bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determination of maintenance coefficients of Saccharomyces cerevisiae cultures with cell recycle by cross‐flow membrane filtration
Authors: Uribelarrea Jean‐Louis , Winter Jacques , Goma Gérard , Pareilleux Alain 
Published: December 2004

AbstractThe fermentation of glucose by a strain of Saccharomyces cerevisiae was studied in a continuous single‐stage process with recycle of the cells via cross‐flow micro‐filtration membranes. Operating conditions were selected such that the culture was not carbon limited and inhibition by ethanol and cell death were minimized.Steady states were obtained for various biomass bleeding rates, i.e., various specific growth rates. From the experimental data, the stoichiometry of the simultaneous reactions, cell growth, ethanol production and maintenance were established using mass and degree of reduction balance relative to substrates (carbon source and oxygen) and products (biomass, ethanol, carbon dioxide etc.), and the growth parameters, yields, and maintenance cofficients were determined. It was shown that the oxygen consumption was not linked to the kinetics of the fermentation. The calculated growth constants were discussed and compared to the currently reported values.
Protein refolding in reversed micelles
Authors: Hagen Anna J. , Hatton T. Alan , Wang Daniel I. C. 
Published: December 2004

AbstractA novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant‐free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.
Protein refolding in reversed micelles: Interactions of the protein with micelle components
Authors: Hagen Anna J. , Hatton T. Alan , Wang Daniel I. C. 
Published: December 2004

AbstractA novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, γ‐interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since γ‐interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.
Hollow fiber supported gas membrane for in situ removal of ammonium during an antibiotic fermentation
Authors: Hecht V. , Bischoff L. , Gerth K. 
Published: December 2004

AbstractTo study the influence of ammonium on an antibiotic cultivation, mass transfer measurements of ammonium through microporous hydrophobic membranes using different stripping methods were carried out and compared. The higher overall mass transfer coefficients for ammonium were obtained with an acid stripping solution compared to water, vacuum, or sweeping air. A hollow fiber module for in situ removal of ammonium during cultivation was designed and operated in an external bypass to a 10‐L fermentor. Compared to a control fermentation, the cell dry mass could be increased 2.6 times and the antibiotic concentration 8 times, if the in situ ammonium removal was in operation.
Enzymatic catalysis in microemulsions: Enzyme reuse and product recovery
Authors: Larsson Karin M. , Adlercreutz Patrick , Mattiasson Bo , Olsson Ulf 
Published: December 2004

AbstractA technique for enzyme reuse and product recovery from enzymatic catalysis in microemulsions is demonstrated. The enzymatic reaction is performed in a homogeneous isotropic microemulsion; AOT (sodium bis‐(2‐ethyl‐ hexyl)sulfosuccinate)/isooctane/buffer or C12E5(penta ethylene glycol dodecyl ether)/heptane/buffer. By small temperature changes the systems are shifted to two phase regions, where an oil‐rich phase, containing the product, coexists with a water‐rich phase containing surfactant and enzyme. The oil‐rich phase may be replaced by an oil solution containing new substrate. Thus, the reaction may be continued and the enzyme reused. This procedure was repeated nine times in the present study. Data on phase behavior in presence and in absence of protein, partitioning of the components and a radioactive‐labelled protein between the phases, and the repeated use of horse liver alcohol dehydrogenase (HLADH) in the microemulsions are presented.
The permeability of EUDRAGIT RL and HEMA–MMA microcapsules to glucose and inulin
Authors: Douglas J. A. , Sefton M. V. 
Published: December 2004

AbstractMeasurement of the rate of glucose diffusion from EUDGRAGIT RL and HEMA‐MMA microcapsules coupled with a Thiele modulus/Biot number analysis of the glucose utilization rate suggests that pancreatic islets and CHO (Chinese hamster ovary) cells (at moderate to high cell densities) should not be adversely affected by the diffusion restrictions associated with these capsule membranes. The mass transfer coefficients for glucose at 20 °C were of the same order of magnitude for both capsules, based on release measurements: ∼5 × 10−6 cm/s for EUDRAGIT RL and ∼2 × 10−6 for HEMA‐MMA. Inulin release from EUDRAGIT RL was slower than for glucose (mass transfer coefficient 14 ± 4 × 10−8 cm/s). The Thiele moduli were much less than 1, either for a single islet at the center of a capsule or CHO cells uniformly distributed throughout a capsule at 10−6 cells/ mL, so that diffusion restrictions within the cells in EUDRAGIT RL or 800 μm HEMA‐MMA capsules should be negligible. The ratio of external to internal diffusion resistance (Biot number) was less than 1, so that at most, only a small diffusion effect on glucose utilization should be expected (i.e., the overall effectiveness factors were greater than 0.8). These calculations were consistent with experimental observation of encapsulated islet behavior but not fully with CHO cell behavior. Permeability restricted cell viability and growth is potentially a major limitation of encapsulated cells; further analysis is warranted.
A structured kinetic model for Zymomonas mobilis ATCC10988
Authors: Veeramallu Uday , Agrawal Pramod 
Published: December 2004

AbstractThe inhibitory effects of glucose and ethanol on Zymomonas mobilis ATCC10988 were isolated through kinetic analysis of transient batch fermentation data. Growth of Z. mobilis was inhibited above a glucose concentration of 80 g/L. Growth was mildly inhibited by ethanol to 50 g/L, and severely inhibited above this concentration. Specific rates of ethanol production and glucose uptake were essentially invariant during batch fermentation. A structured kinetic model was developed, by way of augmentation of the Extended Bottleneck model, to quantify the kinetics of the growth and product formation processes. The model successfully describes the transient batch fermentation of Z. mobilis over a wide range of initial glucose concentration in a semidefined medium.
Balanced nutrient fortification enables high‐density hybridoma cell culture in batch culture
Authors: Jo Eui‐Cheol , Park Hae‐Joon , Park Jong‐Myun , Kim Kyong‐Ho 
Published: December 2004

AbstractCells of an in vitro culture system are not the same as for an in vivo system, metabolically and physiologically; ineffective utilization of nutrients occurs by cells in vitro. Therefore, a simpler approach is needed to examine closely and overcome differences between in vivo and in vitro cells.Recognizing the ineffectiveness of nutrient utilization in vitro, we have constructed, a balanced, fortified high‐density medium based on RPMI 1640 medium previously optimized for relatively low‐density cell culture. The high‐density medium was used to cultivate a hybridoma line in a batch spinner flask culture. In this fortified medium, a hybridoma cell line 2c3.1 was cultivated to near 1 × 107 cells/mL in batch suspension culture. During the culture, glucose, glutamine, and 10 essential amino acids of concentrations five times richer than normal in the medium were almost thoroughly consumed. Combined analysis of these consumption profiles reveals that the balanced, fortified nutrient supply contributes much to cellular activity to overcome the limitations of in vitro cellular growth. Intermediate metabolites, such as ammonium ion and lactic acid, were produced over concentrations reported until now to be inhibitory. This observation suggests that the major conclusive factor against cellular growth over the critical cell density is not so‐called inhibitory metabolites. As a result of the high‐density culture, 5–8 times higher production of a monoclonal antibody for hepatitis B surface antigen (anti‐HBs) was obtained.Active cellular consumption of all the essential nutrients and the corresponding production of MAb strongly support the potential of our approach to overcome the growth limitation of cells in vitro and to obtain high‐density hybridoma cell culture.
Complications encountered using cibacron blue F3G‐A as a ligand for affinity precipitation of lactate dehydrogenase
Authors: Morris John E. , Fisher Rod R. 
Published: December 2004

AbstractThe use of the affinity interaction between Cibacron Blue F3G‐A (CB) and NADH‐dependent enzymes to selectively precipitate these enzymes has been examined. An attempt was made to form crosslinked precipitates of lactate dehydrogenase (LDH) using bis‐ and poly‐CB conjugates. When precipitation was not observed, an examination of the interaction between the enzyme and the conjugated CB was made. Quasielastic light scattering indicated only a slight radius increase, the greatest being from 50 to 130 Å, when a CB‐dextran conjugate was added to a solution of LDH, and no increase when bis‐CB made with a 1, 6‐diaminohexane spacer was added to a similar solution. The results of enzyme inhibition studies showed that conjugated CB bound at the NAD+ site of LDH. Spectral measurements of the conjugated CB below 5 μM were similar to those reported for a stacking interaction that occurs in solutions with CB concentrations above 5 μM We conclude that the conjugated CB is binding to the LDH, but that a competing dye stacking interaction prevents extensive cross‐linking of the LDH, and thus inhibits precipitation.
A hollow‐fiber reactor design for NMR studies of microbial cells
Authors: Briasco Catherine A. , Ross Debra A. , Robertson Channing R. 
Published: December 2004

AbstractA hollow‐fiber membrane reactor was designed and constructed to allow perfusion of entrapped, dense Escherichia coli cells with nutrient medium during examination of cell metabolism using nuclear magnetic resonance (NMR) spectroscopy. Phosphorus‐31 NMR spectra of the perfused cells included peaks for nucleoside di‐ and triphosphates, sugar phosphates, and pH‐sensitive peaks for inorganic phosphate. The observed intensity of the lumenal inorganic phosphate peak was found to depend on flow rate, ruling out the use of this peak as a concentration reference. Absolute intracellular pH values obtained from NMR measurements were found to be accurate to 0.2 pH units due to uncertainties in intracellular ionic concentrations. Relative pH values, however, were found to be sensitive to cell energetic status. The response of E. coli intracellular pH following a shift to carbon starvation medium was monitored with a resolution of 3 min. Use of a hollow‐fiber reactor for cell containment and perfusion during NMR spectroscopy enables metabolic experiments of longer duration and of greater variety than is possible using standard, nonperfused sample tubes.
The role of the plasma membrane fluidity on the shear sensitivity of hybridomas grown under hydrodynamic stress
Authors: Ramírez Octavio T. , Mutharasan R. 
Published: December 2004

AbstractThe role of the plasma membrane fluidity (PMF) on the shear sensitivity of HB‐32 hybridomas to laminar fluid shear was investigated. Steady‐state fluorescence anisotropy (rs) of the cationic fluorescent probe 1‐[4‐(trimethylamino) phenyl]‐6‐phenylhexa‐1,3,5‐triene, was used to evaluate the PMF of whole hybridoma cells. The PMF was manipulated by the addition of the anesthetic benzyl alcohol, by temperature changes and by cholesterol enrichment. The effect of these PMF modifying procedures on the shear sensitivity of HB‐32 was assessed by exposing the cells to defined levels of laminar shear stress in a Couette flow device. Conditions that resulted in lower rs values (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic® F‐68 showed higher values of rs compared to control experiments during various growth phases, suggesting that the protective mechanism of Pluronic F‐68 relies on its ability to decrease the PMF through direct interaction with the plasma membrane. The protective mechanism of serum against turbulent fluid shear is also discussed from analysis of growth and death kinetics of agitated and static cultures at increasing serum levels. The results of this study show that the fluid state of the plasma membrane is important in determining the integrity of hybridomas when exposed to lethal shear levels. It is concluded that increasing membrane fluidity correlates with increasing shear sensitivity.
Determination of growth and coupled nitrification/denitrification by immobilized Thiosphaera pantotropha using measurement and modeling of oxygen profiles
Authors: Hooijmans C. M. , Geraats S. G. M. , van Neil E. W. J. , Robertson L. A. , Heijnen J. J. , Luyben K. Ch. A. M. 
Published: December 2004

AbstractAn oxygen microsensor in combination with mathematical modeling was used to determine the behavior of immobilized Thiosphaera pantotropha. This organism can convert ammonia completely to nitrogen gas under aerobic conditions (coupled nitrification/denitrification) and denitrifies nitrate at highest rates under anaerobic conditions. Immobilization of T. pantotropha can result in aerobic and anaerobic zones inside the biocatalyst particle which will be advantageous for the conversion of ammonia and nitrate from wastewater. However, information of the effects of immobilization on the physiology of T. pantotropha is necessary for the development of such a system. This article gives the extension of a model developed to describe the behavior of chemostat cultures of T. pantotropha so that it can be used for immobilized cells. The original model was based on metabolic reaction equations. Kinetic and diffusion equations have now been added. Experimental verification was carried out using a stirred tank reactor and a Kluyver flask. After immobilization in agarose, the cells were grown in the particles under continuous culture conditions for 3 days. After 24 h the oxygen penetration depth showed a constant value of 100 μ, indicating that a steady state was reached. Scanning electron micrographs showed that large colonies of cells were present in this 100‐μm aerobic layer.From the dynamics of the start‐up phase, several parameters were determined from measurements of the oxygen concentration profiles made every few hours. The profiles simulated by the model were fitted to the measured data. The average value for the maximum specific growth rate was 0.52 h−1, and the maximum oxygen conversion rate was 1.0 mol Cmol−1 h−1. The maximum specific acetate uptake rate was 2.0 mol Cmol−1 h−1, and the Monod constant for acetate was 2.9 × 10−2 mol m−3. The maximum specific nitrification rate was 0.58 × 10−1 mol Cmol−1 h−1, and the amount of oxygen necessary for nitrification was 11% of the total oxygen uptake rate. Most of the kinetic parameters determined for the immobilized cells were in good agreement with those for the suspended cells. Only the maximum specific growth rate was significantly higher, and the maximum specific nitrification rate was some what lower than for suspended cells. The experimental results clearly show that an oxygen microsensor, in combination with mathematical modeling, can successfully be used to elucidate the kinetic behavior of immobilized, oxygen‐consuming, cells.
Moisture‐induced aggregation of lyophilized proteins in the solid state
Authors: Liu W. Robert , Langer Robert , Klibanov Alexander M. 
Published: December 2004

AbstractA critical problem in the storage and delivery of pharmaceutical proteins is their aggregation induced by moisture. A model system has been elaborated and investigated to elucidate the mechanism of this phenomenon. When 10 mg of bovine serum albumin lyophilized from an aqueous solution of pH 7.3 are wetted with just 3 μL of a buffered physiological saline solution and incubated in the solid state at 37°C, the protein progressively loses its solubility in water; e.g., after a 24 h incubation 97% of the protein becomes insoluble. This moisture‐induced aggregation of albumin has been discovered to be due to an intermolecular S—S bond formation via the thiol‐disulfide interchange reaction. The dependence of the extent of the solid‐state aggregation on the amount and mode of addition of moisture and the atmosphere, additives, temperature, and history of the protein powder have been investigated. The moisture‐induced solid‐state aggregation has also been established and studied for three other lyophilized proteins: ovalbumin, glucose oxidase, and β‐lactoglobulin. In all cases, the loss of solubility is caused by thiol‐disulfide interchange either alone or in combination with a conformational (noncovalent) process. The aggregation can be minimized by lyophilizing the proteins from acidic aqueous solutions, by adding inorganic salts, by co‐lyophilizing the proteins with water‐soluble polymers, or by controlling the moisture content at optimal levels.
A structured model for monoclonal antibody synthesis in exponentially growing and stationary phase hybridoma cells
Authors: Bibila T. , Flickinger M. C. 
Published: December 2004

AbstractAn initial structured unsegregated kinetic model describing monoclonal antibody synthesis by a murine hybridoma cell line (9.2.27) grown in 1 liter batch cultures is described. The model is based on the intracellular balances of the heavy and light chain coding mRNAs, the intracellular balances of heavy and light chains and the description of the kinetics of heavy and light chain assembly. Model parameters were varied with specific growth rate in order to account for changes in the rates of antibody synthesis and secretion with entrance of the cells from the exponential into the stationary phase of growth. The parameters were varied based. on experimental data obtained in our laboratory on the variation of total cellular RNA content and the half‐lives of heavy (H) and light (L) chain mRNAs with specific growth rate, and data from other investigators on immunoglobulin synthesis and secretion. The model successfully predicts the experimentally observed decrease in the intracellular heavy and light chain mRNA levels with entrance of 9.2.27 cells from the exponential into the stationary phase of growth, as well as the extracellular accumulation of antibody (IgG2a) during batch culture.
Protein adsorption and leakage in carrier–enzyme systems
Authors: Ulbrich Renate , Golbik Ralph , Schellenberger Alfred 
Published: December 2004

AbstractThe capability of binding enzymes adsorptively to unmodified and silanized silica and glass as well as modified polystyrene carriers was studied for α‐amylase, β‐amylase, and α‐chymotrypsin. In most cases a high percentage of protein was bound very firmly under considerable loss of activity. The leakage of protein from the carriers was studied by measuring the intrinsic protein fluorescence on β‐amylase adsorptively bound to aminopropyl silica, aminomethyl, and hexadecylaminomethyl polystyrene. It was compared with the leakage of β‐amylase covalently bound to the same carriers via glutaraldehyde, trichloro‐triazine, or benzoquinone. In the absence and in the presence of substrate, at 25 and at 60°C, the leakage rates of the adsorptively bound enzymes were not higher than in the covalently bound systems. The poorest binding stability was found in benzoquinone‐coupled β‐amylase derivatives. It is even reduced at higher temperatures, whereas the temperature did not show any remarkable influence on the leakage of the other derivatives. In adsorptively as well as in all the covalently bound systems, the presence of substrate did not promote the protein leakage.
Determination of kinetic parameters related to the piasmid instability: For the recombinant fermentation under repressed condition
Authors: Park Sung Hoon , Ryu Dewey D. Y. , Lee Sun Bok 
Published: December 2004

AbstractThe plasmid stability under the repressed state of cloned gene was studied theoretically as well as experimentally using recombinant E. coli K12ΔH1Δtrp/pPLc23trpA1 as a “host‐vector” model system. The important kinetic parameters studied were the plasmid loss rate (θ) describing the rate at which the plasrnid‐harboring cells lose plas‐mids and the plasmid‐free cells are generated per unit time and the difference in growth rates (Δ) between the two genotypes. These parameters were carefully defined, studied, and compared with other key kinetic parameters involved in the recombinant fermentation to further our understanding of metabolism of recombinants. The ratio of the concentration of plasmid‐free cells to plasmid‐harboring cells (Ω) was introduced, and the mathematical model was derived and used for the determination of the kinetic parameters associated with plasmid instability. These methods developed based on the theoretical considerations were tested experimentally. The results of these methods were compared, and the best method was selected and recommended. The effect of temperature and dilution rate on kinetic parameters θ and Δ were also studied in continuous culture, in order to provide some practical information related to the operation and control of recombinant fermentation processes.