bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative in vivo nuclear magnetic resonance studies of hybridoma metabolism
Authors: Sharfstein Susan T. , Tucker Sean N. , Mancuso Anthony , Blanch Harvey W. , Clark Douglas S. 
Published: December 2004

AbstractCarbon‐13 nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of a murine hybridoma cell line at two feed glutamine concentrations, 4.0 and 1.7 mM. Carbon‐13 labeling patterns were used in conjunction with nutrient uptake rates to calculate the metabolic fluxes through the glycolytic pathway, the pentose shunt, the malate shunt, lipid biosynthesis, and the tricarboxylic acid (TCA) cycle. Decreasing the feed glutamine concentration significantly decreased glutamine uptake but had little effect on glucose metabolism. A significant incrase in antibody productivity occurred upon decreasing the feed glutamine level. The increased antibody productivity in concert with decreased glutamine uptake and no apparent change in glucolytic metabolism suggests that antibody production was not energy limited. Metabolic flux calculations indicate that (1) approximately 92% of the glucose consumed proceeds directly through glycolysis with 8% channeled through the pentose shunt; (2) lipid biosynthesis appears to be greater than malate shunt activity; and (3) considerable exchange occurs between TCA cycle intermediates and amino acid metabolic pools, leading to substantial loss of 13C label from the TCA cycle. These results illustrate that 13NMR spectroscopy is a powerfulf tool in the calculation of metabolic fluxes, particularly for exchange pathways where no net flux occurs. © 1994 John Wiley & Sons, Inc.
Solvation of CBZ–amino acid nitrophenyl esters in organic media and the kinetics of their transesterification by subtilisin
Authors: Reimann Antje , Robb Donald A. , Halling Peter J. 
Published: December 2004

AbstractSubtilisin Carlsberg adsorbed on silica particles has been used to catalyze the transesterification of CBZ–Ala–ONp and CBZ–Leu–ONp with 1–butanol in organic systems preequilibrated to water activity of 0.93. Initial reaction rates are conveniently followed by extraction of the released nitrophenol into an alkaline aqueous phase. Kinetic parameters were determined for varied ester concentrations in toluene, isopropyl ether, and hexane. The effect of solvent on substrate solvation was determined by solubility measurements. Much of the observed effect of solvent on Vm/Km may be accounted for by solvation differences. The residual effect of solvent on Km, after discounting solvation differences, is completely opposite to the apparent trend. © 1994 John Wiley & Sons, Inc.
Metabolic engineering of Pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p‐xylene mixture
Authors: Lee Jang‐Young , Roh Jae‐Rang , Kim Hak‐Sung 
Published: December 2004

AbstractFor the complete biodegradation of a mixture of benzene, toluene, and p―xylene (BTX), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. Toluate―cis‐glycol dehydrogenase in the tol pathway was found to attack benzene―cis―glycol, toluene―cis―glycol, and p―xylene―cis―glycol, which are metabolic intermediates of the tod pathway. Based on this observation, a hybrid strain, Pseudomonase putida TB101, was constructed by introduction of the TOL plasmid pWW0 into P. putida F39/D, a derivative of P. putida F1, which is unable to transform cis―glycol compounds to corresponding catechols. The metabolic flux of BTX into the tod pathway was redirected to the tol pathway at the level of cis―glycol compounds by the action of toluate―cis―glycol dehydrogenase in P. putida TB101, resulting in the simultaneous mineralization of BTX mixture without accumulation of any metabolic intermediates. The profile of specific degradation rates showed a similar pattern as that of the specific growth rate of the microorganism, and the maximum specific degradation rates of benzene, toluene, and p‐xylene were determined to be about 0.27, 0.86, and 2.89 mg/mg biomass/h, respectively. P. putida TB101 is the first reported microorganism that mineralizes BTX mixture simultaneously. © 1994 John Wiley & Sons, Inc.
A novel method to prepare size‐regulated spheroids composed of human dermal fibroblasts
Authors: Yamazaki Manabu , Tsuchida Michiko , Kobayashi Ken‐yu , Takezawa Toshiaki , Mori Yuichi 
Published: December 2004

AbstractA simple method to prepare size‐regulated spheroids has been successfully developed by combining a temperature responsive polymer, poly‐N‐isopropyl‐acrylamide (PNIPAAm), conjugated with collagen and ultraviolet (UV) irradiation with photomasks. The coating layer composed of PNIPAAm conjugated with collagen functions as a cell substratum at 37°C, then when lowering the temperature of culture medium the cells attached to it detach as a self‐supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature LCST; about 30°C, but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. On the other hand, UV effectively immobilized collagen in the coating layer because UV generates crosslinkages in collagen molecules. Crosslinkages were quantitatively introduced by controlling the energy of UV‐irradiation thus the ability of human dermal fibroblasts to attach to and detach from the surface was tightly controlled. When the collagen content in the coating layer was 9 μg/cm2 (collagen ratio, 4.5%), UV‐irradiation energy of 2000 J/m2 was suitable to obtain 100% of the attachability and detachability. However, the cells did not attach to the nonirradiated surface at this collagen content because insufficient collagen was immobilized. Using photomakes to apply UV‐irradiation, it was possible to obtain cell‐adhesive areas(irradiated areas) and nonadhesive areas (nonirradiated areas) on the same surface. Consequently, spheroids of any size and in any number from one dish were prepared. The viability of cells in spheroids 350 μm in diameter was maintained at a high level for 28 days; however, viability of spheroids 800 μm in diameter rapidly decreased for 2 days. The size was very important to maintain the viability. This novel method is useful to develop size‐regulated spheroids for different applications; for example, in toxicology tests. © 1994 John Wiley & Sons, Inc.
Affinity cross‐flow filtration: Purification of IgG with a novel protein a affinity matrix prepared from two‐dimensional protein crystals
Authors: Weiner Christian , SáRa Margit , Dasgupta Gautam , Sleytr Uwe B. 
Published: December 2004

AbstractIn this article, we describe the use of 1‐ to 2‐μm sized affinity microparticles for the isolation and purification of IgG from artificial IgG‐human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross‐flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111‐69, in which the peptidoglycan‐containing layer was completely covered with a hexagonally ordered S‐layer lattice. After crosslinking the S‐layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S‐layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S‐layer protein release under cross‐flow conditions between pH 2 to 12. The IgG‐binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. © 1994 John Wiley & Sons, Inc.
Application of the transition time of metabolic system as a criterion for optimization of metabolic processes
Authors: Torres Néstor V. 
Published: December 2004

AbstractTransition time of metabolic systems in introduced as a suitable optimization criterion for biotechnological processes in which it is desirable to reduce the lag time and minimize the mass contained within the system. Lag time is the time needed for the system to attain the steady state. Results obtained from the sensitivity analysis of this steady state response are presented within the metabolic control analysis and applied to 3 case studies. In all of them the information provided by the transition time control profile allows the implementation of a strategy for biotechnological manipulations aimed at the improvement of the process. © 1994 John Wiley & Sons, Inc.
Transient responses of hybridoma cells in continuous culture to step changes in amino acid and vitamin concentrations
Authors: Hiller G. W. , Clark D. S. , Blanch H. W. 
Published: December 2004

AbstractThe effects of step‐change increase in the concentrations of amino acids and vitamins on the metabolism, growth, and antibody productivity of a murine hybridoma cell line grown in continuous culture on serum‐free medium are presented. Additions of the amino acids cysteine with methionine, tryptophan, and isoleucine with valine and vitamin B12 (as cyanocobalamin) resulted in significant increases in viable cell concentrations. Additions of aspartate with asparagine, and threonine with vitamin B1 (as thiamine hydrochloride) resulted in significant increases in final antibody concentrations. Substantial decrease in the fraction of amino acid nitrogen excreted as ammonia occurred upon supplementation with three times the normal concentrations of branched chain amino acids. Decreases in the fraction of amino acid nitrogen converted to ammonia were paralleled by increases in the fraction converted to alanine. © 1994 John Wiley & Sons, Inc.
Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A
Authors: Wilson S.‐A. , Daniel R. M. , Peek K. 
Published: December 2004

AbstractA proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac‐Phe‐OEt and Bz‐Ala‐OMe. Tur‐NH2, Trp‐NH2, Leu‐pNA, and Val‐pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz‐ala‐Tyr‐NH2 was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz–ala‐OMe, 25 mM Tyr‐NH2, 70°C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz‐Ala‐OMe and 615 mM Tyr‐NH2 at 40°C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; ‐βNA and ‐pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield. © 1994 John Wiley & Sons, Inc.
An on‐line sampling system for fermentation monitoring Using membrane inlet mass spectrometry (MIMS): Application to phenoxyacetic acid monitoring in penicillin fermentation
Authors: Hansen Ken Flemming , Lauritsen Frants R. , Degn Hans 
Published: December 2004

AbstractA sampling system for on‐line monitoring of organic compounds of low volatility in complex fermentation media uses membrane inlet mass spectrometry (MIMS). A Syringe pump draws a continuous flow of microfiltered broth from the reactor and circulates it after acidification through a membrane inlet, in which a membrane is the only interface between the sample and the high vacuum of a mass spectrometer. All operations run automatically, i.e., sampling, acidification measurement, and calibration. The on‐stream acidification enables MIMS monitoring of carboxylic acids, as they must be undissociated in order to pass the hydrophobic membrane. The performance of the monitoring system was tested by measurements of standard solutions of phenoxyacetic acid (POAA, the sie chain precursor of penicillin‐V) as well as on POAA during 200 h penicillin‐V fermentation. During the entire fermentation POAA was monitored n low millimolar concentrations with high accuracy and fast response to step changes in POAA concentration. Tandem mass spectrometry (MS/MS) allowed direct identification of peaks in the mass spectrum of the broth that were not accounted for by POAA. These peaks were identified as SO2 and SCO. © 1994 John Wiley & Sons, Inc.
Effects of oxygen and nutrients limitation on ajmalicine production and related enzyme activities in high density cultures of Catharanthus roseus
Authors: Schlatmann J. E. , Moreno P. R. H. , Vinke J. L. , ten Hoopen H. J. G. , Verpoorte R. , Heijnen J. J. 
Published: December 2004

AbstractOxygen and nutrient limitation was investigated in order to identify the origin of a lower specific ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. © 1994 John Wiley & Sons, Inc.
Transport of bacteria in porous media: I. An experimental investigation
Authors: Sarkar A. K. , Georgiou George , Sharma Mukul M. 
Published: December 2004

AbstractThe convective transport of concentrated suspension of bacteria in porous media is of interest for several processes such as microbial enhanced oil recovery and in situ bioremediation. The parameters which affect the transport of the bacterium Bacillus licheniformis JF‐2, a candidate microorganism for microbial enhanced oil recovery, were investigated experimentally in sandpacks. Bacteria retention and permeability reduction occurred primarily in the first few centimeters upon entering the porous medium. In downstream sections of the sandpack, the permeability reduction was low, even in cases in which high cell concentrations (108 cfu/mL) were detected in the effluent. The effect of (i) addition of a dispersant, (ii) linear velocity of injection, (iii) cell concentration, (iv) salinity (v) temperature, and (vi) the presence of a residual oleic phase were determined experimentally. A lower reduction in permeability and a higher effluent bacterial concentration were obtained in the presence of dispersant, high injection velocities, low salinities, and at a higher temperature. Macroscopic measurements at different linear velocities and in the presence or absence of dispersants suggest that the formation of reversible microaggregates and multiparticle hydrodynamic exclusion may be the primary mechanisms for bacterial retention and permeability reduction. © 1994 John Wiley & Sons, Inc.
Symmetric enzyme distribution in asymmetric UF polysulfone membranes
Authors: Gille Michael , Staude Eberhard 
Published: December 2004

AbstractInvertase as well as as amyloglucosidase were immobilized within asymmetyric ultrafiltration membranes that were prepared from polysulfone or homogeneously modified polysulfone. The chemical modification was carried out by sulfonation and halomethylation. This additional change of the surface properties of the capillaries within the membrane offers the possibilities for various types of enzyme fixation, namely adsorption, charge interactions, or covalent bonding. By variation of the immobilization conditions the distribution of the enzyme could be adjusted over the membrane's cross section. At a distinct enzyme concentration in the loading solution a homogeneous enzyme distribution within the membrane could be verified. This was shown by diffusion experiments. Under ultrafiltration conditions using a solution that contains membrane‐impermeable macromolecules as well as a membrane‐permeable solute like saccharose the residence time within the membrane was increased due to gel formation atop the membrane yet the kinetic was no affected. The nonpermeable soluble starch was not reacted by the amyloglucosidase membrane, indicating that the skin layer was free of enzymes. © 1994 John Wiley & Sons, Inc.
Surfactant‐induced breakthrough effects during the operation of two‐phase biocatalytic membrane reactors
Authors: Vaidya A. M. , Halling P. J. , Bell G. 
Published: December 2004

AbstractSurface‐active components, both reactants and products, are frequently encountered in two‐phase, aqueous‐organic, biocatalytic reactions, When such reaction are carried out in a membrane reactor, employing a membrane selectively wetted by one of the two reactants, changes in the content of these surfactants– as a consequence of the progress of the reaction–can lead to wetting transitions at the two membrane‐liquid interfaces as a result of adsorption of the tenside. This can lead to a decrease in the pressure required to cause the, initially, nonwetting phase to break through the membrane. Such effects render difficult the operation of two‐phase membrane bioreactors. Hence, it is necessary to make a careful selection of the membrane material and type by considering factors such as UF versus MF and low MWCO versus high MWCO to enable the reactor to be operated without breakthrough, but without significantly compromising the reaction rates that can be maintained.The phenomena leading to breakthrough effects are discussed in this paper, and experimental results for the hydrolysis of ethyl laurate by lipase from Candida rugosa in a batch flat sheet membrane reactor are presented with the reactor operated with a variety of membranes. An experimental result showing the decrease in the pressure required to cause breakthrough of the organic phase (for the system ethyl laurate‐lauric acid–water) as the content of the highly surface‐active lauric acid in the organic phase is increased is also presented for an asymmetric, hydrophilic meta‐aramid ultrafiltration membrane. © 1994 John Wiley & Sons, Inc.
Closed‐loop control of fed‐batch cultures of recombinant Escherichia coli using on‐line HPLC
Authors: Turner Claire , Gregory Malcolm E. , Thornhill Nina F. 
Published: December 2004

AbstractThis article describes a fully automated system for the on‐line monitoring and closed‐loop control of a fed‐batch fermentation of recombinant Escherichia coli, and presents two case studies of its used in limiting production of unwanted byproducts such as acetic in fed‐batch fermentations. The system had two components. The first components, on‐line monitoring, comprised an aseptic sampling device, a microcentrifuge, and HPLC System. These instruments removed a Sample from a fermentor, spun it at high speed to separate solid and liquid components, and then automatically injected the supernatant onto an HPLC column for analysis. The second component consisted of control algorithms programmed using the LabView visual programming environment in a control computer that was linked via a remote components were linked so that results from the on‐line HPLC were captured and used by the control algorithm was designed to demonstrate coarse feedback control to confirm the operability of the controller. The second case study showed how the system could be used in a more sophisticated feedings strategy providing fine control and limiting acetate concentration to a low level throughout the fermentation. © 1994 John Wiley & Sons, Inc.
Oxygen requirements and mass transfer in hairy‐root culture
Authors: Yu Shaoxiong , Doran Pauline M. 
Published: December 2004

AbstractOxygen mass transfer in clumps of Atropa belladonna hairy roots was investigated as a function of root density and external flow conditions. Convection was the dominant mechanism for mass transfer into root clumps 3.5 to 5.0 cm in diameter; Peclet numbers inside the clumps ranged from 1.4 × 103 to 7.1 × 104 for external superficial flow velocities between 0.4 and 1.4 cm s−1. Local dissolved‐oxygen levels and rates of oxygen uptake were measured in aflow chamber and in bubble column and stirred bioreactors. When air was used as oxygen source, intraclump dissolved‐oxygen tensions ranged from90% to 100% air saturation at high external flow velocity andlow root density, to less than 20% air saturation in dense root clumps. Specific oxygen‐uptake rate declined with increasing root density. When external boundary layers around individual roots were eliminated byforcing liquid through the clumps at superficial velocities between 0.2 and1.0 cm s−1, internal dissolved‐oxygen tension was maintained at 95% to 100% air saturation and rate of oxygen uptake at 1.6 × 10−6 g g−1 s−1 dry weight. Liquid culture of single A. belladonna hairy roots was used to investigate the effect of dissolved‐oxygen tensionon root growth and morphology. Total root length and number of root tips increased exponentially at oxygen tensions between 70% and 100%air saturation. Specific growth rate increased with oxygen tension up to 100% air saturation; this result demonstrates that hairy roots aeratedwithout oxygen supplementation are likely to be oxygenlimited. No growth occurred at 50% air saturation. Growth of hairy roots proceeded with an average length per tip of about 1 cm; this value was essentially independent of dissolved‐oxygen tension between 70% and 100% air saturation. © 1994 John Wiley & Sons, Inc.