摘要:

AbstractRecombinant bacterial systems exhibit limited capacities for heterologous protein production. As seen with this and other systems, cloned‐gene protein production reaches an upper limit despite further increases in gene dosage. A series of closely related plasmids which contain mutations affecting their copy number has been used to investigate the macromolecular impediments to enhanced recombinant protein production. Within a common host, HB101, the level of the ampicillin resistance gene,bla, was varied using five plasmids which differ solely in their replication machinery. Separate fermentations were conducted in which the plasmid copy number was varied from 0 to over 400 while the specific growth rate was fixed at 0.6 h−1for each chemostat cultivation. The effects of constitutive expression of theblagene as copy number was elevated were then determined using pulse‐labelling and RNA–DNA hybridizations. Specifically, the steady‐state level, synthesis rate, and stability of β‐lactamase messenger RNA and ribosomal RNA were determined as a function of copy number. The results indicate that as copy number rises, both β‐lactamase mRNA synthesis rates and steady‐state mRNA levels increase. Therefore, β‐lactamase production in these strains does not appear to be limited by the level of β‐lactamase mRNA. However, as the copy number was amplified, the stability of rRNA decreased to the point that steady‐state levels of rRNA decreased. These data indicate that a limitation develops within the translational capacity of the cell at high levels of cloned‐gene expression. The results suggest that strategies designed to enhance recombinant protein expression should include manipulation of translati

ISSN:0006-3592    

DOI:10.1002/bit.260360902

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractA hollow‐fiber membrane reactor was designed and constructed to allow perfusion of entrapped, denseEscherichia colicells 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 ofE. coliintracellular 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 sam

ISSN:0006-3592    

DOI:10.1002/bit.260360903

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractEscherichia colicells were immobilized and grown in hollow‐fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using35S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 °C were shown to be severe, with regions of starved cells occupying up to 80% of the cell‐containing region. Phosphorus‐31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume‐averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur‐35 autoradiographs of a reactor operated at 16°C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near‐normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopi

ISSN:0006-3592    

DOI:10.1002/bit.260360904

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThe heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high‐molecular‐weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell‐side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 × 104D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell‐side convective flow and its influence on the inoculum and high‐molecular‐weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have

ISSN:0006-3592    

DOI:10.1002/bit.260360905

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

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 lowerrsvalues (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic® F‐68 showed higher values ofrscompared 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 increa

ISSN:0006-3592    

DOI:10.1002/bit.260360906

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractThiosphaera pantotrophais capable of aerobic heterotrophic nitrification and both aerobic and anaerobic denitrification. These phenomena have been studied in acetate‐limited aerobic and anaerobic continuous cultures supplied with ammonia and nitrate. The internal reaction rates were defined, based on biochemical knowledge. The observable external conversion rates are related through a linear equation on the basis of the specified internal reaction rates. The linear equation is a Pirt relation extended for microbial systems with multiple electron donors (acetate and ammonia) and electron acceptors (oxygen and nitrate). The coefficients in this equation were estimated from the continuous culture measurements, and are composed of parameters involved in ATP production and consumption by the microorganism. It is shown that with realistic values for these parameters, the metabolically structured model describes the aerobic as well as the anaerobic experiment

ISSN:0006-3592    

DOI:10.1002/bit.260360907

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractAn oxygen microsensor in combination with mathematical modeling was used to determine the behavior of immobilizedThiosphaera 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 ofT. pantotrophacan 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 ofT. pantotrophais necessary for the development of such a system. This article gives the extension of a model developed to describe the behavior of chemostat cultures ofT. pantotrophaso 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−1h−1. The maximum specific acetate uptake rate was 2.0 mol Cmol−1h−1, and the Monod constant for acetate was 2.9 × 10−2mol m−3. The maximum specific nitrification rate was 0.58 × 10−1mol Cmol−1h−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 immobiliz

ISSN:0006-3592    

DOI:10.1002/bit.260360908

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractA new type of bioreactor, in which two liquid phases are used, is introduced and a model description of its hydrodynamic behavior is given. The model is based on the two‐phase drift‐flux model of Zuber and Findlay and on a friction coefficient derived from one‐phase flow theory. Drop sizes, necessary for model calculations, were estimated from literature correlations and photographically verified. The model predicts the continuous‐phase circulation velocity and dispersed‐phase hold‐up within an accuracy of 5% for a pilot‐plant reactor, and within 10% for a lab‐scale reactor. Only at very low ranges, especially on lab scale, is this accura

ISSN:0006-3592    

DOI:10.1002/bit.260360909

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

摘要:

AbstractFoam fractionation of bovine serum albumin (BSA) was studied as a model system for potato wastewater. The effects of feed concentration, superficial gas velocity, feed flow rate, bubble size, pH, and ionic strength on the enrichment and recovery of BSA were investigated in a single‐stage continuous foam fractionation column. Enrichments ranged from 1.5 to 6.0 and recoveries from 5 to 85%. The feed concentrations were varied from 0.01 to 0.2 wt %, and enrichments were found to increase with lower feed concentrations. Enrichments also increased with lower superficial gas velocities and larger bubble sizes. At sufficiently low feed flow rates, enrichment was found to increase with an increase in the flow rate, eventually becoming insensitive to the feed flow rate at higher values. The pH was varied from 3.5 to 7.0 and ionic strength from 0.001Mto 0.2M. The effects of pH and ionic strength were found to be coupled with bubble size. A minimum bubble size was found at pH 4.8, the isoelectric point of BSA, resulting in a minimum in the enrichment. Bubble size, and thus enrichment, was found to increase as the ionic strength decreased from 0.2Mto 0.01M. Previous models1,2for the hydrodynamics of foam column were extended for a singlestage continuous foam fractionation column for the prediction of enrichment and recovery. The model assumed adsorption equilibrium, infinite surface viscosity, and bubbles of the same size. Though coalescence was formally accounted for in the model by considering bubble size as a function of foam height, calculations for the experimental runs were performed only for the case of no coalescence. Quantitative predictions of enrichment and recovery could not be made with a single representative bubble size because of the broad inlet bubble size distribution as well as broadening of the distribution as a result of coalescence. The experimental enrichments were higher and recoveries were lower than the model predictions, the discrepancy being more pronounced at lower feed concentrations because of increased coalescence. The higher enrichments are due to the predominant effect of internal reflux as a result of coalescence whereas the lower recoveries are a result of detrimental effects of broadening bubble size distribution

ISSN:0006-3592    

DOI:10.1002/bit.260360910

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY

ISSN:0006-3592    

DOI:10.1002/bit.260360911

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1990

数据来源: WILEY