The purification of pepsinogen from the stomach of red perch using aqueous two phase systems (ATPS) formed by polyethylene glycol (PEG) and salt at 4°C was optimized. Salt type, salt concentration, PEG molecular weight and PEG concentration had significant effects on total volume (TV), volume ratio (VR), enzyme activity (AE), protein content (CP), specific activity (SA), purification fold (PF) and recovery yield (RY). (NH4)2SO4 at 15% w/w concentration was selected as the optimum salt type and concentration. PEG 1500 at 18% w/w concentration was selected as the optimum PEG molecular weight and concentration. 15% (NH4)2SO4-18% PEG 1500, the optimal ATPS, was compared with ammonium sulfate fractionation (ASF). ATPS gave better partition of pepsinogen (SA of 5.40 U/mg, PF of 5.20 and RY of 86.6%) than ASF (SA of 2.55 U/mg, PF of 2.46, RY of 70.4%). / This is the electronic copy of partition of pepsinogen in aqueous two phase system method.

Counter current chromatography (CCC) is a form of liquid liquid chromatography, which the Brunel Institute for Bioengineering (BIB) team have developed to process scale. In this thesis, its application has been successfully extended to the rapid, scalable purification of monoclonal antibodies (mAb) from mammalian cell culture, using aqueous two-phase systems (ATPS) of inorganic salts and polymer. A polyethylene glycol (PEG) and sodium citrate system was found to be the most appropriate by robotic phase system selection. The search for an economical alternative to protein A HPLC is a substantial bioprocessing concern; in this work CCC has been investigated. Initial studies showed that unpredictably, despite separation from impurities being achieved, some loss in the IgG‘s ability to bind to Protein A was seen, as confirmed by Protein A BiaCore analysis. CCC machines were seen to adversely affect IgG functionality. This led to a systematic investigation of the effect of CCC phase mixing on IgG functionality in a number of different CCC instruments, allowing direct comparisons of modes of CCC (hydrodynamic and hydrostatic CCC) and their associated mixing (wave-like and cascade, respectively). The varying g forces produced within the CCC column were determined using a recently developed model to calculate g force range. The effect of interfacial tension was also studied using a custom built 'g' shaker. The optimum CCC mode was identified to be the non synchronous CCC, operated in a hydrodynamic mode but allowing bobbin to rotor speed (Pr ratio) to be controlled independently. In a normal synchronous J type centrifuge a Pr of 1 is fixed, this is where the bobbin and rotor speed are identical I.e. one bobbin rotation (where mixing occurs) to one rotor revolution (where settling occurs). Constraints were seen with this 1:1 ratio and the separation of mAb using ATPS. This work has shown with the use of the non synchronous CCC at a Pr of 0.33, mixing is reduced and rotor rotations increased. Consequently the associated g force range is decreased. Furthermore, by the extension of settling time, the clear separation of the mAb from impurities has been achieved with retention of biological activity. This thesis demonstrates the importance of settling time for ATPS in phase separation and documents the fundamental requirements for the successful separation of biologics. Purified non synchronous CCC samples have additionally undergone rigorous quality control testing at Lonza Biologics by their purification scientists. This work has ultimately showed that with optimisation, the non synchronous CCC can be used to produce biological samples that are of industry standard.

No description available.

A procedure for the preparation of spinach leaf mitochondria was developed. The procedure combines differential centrifugation, partition in dextran- polyethyleneglycol two-phase system and Percoli density gradient centri- fugation. The different steps separate the material mainly according to size, surface properties and density, respectively. No chlorophyll was present in the final mitochondrial preparation and the mitochondria were also markedly enriched relative to peroxisomes and microsomes as esti­mated from the recovery of marker enzymes. The latency of enzyme activities was used to study the apparent intactness of the mitochondrial membranes. These measurements showed that both the inner and outer mitochondrial membranes were more than 90 % intact. The mitochondria were also functionally intact since the coupling between respiration and oxidative phosphorylation was retained. The purity of the preparation made it possible to study cytochromes from leaf mitochondria. The cytochrome content of stalk and leaf mitochondria was measured in order to compare mitochondria from photosynthesizing and non-photosynthesizing tissue. The measurements were performed by difference spectroscopy both at room temperature and at liquid nitrogen temperature. Qualitatively the cytochrome content in mitochondria from stalks and leaves was identical. Quantiatively leaf mitochondria contained,on a protein basis, only half the amount of the different cytochromes as compared to stalk mitochondria. The relative content of the different cytochromes was, however, similar suggesting that the composition of the respiratory chain was the same. The photorespiratory conversion of glycine to serine takes place in the mitochondria and involves oxidative decarboxylation of glycine. The ability to oxidize glycine via the respiratory chain was present in spinach leaf mitochondria, but absent in mitochondria prepared from roots, stalks and leaf veins from the same plants. This confirmed the specific localization of the glycine oxidizing activity to photosyntheticaliy active tissue, as suggested by studies with other plant material. The conversion of glycine to serine is a complex reaction depending on the combined action of two enzymes: glycine decarboxylase and serine hydroxymethyltransferase. The effect of inhibitors on the serine hydroxy­methyl transferase activity and the rate of the glycine bicarbonate exchange reaction associated with glycine decarboxylase was studied. These reactions represent partial steps in the conversion of glycine to serine and the aim was to investigate the site of inhibition for the different inhibitors, namely, isonicotinyl hydrazide (a pyridoxa!phosphate antagonist), amino- acetonitrile, glycinehydroxamate (glycine analogues) and cyanide. The results showed that these inhibitors had a complex pattern of inhibition. The same inhibitor affected more than one site and often with an apparently different mechanism. It was, however, found that aminoacetonitrile at low concentrations specifically inhibited glycine decarboxylase and that cyanide specifically inhibited serine hydroxymethyltransferase. / digitalisering@umu

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-07-31Bitstream added on 2014-06-13T20:56:21Z : No. of bitstreams: 1 machado_acr_me_jabo.pdf: 286825 bytes, checksum: f2ef2df876cf8ed795033e0d4e37d7da (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A busca por meios de cultura alternativos e métodos de produção, são aspectos importantes para viabilizar a utilização de fungos entomopatogênicos em larga escala. O presente trabalho avaliou, em ensaios distintos, a produção dos isolados JAB 02 e JAB 45 de Lecanicillium lecanii em meios líquidos obtidos de resíduos e subprodutos agroindustriais (água do cozimento de arroz e da prensa da mandioca, vinhaça, leite de levedura e melaço de cana-de-açúcar, soro de queijo e milhocina), e em misturas de substratos sólidos obtidos de diferentes grãos e derivados (trigo grosso e farelos de trigo e de soja com lentilha, painço, sorgo, trigo em grão, quirela de milho). A determinação dos meios mais eficientes para produção dos isolados foi baseada na avaliação da produção de conídios e de biomassa nos meios líquidos e a produção e viabilidade de conídios obtidos nos meios sólidos. Para ambos os isolados, a produção em meios líquidos favoreceu a formação de biomassa micelial, obtendo¬se os maiores valores nos meios a base de água da prensa da mandioca, soro de queijo, milhocina@ e melaço de cana-de-açúcar. Os meios sólidos que proporcionaram melhores resultados para o isolado JAB 02 foram obtidos da mistura de trigo grosso e de farelo de trigo com lentilha e com sorgo. Para JAB 45, obtiveram-se as maiores produções de conídios usando a mistura de trigo grosso com lentilha, e de farelo de trigo com painço, lentilha e trigo em grão, destacando¬se a última no qual a produção foi de 1,05 x 108 con g substrato-1. Os meios líquidos e sólidos que proporcionaram os melhores resultados foram, posteriormente, combinados em um sistema bifásico de cultivo Neste ensaio foram avaliadas a esporulação e viabilidade de conídios e o rendimento do processo. O método de cultivo não influenciou... / The search for alternative media and production methods are important aspects for the large scale use feasibility of entomopathogenic fungi. The present work evaluated the production of the isolates JAB 02 and JAB 45 of Lecanicillium lecanii, in liquid media obtained from agroindustrial residuais and byproducts (water of cooked rice and cassava bran, sugarcane vinasse, years cream, sugar cane molasses, cheese whey and milhocina@) and in combination with solid substracts obtained from different grains and its derivates (ground wheat and wheat and soybean bran's with lentil, Italian millet sorghum, wheat grain, cracked corn). The most favorable media were combined in a two-phase system, where the fungus was initially cultivated in a liquid medium and then the micelial mass was transferred to a solid medium. The determination of the most efficient production media for the isolates was based, in the liquid media, on the evaluation of conidia and biomass production, and, in the solid media, on conidia production and viability. For both isolates, the production in liquid media favored large quantity of micelial biomass, emphasizing the media based on water from cassava bran, cheese whey, milhocina@ and sugar cane moi asses. The solid media that proportionated better results, for JAB 02, were obtained from the mixture of ground wheat and wheat barn with lentil and sorghum. For JAB 45, the biggest production of conidia were obtained by the mixture of ground wheat with lentil and wheat bran combined with Italian millet, lentil and wheat grain and with lentil, emphasizing the last one with the production of 1,05 x 108 con 9 substracr1. The liquid and solid media that proportionated better results were afterwards combined in a two-phase system. In this system the conidia sporulation and viability and process income were evaluated. For JAB 02 the cultivation method did not influence the conidia... (Complete abstract click electronic access below)

Orientador: Antônio Carlos Monteiro / Banca: Maria Silvia Pereira Leite / Banca: Inajá Marchizeli Wenzel / Resumo: A busca por meios de cultura alternativos e métodos de produção, são aspectos importantes para viabilizar a utilização de fungos entomopatogênicos em larga escala. O presente trabalho avaliou, em ensaios distintos, a produção dos isolados JAB 02 e JAB 45 de Lecanicillium lecanii em meios líquidos obtidos de resíduos e subprodutos agroindustriais (água do cozimento de arroz e da prensa da mandioca, vinhaça, leite de levedura e melaço de cana-de-açúcar, soro de queijo e milhocina), e em misturas de substratos sólidos obtidos de diferentes grãos e derivados (trigo grosso e farelos de trigo e de soja com lentilha, painço, sorgo, trigo em grão, quirela de milho). A determinação dos meios mais eficientes para produção dos isolados foi baseada na avaliação da produção de conídios e de biomassa nos meios líquidos e a produção e viabilidade de conídios obtidos nos meios sólidos. Para ambos os isolados, a produção em meios líquidos favoreceu a formação de biomassa micelial, obtendo¬se os maiores valores nos meios a base de água da prensa da mandioca, soro de queijo, milhocina@ e melaço de cana-de-açúcar. Os meios sólidos que proporcionaram melhores resultados para o isolado JAB 02 foram obtidos da mistura de trigo grosso e de farelo de trigo com lentilha e com sorgo. Para JAB 45, obtiveram-se as maiores produções de conídios usando a mistura de trigo grosso com lentilha, e de farelo de trigo com painço, lentilha e trigo em grão, destacando¬se a última no qual a produção foi de 1,05 x 108 con g substrato-1. Os meios líquidos e sólidos que proporcionaram os melhores resultados foram, posteriormente, combinados em um sistema bifásico de cultivo Neste ensaio foram avaliadas a esporulação e viabilidade de conídios e o rendimento do processo. O método de cultivo não influenciou... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The search for alternative media and production methods are important aspects for the large scale use feasibility of entomopathogenic fungi. The present work evaluated the production of the isolates JAB 02 and JAB 45 of Lecanicillium lecanii, in liquid media obtained from agroindustrial residuais and byproducts (water of cooked rice and cassava bran, sugarcane vinasse, years cream, sugar cane molasses, cheese whey and milhocina@) and in combination with solid substracts obtained from different grains and its derivates (ground wheat and wheat and soybean bran's with lentil, Italian millet sorghum, wheat grain, cracked corn). The most favorable media were combined in a two-phase system, where the fungus was initially cultivated in a liquid medium and then the micelial mass was transferred to a solid medium. The determination of the most efficient production media for the isolates was based, in the liquid media, on the evaluation of conidia and biomass production, and, in the solid media, on conidia production and viability. For both isolates, the production in liquid media favored large quantity of micelial biomass, emphasizing the media based on water from cassava bran, cheese whey, milhocina@ and sugar cane moi asses. The solid media that proportionated better results, for JAB 02, were obtained from the mixture of ground wheat and wheat barn with lentil and sorghum. For JAB 45, the biggest production of conidia were obtained by the mixture of ground wheat with lentil and wheat bran combined with Italian millet, lentil and wheat grain and with lentil, emphasizing the last one with the production of 1,05 x 108 con 9 substracr1. The liquid and solid media that proportionated better results were afterwards combined in a two-phase system. In this system the conidia sporulation and viability and process income were evaluated. For JAB 02 the cultivation method did not influence the conidia... (Complete abstract click electronic access below) / Mestre

Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-22T03:53:34Z No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Rejected by Gilmar Barros (gilmargomesdebarros@gmail.com), reason: Faltou colocar o nome do co-orientador. on 2016-04-22T14:51:38Z (GMT) / Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-25T19:34:12Z No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Rejected by dayse paz (daysepaz@hotmail.com), reason: Tens que incluir co-orientador ele segue sendo o segundo. Atenciosamente Equipe RI-FURG on 2016-04-26T14:45:06Z (GMT) / Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-26T15:32:29Z No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Approved for entry into archive by dayse paz (daysepaz@hotmail.com) on 2016-04-26T15:37:03Z (GMT) No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) / Made available in DSpace on 2016-04-26T15:37:03Z (GMT). No. of bitstreams: 1 silvana menoncin - purificao de lipases produzidas por fungo utilizando sistema.pdf: 1423756 bytes, checksum: ab26bd30dc4f0e48f90bdb7c74c6916b (MD5) Previous issue date: 2011 / A purificação de proteínas consiste em um dos desafios da área de Bioprocessos. O grau de pureza requerido para enzimas está diretamente relacionado com a aplicação na qual estas serão empregadas. Os sistemas aquosos bifásicos (SAB) e o processo de separação por membranas (UF) vêm sendo amplamente estudados na purificação de diversas proteínas, por apresentarem algumas vantagens sobre os processos cromatográficos, como facilidade de escalonamento e baixo custo. No entanto, a purificação de lipases por SAB ainda é pouco reportada. Neste sentido, o foco deste trabalho foi avaliar a utilização da melhor combinação do SAB formado por polietilenoglicol (PEG) e tampão fosfato de potássio e a combinação com o processo de separação por membrana, na purificação de lipase de Penicillium crustosum produzida por fermentação em estado sólido. O SAB se apresentou como uma técnica potencial para a separação e purificação da lipase obtendo-se fator de purificação 5,8 no sistema composto de 20% PEG 6000, 7,0% fosfato de potássio pH 8. A técnica do planejamento experimental foi utilizada como uma estratégia para a maximização da purificação, no qual foram avaliadas as concentrações de polietilenoglicol (PEG), fosfato de potássio e NaCl em sistemas compostos por PEG 6000 fosfato de potássio pH 8. O sistema 20% PEG 6000, 5,8% fosfato de potássio pH 8 com acréscimo de 4,8% de NaCl apresentou recuperação da atividade de hidrólise de 53,8% e o maior fator de purificação 10,5. O sistema composto de 20% PEG 6000, 5,8% de fosfato de potássio sem o acréscimo de NaCl foi o que apresentou maior atividade de transesterificação e fator de purificação, 8,5 U/mg e 28,3 vezes, respectivamente. Membranas comerciais com massa molecular de corte de 30 e 60 kDa foram testadas. Com acréscimo de 0,09 mol/L de NaCl o fator de purificação aumentou de 0,8 para 1,4 vezes. Quando combinou-se o sistema aquoso bifásico e separação por membrana, o fator de purificação em termos de atividade de hidrólise diminuiu, mas com relação à de atividade de transesterificação aumentou, alcançando valores em torno de 39 vezes para os sistemas compostos de 11,6% PEG 6000, 10% fosfato de potássio e 1,2% NaCl e 38,3 para o sistema formado por 16% PEG 6000, 8% fosfato de potássio e 4,8% NaCl. / The purification of proteins is one of the challenges in the area of Bioprocesses. The purity degree required for enzymes is directly related to the application in which they are employed. Aqueous two-phase systems (ATPS) and membrane separation process have been investigated in the purification of several proteins, which present some advantages over the chromatographic processes, such as easy scale-up and low cost. However, the purification of lipases by ATPS has been little reported. In this sense, the focus of this study was to evaluate the use of the best combination of the ATPS consisting of polyethylene glycol (PEG) and potassium phosphate buffer and the combination with membrane separation process for the lipases purification from Penicillium crustosum produced by solid state fermentation. The ATPS is a potential technique for the separation and purification of such lipases, resulting in a purification factor 5.8 using the system composed of 20% PEG 6000, 7.0% potassium phosphate, pH 8. The technique of experimental design was used as a strategy for maximizing the purification, in which we assessed the concentrations of polyethylene glycol (PEG), potassium phosphate and sodium chloride in systems composed of PEG 6000 at pH 8. The system 20% PEG 6000, 5.8% potassium phosphate at pH 8, and 4.8% NaCl showed a recovery of the activity (hydrolysis) of 53.8% and yielded the highest purification factor 10.5, based on hydrolytic activity. The system made up of 20% PEG 6000, 5.8% potassium phosphate without the addition of NaCl showed highest activity of transesterification and purification factor, 8.5 U/mg and 28,3, respectively. Commercial membranes with molecular weight cutoff of 30 and 60 kDa were tested. That addition of 0.09 mol/L NaCl to the enzymatic extract in the feed increased the purification factor from 0.8 to 1.4. When the processes of ATPS and UF were combined, the purification factor in terms of hydrolytic activity decreased, but increased in terms of transesterification activity, reaching values around 39 for PF in the systems composed of 11.6% PEG 6000 10% potassium phosphate and 38,3 in the systems composed of 1.2% NaCl and 16% PEG 6000, 8% potassium phosphate and 4.8% NaCl.

No description available.

近年発酵生産が注目されているコハク酸の抽出分離法についてイオン液体を含む液膜法及び水性２相抽出法を用いて研究した．イオン液体を含む含浸型液膜により，受容相に塩酸水溶液を用いることでコハク酸の上り坂輸送を達成できた．次にイオン液体を含む高分子溶媒膜によるコハク酸の透過を検討した．最適な条件は含浸液膜と異なり，透過にはコハク酸の各化学種が関与していた．最後に水溶性有機溶媒やイオン液体を用いた水性２相抽出法によるコハク酸の抽出を行った．塩および糖を相分離剤とすることで，発酵液で想定されるコハク酸の80％以上の抽出が達成できた． / Succinic acid is widely used in many industrial sectors; pharmacy, textile, food, etc. The bio-production of succinic acid from renewable resource was desired because the petrochemical production of succinic acid became costly. In this study, the extraction and separation techniques of succinic acid by liquid membranes (LMs) and aqueous two-phase system (ATPS) containing ionic liquids (ILs) were exploited. The permeation mechanisms and optical conditions were elucidated. / 博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24814号 / 工博第5157号 / 新制||工||1985(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 山子 茂, 教授 辻井 敬亘, 教授 大内 誠 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM