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Effects Of Bioreactor Operation Parameters On Intracellular Reaction Rate Distribution In Beta-lactamase Production By Bacillus SpeciesArifoglu, Muge 01 August 2004 (has links) (PDF)
In this study, the effects of oxygen transfer (OT) on beta-lactamase production and on intracellular reaction rates were investigated with Bacillus licheniformis ATCC 2597. In order to clarify the oxygen transfer effects on the production of beta-lactamase, firstly a glucose based defined medium was designed and using this medium, the effects of bioreactor operation parameters, i.e., pH and temperature, on beta-lactamase activity and cell formation were investigated in laboratory scale batch-bioreactors using shake bioreactors having V=33 ml working volumes. Among the investigated bioprocess conditions, the highest beta-lactamase activity was obtained as A=115 U cm-3, in the medium with 7.0 kg m-3 glucose, 7.1 kg m-3 (NH4)2HPO4 and the salt solution, at pH0=7.5, T=37C, N=200 min-1. At the optimum conditions found in laboratory scale the effects of OT on cell generation, substrate consumption, product (beta-lactamase) and by-products formations were investigated at three different air inlet (Q0/ VR = 0.2, 0.5 and 1 vvm) and at three agitation rates (N=250, 500, 750 min-1) in V = 3.0 dm3 batch bioreactors consisting of temperature, pH, foam, stirring rate and dissolved oxygen controls. Along with the fermentation, cell, substrate and by-product concentrations, beta-lactamase activity, yield coefficients, specific rates,
oxygen uptake rates and the liquid phase mass transfer coefficient values were determined. The highest beta-lactamase activity was obtained at 0.5 vvm 500 min-1 and at 0.2 vvm 500 min-1 conditions as ca. A=90 U cm-3 while the highest cell concentration was obtained as Cx=0.67 kg m-3 at 0.5 vvm 750 min-1 and at 0.2 vvm 750 min-1 conditions. KLa, increased with the increase in the agitation and aeration rates and its values varied between 0.007-0.044 s-1 and oxygen uptake rate varied between 0.4-1.6 mol m-3 s-1. Finally, the influence of OT conditions on the intracellular reaction rates was investigated using metabolic flux analysis to evaluate the effects of oxygen on the metabolism.
Keywords: beta-lactamase, production, Bacillus, oxygen transfer, metabolic flux analysis
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IMPROVING THE CELLULAR ECONOMY OF STREPTOCOCCUS ZOOEPIDEMICUS THROUGH METABOLIC ENGINEERINGFong Chong, Barrie Unknown Date (has links)
Hyaluronic acid (HA) is a high molecular weight polysaccharide that is mainly produced by animals and certain bacteria. This polymer is biocompatible and possesses desirable rheological properties that are accentuated by high molecular weight. Diverse therapeutic applications have developed which harness these features. Pharmaceutical grade HA is mostly extracted from animal tissue. The HA derived from this source is suitable for most pharmaceutical preparations but there is growing pressure to avoid animal tissue products. This has provided the incentive to expand microbial-based HA manufacturing. However, the inherent low molecular weight of the polymer derived via this route has hampered widespread acceptance of microbial HA. This thesis examined the ramifications of improving the cellular economy of the HA-producing, gram-positive bacterium, Streptococcus zooepidemicus. Improved cellular economy is believed to be a prerequisite for achieving superior HA yields and molecular weights in this microorganism. This work examined the metabolic variation that accompanied the shift to more efficient modes of carbon utilization. In particular the effect of different sugar sources, uncoupling growth and polymer formation, and changes to the cellular oxidoreduction capacity were studied in more detail. This study utilized different sugar sources to enhance the recovery of energy. Fermenting glucose, fructose and maltose produced contrasting patterns of growth and HA formation. Culturing the organism in maltose caused a shift towards energy-efficient heterofermentative metabolism. Maltose-cultured cells displayed a biphasic pattern of metabolism. The first stage corresponded to a growth phase in which biomass synthesis profited from the increased energy yield. The second stage corresponded to an arginine-deficient stationary phase where the majority of the HA was formed. The fermentation rate was slower during stationary phase but continued to support HA biosynthesis. This bisphasic metabolism proved to be beneficial. A protracted stationary phase led to higher molecular weight HA. Fructose was unable to sustain a comparable polymer yield or molecular weight as glucose or maltose. There was evidence that the arginine deiminase pathway was responsible for the premature depletion of arginine in maltose-fermenting cultures. The accumulation of biomass exhibited a concentration-dependent response to the amount of glutamine in the medium. A second arginine transporter possessing a low affinity for glutamine could explain this phenomenon. Arginine consumption was slower when the glutamine level was elevated. This may indicate competition for a common transmembrane carrier. An elevated energetic yield and ATP formation rate were features of aerobic maltose metabolism. The relative improvement in biomass and HA yields were substantially greater for cultures fermenting maltose compared to glucose. However, no improvement in molecular weight compared to glucose was observed. A major factor contributing to the success of aerobic maltose fermentation was the particularly high NADH oxidase flux. This enzyme reoxidizes reduction equivalents in a reaction that is physically decoupled from the production of reduced metabolic products. Less lactate and ethanol accumulated in the presence of high NADH oxidase levels but acetate production was stimulated leading to an improved energetic yield. This result prompted an investigation into the effect of elevating the NADH oxidase level. The native NADH oxidase gene was sequenced and cloned into an inducible expression plasmid and introduced into S. zooepidemicus. Overproduction of this enzyme led to the desired improvement in ATP yield. A significant improvement in biomass yield was demonstrated. HA yield and molecular weight were not affected. Lactate and acetate were the main fermentation products. At high induction levels the quantity of lactate and acetate approached limiting levels and pyruvate overflow was more pronounced. This was attributed to insufficient flux capacity of the pyruvate dehydrogenase enzyme complex. The application of metabolic engineering to S. zooepidemicus has provided some insight into the regulation of energy metabolism in this microorganism and its relationship to HA synthesis. This study has observed that the specific rate of HA synthesis is correlated to the sugar uptake rate but is unaffected by the ATP yield. Under present conditions the formation of HA is not limited by the availability of energy. Nonetheless, microbial HA production will benefit from maximizing energetic yield. It was demonstrated that less catabolic carbon was expended to support biomass formation if the energetic yield was high. Therefore more residual carbon was available for HA synthesis.
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Desenvolvimento de uma ferramenta computacional para a análise de fluxos metabólicos empregando carbono marcado. / Development of a computational tool for metabolic flux analysis with labeled carbon.Rafael David de Oliveira 11 October 2017 (has links)
A 13C-Análise de Fluxos Metabólicos (13C-MFA) tornou-se uma técnica de alta precisão para estimar fluxos metabólicos e obter informações importantes sobre o metabolismo. Este método consiste em procedimentos experimentais, técnicas de medição e em cálculos para análise de dados. Neste contexto, os grupos de pesquisa de engenharia metabólica necessitam de ferramentas computacionais precisas e adequadas aos seus objetos de estudo. No presente trabalho, foi construída uma ferramenta computacional na plataforma MATLAB que executa cálculos de 13C-MFA, com balanços de metabólitos e cumômeros. Além disso, um módulo para estimar os fluxos metabólicos e um módulo para quantificar as incertezas das estimativas também foram implementados. O programa foi validado com dados presentes na literatura e aplicado a estudos de caso. Na estimação de fluxos de Pseudomonas sp. LFM046, identificou-se que esse micro-organismo possivelmente utiliza a Via das Pentoses em conjunto com a Via Entner-Doudoroff para a biossíntese de Polihidroxialcanoato (PHA). No design ótimo de experimentos para uma rede genérica de Pseudomonas, identificou-se a glicose marcada no átomo cinco como um substrato que permitirá determinar o fluxo na Via das Pentoses com menor incerteza. / 13C-Metabolic Flux Analysis (13C-MFA) has become a high-precision technique to estimate metabolic fluxes and get insights into metabolism. This method consists of experimental procedures, measurement techniques and data analysis calculations. In this context, metabolic engineering research groups demand accurate and suitable computational tools to perform the calculations. A computational tool was implemented in MATLAB platform that performs 13C-MFA calculation, using metabolite and cumomer balances, as well as a module to estimate the fluxes and a module to quantify their uncertainty. The program was validated with some classical cases from literature. From the flux estimates of Pseudomonas sp. LFM046, it was identified that the microorganism possibly uses the Pentose Phosphate Pathway along with the Entner-Doudoroff Pathway for Polyhydroxyalkanoate (PHA) biosynthesis. From the optimal experimental design for a generic Pseudomonas network, it was possible to conclude that glucose labeled at atom five is the best option to determine the flux in the Pentose Phosphate Pathway with smaller uncertainty.
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Energetic Costs of AhR Activation in Rainbow Trout (Oncorhynchus mykiss) HepatocytesNault, Rance January 2011 (has links)
Aquatic organisms in response to toxic insults from environmental pollutants activate defence systems including the aryl hydrocarbon receptor (AhR) in an attempt to metabolize and excrete these toxicants and their metabolites. These detoxification mechanisms however may come with certain energetic costs. I hypothesize that the activation of the AhR by β-Naphthoflavone (β-NF), a model AhR agonist, results in increased energetic costs requiring metabolic reorganization in rainbow trout hepatocytes. While the results obtained suggest that there are no significant energetic costs of AhR activation, analysis of enzyme activities suggests possible metabolic reorganization. This study also showed significant changes in cellular processes in hepatocytes over the incubation periods which previously were not reported. Furthermore, for the first time in fish hepatocytes, metabolic flux analysis (MFA) was used to examine intra-cellular metabolism, the applicability of which is discussed.
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Continuous production of succinic acid by Actinobacillus succinogenes : steady state metabolic flux variationBradfield, M.F.A. (Michael Ford Alexander) January 2013 (has links)
Continuous fermentations were performed in a novel external-recycle, biofilm reactor using D-glucose and CO2 as carbon substrates. Corn steep liquor (CSL) and yeast extract (YE) served as nitrogen sources.
In anaerobic fermentations using medium containing CSL and YE, succinic acid (SA) yields were found to be an increasing function of glucose consumption. The ratio of SA to the major by-product, acetic acid (YAASA), increased from 2.4 g g-1 at a glucose consumption of 15 g L-1, to 5.7 g g-1 at a glucose consumption of 46 g L-1. For medium containing no CSL, YAASA remained near 1.97 g g-1, exceeding this for cases where biofilm grown on CSL-containing medium was present.
The ratio of formic acid to acetic acid (YAAFA), for CSL-containing medium, decreased from an equimolar value (0.77 g g-1) at a glucose consumption of 10 g L-1 to zero at 46 g L-1 glucose consumed. In contrast, YAAFA for YE-only medium remained at 0.77 g g-1. Therefore, pyruvate was metabolised solely by pyruvate-formate lyase when no CSL was present.
The highest SA yield obtained on glucose, SA titre and SA productivity were 0.91 g g-1, 48.5 g L-1 and 9.4 g L-1 h-1, respectively, all for medium containing CSL. Medium that included CSL significantly outperformed medium that excluded CSL, achieving 64%, 21% and 203% greater SA titres, yields on glucose and productivities respectively.
Metabolic flux analyses based on the established C3 and C4 metabolic pathways of Actinobacillus succinogenes revealed that the increase in YAASA, for CSL-containing fermentations, could not be attributed to the decrease in formate and biomass formation, and that an additional source of reducing power was present. The fraction of reducing power (NADH) unaccounted for increased with glucose consumption, suggesting that the maintenance or non-growth metabolism encountered at higher SA titres differs from the growth metabolism. It is postulated that the additional reducing power originates from an active pentose phosphate pathway in non-growing cells or from an undetected component(s) in the fermentation medium. No major metabolic flux variations were found in fermentations that excluded CSL. / Dissertation (MEng)--University of Pretoria, 2013. / gm2014 / Chemical Engineering / unrestricted
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Desenvolvimento de processo de produção de polihidroxibutirato a partir da xilose empregando técnicas de engenharia evolutiva e bioprocessos. / Development of polyhydroxybutyrate production from xylose employing evolutionary engineering techniques and bioprocesses.Gómez, Carlos Andrés Fajardo 26 May 2015 (has links)
O trabalho é proposto visando melhorar o consumo de xilose na bactéria Burkholderia sacchari utilizando o acúmulo de PHB como modelo de produção Foi desenvolvido um processo de evolução por meio da aplicação de feast and famine e Cultivos sequenciais em fase exponencial. Foi obtida uma linhagem mutante com uma velocidade especifica de crescimento de 0,24 h -1. Foi feita uma análise de fluxos metabólicos da qual foi possível concluir que o metabolismo da xilose acontecia em sua maioria pela VP junto com a ED. Foi feito um ensaio de acumulo com carbono marcado utilizando uma solução de xilose, de 20:80 de xilose marcada 13C em todos os carbonos e xilose não marcado, para determinar quais seriam as possíveis vias metabólicas no uso da xilose por parte de B. sacharia LFM 101 e da linhagem evoluída BSEV11. Foi determinado que houve embaralhamento de carbonos, fato que só acontece quando o metabolismo da xilose e feito pela VP junto com a via ED, assim foi possível conferir a via ED como principal via para o metabolismo da xilose em B. sacchari LFM 101. / To evaluate the possibilities of improving the productivity of PHA production from xylose, evolutionary engineering techniques were applied to B. sacchari to select cells with maximum specific growth rates (max) higher than the wild type. Metabolic flux analysis was also performed to evaluate the fluxes through central pathways and the possibility of further improvements by modifying fluxes rates. The evolved strain reached a max of 0.24 ± 0.01 h-1 at the end of the evolutionary process. Strains were submitted to bioreactor experiments. A metabolic network of the strain was usedn to determine the possible distribution of metabolic fluxes. A total of 19 elementary modes were obtained. It was concluded that the metabolism of xylose occurred mostly by VP along with the ED. The ED pathway has the major activity going on in a cyclic way. It was also performed a 13C labeled xylose assay, in which it was possibly to confirm the obtained results from the metabolic flux analyses.
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Bioprocess Development For Therapeutical Protein ProductionCelik Akdur, Eda 01 December 2008 (has links) (PDF)
In this study, it was aimed to develop a bioprocess using the Pichia pastoris expression system as an alternative to the mammalian system used in industry, for production of the therapeutically important glycoprotein, erythropoietin, and to form stoichiometric and kinetic models.
Firstly, the human EPO gene, fused with a polyhistidine-tag and factor-Xa protease target site, in which cleavage produces the native termini of EPO, was integrated to AOX1 locus of P. pastoris. The Mut+ strain having the highest rHuEPO production capacity was selected. The glycosylation profile of rHuEPO was characterized by MALDI-ToF MS and Western blotting. The native polypeptide form of human EPO was obtained for the first time in P. pastoris expression system, after affinity-purification, deglycosylation and factor-Xa protease digestion.
Thereafter, effects of medium components and pH on rHuEPO production and cell growth were investigated in laboratory-scale bioreactors. Sorbitol was shown to increase production efficiency when added as a co-substrate. Moreover, a cheap alternative nutrient, the byproduct of biodiesel industry, crude-glycerol, was suggested for the first time for P. pastoris fermentations. Furthermore, methanol feeding strategy was investigated in fed-batch pilot-scale bioreactors, producing 70 g L-1 biomass and 130 mg L-1 rHuEPO at t=24h.
Moreover, metabolic flux analysis by using the stoichiometric model formed, which consisted of m=102 metabolites and n=141 reactions, proved useful in further understanding the P. pastoris metabolism. Finally, the first structured kinetic model formed for r-protein production with P. pastoris successfully predicted cell growth, substrate consumption and r-product production rates, where rHuEPO production kinetics was associated with AOX production and proteolytic degradation.
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Cryopreservation effects on a pancreatic substitute comprised of beta cells or recombinant myoblasts encapsulated in non-adhesive and adhesive alginate hydrogelsAhmad, Hajira Fatima 05 September 2012 (has links)
For clinical translation of a pancreatic substitute, long-term storage is essential, and cryopreservation is a promising means to achieve this goal. The two main cryopreservation methods are conventional freezing and vitrification, or ice-free cryopreservation. However, as both methods have their potential drawbacks for cryopreservation of a pancreatic substitute, they must be systematically evaluated in order to determine the appropriate method of cryopreservation. Furthermore, previous studies have indicated benefits to encapsulation in 3-D adhesive environments for pancreatic substitutes and that adhesion affects cell response to cryopreservation. Thus, the overall goal of this thesis was to investigate cryopreservation effects on model pancreatic substitutes consisting of cells encapsulated in non-adhesive and adhesive 3-D alginate hydrogels. Murine insulinoma betaTC-tet cells encapsulated in unmodified alginate hydrogels were chosen as the model pancreatic substitute in a non-adhesive 3-D environment. Murine myoblast C2C12 cells, stably transfected to secrete insulin, encapsulated in partially oxidized, RGD-modified alginate hydrogels were chosen as the model pancreatic substitute in a 3-D adhesive environment. With respect to cryopreservation effects on intermediary metabolism of betaTC-tet cells encapsulated in unmodified alginate, results indicate that relative carbon flow through the tricarboxylic acid cycle pathways examined is unaffected by cryopreservation. Additionally, insulin secretory function is maintained in Frozen constructs. However, vitrification by a cryopreservation cocktail referred to as DPS causes impairment in insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. Results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response to cryopreservation after vitrification or freezing. Although there are differences in metabolic activity and insulin secretion immediately post-warming for DPS-vitrified RGD-encapsulated Stable C2C12 cells relative to Fresh controls, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate.
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Desenvolvimento de processo de produção de polihidroxibutirato a partir da xilose empregando técnicas de engenharia evolutiva e bioprocessos. / Development of polyhydroxybutyrate production from xylose employing evolutionary engineering techniques and bioprocesses.Carlos Andrés Fajardo Gómez 26 May 2015 (has links)
O trabalho é proposto visando melhorar o consumo de xilose na bactéria Burkholderia sacchari utilizando o acúmulo de PHB como modelo de produção Foi desenvolvido um processo de evolução por meio da aplicação de feast and famine e Cultivos sequenciais em fase exponencial. Foi obtida uma linhagem mutante com uma velocidade especifica de crescimento de 0,24 h -1. Foi feita uma análise de fluxos metabólicos da qual foi possível concluir que o metabolismo da xilose acontecia em sua maioria pela VP junto com a ED. Foi feito um ensaio de acumulo com carbono marcado utilizando uma solução de xilose, de 20:80 de xilose marcada 13C em todos os carbonos e xilose não marcado, para determinar quais seriam as possíveis vias metabólicas no uso da xilose por parte de B. sacharia LFM 101 e da linhagem evoluída BSEV11. Foi determinado que houve embaralhamento de carbonos, fato que só acontece quando o metabolismo da xilose e feito pela VP junto com a via ED, assim foi possível conferir a via ED como principal via para o metabolismo da xilose em B. sacchari LFM 101. / To evaluate the possibilities of improving the productivity of PHA production from xylose, evolutionary engineering techniques were applied to B. sacchari to select cells with maximum specific growth rates (max) higher than the wild type. Metabolic flux analysis was also performed to evaluate the fluxes through central pathways and the possibility of further improvements by modifying fluxes rates. The evolved strain reached a max of 0.24 ± 0.01 h-1 at the end of the evolutionary process. Strains were submitted to bioreactor experiments. A metabolic network of the strain was usedn to determine the possible distribution of metabolic fluxes. A total of 19 elementary modes were obtained. It was concluded that the metabolism of xylose occurred mostly by VP along with the ED. The ED pathway has the major activity going on in a cyclic way. It was also performed a 13C labeled xylose assay, in which it was possibly to confirm the obtained results from the metabolic flux analyses.
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An Integrative Genome-Based Metabolic Network Map of Saccharomyces Cerevisiae on Cytoscape: Toward Developing A Comprehensive ModelHamidi, Aram 03 1900 (has links)
Indiana University-Purdue University Indianapolis (IUPUI) / Metabolic flux analyses and their more comprehensive forms, genome-scale metabolic networks (GSMNs), have gained tremendous attention in industrial and medical research. Saccharomyces cerevisiae (S. cerevisiae) is one of the organisms that has had its GSMN subjected to multiple frequent updates. The objective of this study is to develop a visualization tool for the GSMN of S. cerevisiae for educational and research purposes. This visualization tool is called the Master Metabolic Map of Saccharomyces cerevisiae (MMMSC). In this study, a metabolic database of S. cerevisiae developed by us was transferred to Cytoscape, a useful and efficient bioinformatics software platform for visualizing molecular networks. After the MMMSC was created, nodes, representing metabolites and enzymes, and edges, representing the chemical reactions that connect the nodes, were curated manually to develop a metabolic visualization map of the whole metabolic system of S. cerevisiae (Figure 4). In the discussion, examples are provided regarding possible applications of MMMSC to predict possible effects of the manipulation of the S. cerevisiae metabolome for industrial and medical purposes. Ultimately, it is concluded that further work is needed to complete the metabolic database of S. cerevisiae and the related MMMSC. In future studies, these tools may be integrated with other omics and other approaches, especially the directed-evolution approach, to increase cost and time efficiency of future research and to find solutions for complex and, thus far, poorly managed environmental and health problems.
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