a fitted second order model. The canonic and surface response analysis showed a maximum specific growth rate of 0.22 h-1 obtained when glucose and amonium acetate concentration, were 4.2 , 2.5 g/L, respectively, and specific cefalosporin C production rate of 1.12 mgCPC/gcel.h when sucrose and DL-methionine concentration, were 10.5, 2.5 g/L, respectively, for a celular concentration of 14.0 g/L. The experimental design showed to be very efficcient in evaluating the bioprocess, as long as high values of µmax e µp were obtained in comparison with literature data. The feedforward neural network (FNN) was employed for modelling and simulation the effect of glucose, amonium acetate, DL-methionine, phosphorus, oleic acid and salt solution on the maximum specific growth rate. The network topologies were used with 6 neurons in the input layer, 22 hidden layer and 1 output layer. The data base for training the FNN was obtained from results of the factorial designs processed with a backpropagation learning algorithm. The surfaces response obtained by simulations for all range of glucose and amonium acetate concentrations showed inhibitory effect for the two substrates on the maximum specific growth rate. The results of the simulation are in good agreement with the experimental and statistical model results.
Neste trabalho verificou-se o efeito das variáveis concentração de glicose, acetato de amônio, DL-metionina, fósforo, ácido oléico e solução de sais na velocidade específica máxima de crescimento (µmax), assim como sacarose e DL-metionina na velocidade específica de produção do antibiótico cefalosporina C (µp), pelo fungo Cephalosporium acremonium ATCC 48272, usando como ferramenta estatística a técnica de planejamento experimental e a análise por superfície de resposta. Na otimização destas variáveis, os ensaios foram conduzidos de acordo com os planejamentos fatoriais, fracionário, completo e composto central, em fermentações em incubador rotatório, variando as concentrações iniciais dos nutrientes do meio, a 28 oC, pH 7,0+0,1, com agitação de 250 rpm. Para ambos os casos, um modelo ajustado de 2a ordem foi o que melhor correlacionou as variáveis. Os resultados da otimização mostraram que uma velocidade máxima de crescimento de 0,22 h-1 pode ser atingida para uma concentração de glicose e acetato de amônio em torno de 4,20 e 2,5 g/L, respectivamente, e uma velocidade específica de produção de cefalosporina C de 1,12 mgCPC/gcel.h para uma concentração de sacarose de 10,5 g/L e DL-metionina 2,5 g/L com uma massa celular média, para produção, de 14,0 g/L. O planejamento estatístico mostrou-se uma ferramenta bastante eficiente para avaliação do bioprocesso, pois com a realização dos ensaios propostos, foi possível obter valores de µmax e µp superiores aos encontrados na literatura. Desenvolveu-se um modelo baseado na técnica de redes neurais para a simulação de todo o domínio das concentrações de glicose, acetato de amônio, DL-metionina, fósforo, ácido oléico e solução de sais na velocidade específica máxima de crescimento. A rede que melhor representou os valores experimentais foi uma do tipo "feedforward", com três camadas e 22 neurônios na camada oculta, treinada com o algoritmo de retropropagação, por um conjunto de resultados obtidos através de ensaios do planejamento estatístico. As superfícies obtidas pela simulação em amplo domínio de concentrações de glicose e acetato de amônio, mostraram que o crescimento do microrganismo segue um modelo cinético de inibição por este dois nutrientes, prevendo altos valores de µmax para concentrações de glicose e acetato de amônio, comparáveis com os obtidos pelo modelo estatístico.

Yap Hong Kin.
"April 2004."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2004.
Includes bibliographical references (p. 102-116)
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

碩士
明新科技大學
化學工程研究所
96
This study, at first, investigated the morphology evolution of the aerobic pure culture Acremonium chrysogenum (ATCC 48272), by using five different kinds and concentrations of culture media with/without an inducer. The observation shows that the methiolene-containing media has the best inducing effect of culture mutation. Under which A. chrysogenum swelled up in the beginning 6 to 8 hours of the cultivation, and new spores were secreted from parts of the microorganism. Second, the experiments were carried out to investigate the influence of various operating variables, including temperature, pH, agitation rate, inducer concentration, and mycelium outset density, on the product yield. The experimental results show that the optimal operating variables are as follows: 530 rpm, pH 5.89, 31.85ºC, DO 40%, 6.4g/L methionine, and 43.5% of inoculation (v/v). The experimental results show that a maximum concentration of 12.24g/L Cephalosporin C can be achieved under the optimal operating condition. A maximum concentration of 10.11g/L Cephalosporin C can be achieved at 30oC, 4.72g/L was achieved at 600rpm of the agitation rate, 6.29g/L was achieved at 6g/L methionine, and 9.57g/L was achieved at 30% mycelium outset density. Eventually, the fermentation broth was cooperated with PALL cross-flow microfiltration system to concentrate and recover the product. It was filtered through 5 �慆 filter paper and 0.45 �慆 as well as 0.2 �慆 of microfiltration (MF) membrane. The filtrate was then filtered by a 1K Dalton of ultrafiltration (UF) membrane to exclude the huge molecules (such as enzymes). Finally, the filtrate solution was transformed to be the Zn-salt crystal by added with trace ZnSO4•7H2O solution. The concentration of Cephalosporin C was dramatically enhanced from 4.72g/L to 25.16g/L, successfully by using the membrane separation, the effective concentration and purification procedure.

The method was developed to determine simultaneously 19 drugs of three classes (seven Penicillins and Cephalosporins, and five Quinolones) regulated by European legislation 2377/90/EC in cow milk using LC-MS/MS. To make the sample preparation as simple as possible deproteinization step using organic solvent was eliminated. SPE clean up and concentration was done using OASIS HLB cartridge. The separation of 19 antibiotics was achieved in 11 minutes using C8 column with gradient elution. MS data was acquired on MRM mode with two transitions for each compound. Validation procedure was conducted based on European directive 2002/657/EC. 89% of drugs presented recoveries higher than 65 % with exception of AMOX (57%) and DAN (38 %). Repeatability values expressed as relative standard deviation, (RSDs) were not more than 15 %. LODs values ranged from 0.03 (NAFC) to 0.5 μg/kg (PER) and LOQs from 0.1 (NAFC, PENG, CIP, DAN, and ENR) to 1.25 μg/kg (PER). The method was applied to 49 real samples. 37% of the sample was found to be non-compliant with an error probability of 􀉴. Of all samples analysed, 14 % of samples contained AMOX and 16 % contained PENG. AMOX and PENG was the most common residue found in milk sample.