bluuebunny/crossref_metadata_2025_split huggingface dataset:
Stability and reaction characteristics of reverse‐phase evaporation vesicles (REVs) as enzyme containers
Authors: Sada Eizo , Katoh Shigeo , Terashima Masaaki , Shiraga Hiroshi , Miura Yasushi 
Published: December 2004

AbstractThe ion‐dependent enzymes, activities of which are affected by ions, were entrapped in the inside aqueous phase of reverse‐phase evaporation vesicles (REVs) to protect the enzyme activities from inhibitory cations. Their activities were still preserved in the presence of the inhibitory cations because the permeabilities of the inhibitory cations through the lipid membranes of REVs were much lower than those of substrates and products. The REVs were relatively stable in hypertonic condition, while they were unstable in hypotonic condition. Changes in osmotic pressure difference largely affected solute permeabilities of REVs. The shrinking and swelling of REVs resulting from osmotic pressure differences led to these changes in stabilities and solute permeabilities. Although the entrapment of the enzyme into REVs showed good protective effects against divalent cations, it was not effective against univalent cations. The measurements of cation permeabilities revealed that the enzyme trapped in the bilayer regions of REVs acts as a selective channel for the univalent cations. The REVs containing the enzyme could be used without any activity losses in a hollow fiber module.
Polarographic measurement of oxygen uptake using lipoxygenase in reverse micelles
Authors: Hochkoeppler Alejandro , Palmieri Sandro 
Published: December 2004

AbstractLipoxygenase‐catalyzed linoleic acid peroxidation was chosen as a model system to study the applicability of oxygraphy to monitor the oxygen uptake in organic solvents containing reverse micelles. Care was taken to control the oxygen back transfer from the atmosphere to the sample micellar solution, resulting in a significant improvement of electrode response. Under these conditions, lipoxygenase activity was linear up to 100 μg of enzyme. Given the quality of the calibration curve and the good correlation between lipoxygenase and ascorbate oxidase, the described technique is proposed as an alternative method for determining lipoxygenase activity in reverse micelles. The reliability of this technique was confirmed by the good agreement between polarography and classic spectrophotometry in kinetic studies. Preliminary experiments carried out on soybean cells solubilized in a Tween 85‐isopropylpalmitate system demonstrated that a light‐dependent oxygen uptake can be measured. The authors propose that the Clark‐type electrode be employed to study both the activity of oxidasic enzymes in reverse micelles and cell viability and physiology in organic solvents.
Depression of protein synthetic capacity due to cloned‐gene expression in E. coli
Authors: Wood Thomas K. , Peretti Steven W. 
Published: December 2004

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−1 for each chemostat cultivation. The effects of constitutive expression of the bla gene 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 translation as well as transcription.
Minimal nutritional requirements for immobilized yeast
Authors: Chen C. , Dale M. C. , Okos M. R. 
Published: December 2004

AbstractThe effect of reduced nutritional levels (particularly nitrogen source) for immobilized K. fragilis type yeast were studied using a trickle flow, “differential” plug flow type reactor with cells immobilized by adsorption onto an absorbant packing matrix. Minimizing nutrient levels in a feed stream to an immobilized cell reactor (ICR) might have the benefits of reducing cell growth and clogging problems in the ICR, reducing feed preparation costs, as well as reducing effluent disposal costs. In this study step changes in test feed medium nutrient compositions were introduced to the ICR, followed by a return to a basal medium. Gas evolution rates were monitored and logged on a continuous basis, and effluent cell density was used as an indicator of cell growth rate of the immobilized cell mass. Startup of the reactor using a YEP medium showed a rapid buildup of cells in the reactor during the initial 110 h operation. The population density then stabilized at 1.6 × 1011 cells/g sponge. A defined medium containing a complex mix of essential nutrients with an inorganic nitrogen source (ammonium sulfate) was able to maintain 90% of the productivity in the ICR as compared to the YEP medium, but proved unable to promote growth of the immobilized cell mass during startup. Experiments on reduced ammonium sulfate in the defined medium, and reduced yeast extract and peptone in YEP medium indicated that stable productivity could be maintained for extended periods (80 h) in the complete absence of any nutrients besides a few salts (potassium phosphate and magnesium sulfate). It was found that productivity rates dropped by 35–65% from maximal values as nitrogenous nutrients were eliminated from the test mediums, while growth rates (as determined by shed cell density from the reactor) dropped by 75–95%. Thus, nutritional deficiencies largely decoupled growth and productivity of the immobilized yeast which suggests productivity is both growth‐ and non‐growth‐associated for the immobilized cells. A yeast extract concentration of 0.375 g/L with or without 1 g/L ammonium sulfate was determined to be the minimum level which gave a sustained increase in productivity rates as compared to the nutritionally deficient salt medium. This represents a 94% reduction in complex nitrogenous nutrient levels compared to standard YEP batch medium (3 g/L YE and 3.5 g/L peptone).
Physiological, biochemical, and mathematical studies of micro‐aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. III: Mathematical model of cellular energetics and catabolism
Authors: Grosz Ron , Stephanopoulos Gregory 
Published: December 2004

AbstractMathematical models of the catabolic pathways, the utilization and waste of ATP, and the factors affecting yeast growth in a micro‐aerobic chemostat are presented. The models incorporate the intracellular metabolite and enzyme activity assays performed in Part II to explain the unusual macroscopic chemostat behaviors reported in Part I. The catabolic model successfully predicts a maximum in the specific ethanol productivity as a function of the intracellular ATP concentration. The ATP balance model enables the prediction of the intracellular ATP concentration and the ATP yield for given dissolved oxygen concentrations. Finally, in the context of a growth model, singularity theory provides a framework to explain the transition observed in Part I between hysteresis and the monotonic biomass versus oxygenation profiles in response to changes in the nutrient composition. The models serve to organize data and to concretely express proposed metabolic mechanisms and cause–effect hypotheses. The model is only applicable to the micro‐aerobic and excess glucose conditions encountered in this study.
Analysis of E. coli phoA‐lacZ fusion gene expression inserted into a multicopy plasmid and host cell's chromosome
Authors: Shin Pyong K. , Seo Jin‐Ho 
Published: December 2004

AbstractThe production of β‐galactosidase from the E. coli phoA‐lacZ fusion gene was studied to compare the gene expression behavior of two cloning methods: insertion to multicopy plasmids and integration into host cell's chromosome. The chromosome‐integrating strain showed more tight control of fusion gene expression levels than the plasmid‐containing strain. A 100‐fold enhancement of specific β‐galactosidase activity in the former strain was achieved in response to changes of initial inorganic phosphate concentration from 1 to 0.1 mM, whereas a 26‐fold increase was observed in the latter strain. The low degree of overexpression in the plasmid‐bearing cells was due to a combination of factors including leaky expression in repressed conditions and limitation of biosynthetic machinery in derepressed conditions. In a mixture of inorganic and organic phosphates, inorganic phosphate levels in the medium exhibited oscillatory behavior. The oscillation of inorganic phosphate is attributed to selective usage of inorganic phosphate followed by hydrolysis of organic phosphate to inorganic by alkaline phosphatase. The fluctuation of inorganic phosphate levels also caused the oscillation of β‐galactosidase activity.
Lewis cell studies to determine reactor design data for two‐liquid‐phase bacterial and enzymic reactions
Authors: Woodley J. M. , Brazier A. J. , Lilly M. D. 
Published: December 2004

AbstractSubstrate transfer rates from organic to aqueous phases were measured in the presence and absence of biocatalyst in the reaction medium, using modified Lewis cells. These measurements, in combination with intrinsic aqueous phase biocatalytic reaction kinetics, were used to confirm that benzyl acetate hydrolysis by pig liver esterase and toluene oxidation by a strain of Pseudomonas putida occur uniformly throughout the bulk of the aqueous phase. Such data may be used to provide a basis for two‐liquid‐phase biocatalytic reactor design.
NADH fluorescence and oxygen uptake responses of hybridoma cultures to substrate pulse and step changes
Authors: Siano Steven A. , Mutharasan R. 
Published: December 2004

AbstractA fiber‐optic probe was interfaced to an analytical spectrofluorophotometeru and used to measure NAD(P)H fluorescence of hybridoma cells in a bioreactor. NAD(P)H fluorescence was found to qualitatively represent metabolic state during various induced metabolic transitions. NAD(P)H fluorescence increased immediately following aerobic–anaerobic transitions, and decreased immediately upon transition back to aerobic metabolism. Pulsing of glucose to glucose‐depleted cultures caused NAD(P)H fluorescence to first increase immediately after the pulse, and then decrease gradually before reaching a constant level. Pulsing of glutamine to glutamine‐depleted cultures resulted in a gradual increase in NAD(P)H fluorescence which lagged a simultaneous increase in oxygen uptake. ATP production and oxygen uptake also varied with metabolic state. The decrease in oxidative phosphorylation following transition from aerobic to anaerobic metabolism was found to be only partially compensated by the concomitant increase in substrate‐level phosphorylation, as shown by decreases of 35–52% in calculated total specific ATP production rates. The specific oxygen uptake rate decreased by 6–38% following glucose pulses of between 0.2 and 0.5 g/L, respectively, and by 50% following glutamine depletion. Subsequent pulsing of glutamine after depletion caused oxygen uptake to increase by 50%.
A new method for direct selection of plasmid‐free segregants using mercury hypersensitivity as a phenotypic marker of bacterial plasmids
Authors: Mori Tsukasa , Kaga Takayuki , Toda Kiyoshi , Nakahara Hideomi , Ohtake Hisao 
Published: December 2004

AbstractA new method was developed for direct selection of plasmid‐free segregants using mercury hypersensitivity (Hgss) as a phenotypic marker of bacterial plasmids. The Hgss marker originated from the 4.8‐kb EcoRI fragment H of the R‐factor R100. Since the EcoRI fragment H spans the majority of the mercury resistance operon (mer), but lacks the intact merA gene coding for the mercury reductase enzyme, this fragment conferred the Hgss phenotype. The Hgss marker was introduced into high‐copy‐number plasmids pUC18, pBR322, and pHSG298. Segregational loss of the Hgss plasmids caused a significant increase of resistance to Hg2+, and this allowed direct selection of plasmid‐free segregants on nutrient agars containing 1–2 μg HgCl2 ml−1. Plasmid‐loss segregants were estimated to appear at frequencies ranging from 10−3 to 10−7 for the tested high‐copy‐number plasmids. THe Hgss marker proved to be useful for direct selection of plasmid‐free segregants from a mixed population of plasmid‐harboring and plasmid‐free cells.
Mass transfer characterization of an airlift probe for oxygenating and mixing cell suspensions in an NMR spectrometer
Authors: Kramer Hans W. , Bailey James E. 
Published: December 2004

AbstractThe oxygen transfer characteristics of a 20‐mm O.D. airlift contactor fitted with an oxygen microelectrode were determined by steady‐state sulfite oxidation measurements. The volumetric mass transfer coefficient kLa was proportional to sparging power input per unit volume raised to a power which varied from 0.41 in water (coalescing bubbles) to 0.76 in NaCl solutions (noncoalescing bubbles). The highest observed kLa value was 0.012 s−1 which is sufficient to aerate Escherichia coli in an NMR spectrometer at moderate to high cell densities, depending on the physiological state of the cells.
Recombinant protein excretion in Escherichia coli JM103[pUC8]: Effects of plasmid content, ethylenediaminetetraacetate, and phenethyl alcohol on cell membrane permeability
Authors: Ryan Wen , Parulekar Satish J. 
Published: December 2004

AbstractThe presence of a high copy number plasmid (pUC8) was found to affect integrity of the cell envelope of Escherichia coli JM103, causing in turn significant release of the plasmid‐encoded protein (β‐lactamase). The alterations in cell membrane permeability were evident from the increased susceptibility of recombinant cells to deoxycholic acid and methylene blue, which did not have appreciable effect on plasmid‐free cells. The deteriorated cell membrane structure also resulted in a substantial reduction in specific growth rate and mass yield of plasmid‐bearing cells. Further enhancement in β‐lactamase excretion was achieved by permeabilizing cell membrane with ethylenediaminetetraacetate (EDTA) and phenethyl alcohol (PEA). Unlike other commonly used physical and chemical methods for releasing the enzymes accumulated in the cells, application of EDTA and PEA at appropriate concentrations neither led to cell death nor interrupted synthesis of the plasmid‐encoded protein. While in situ application of PEA was complicated due to interference with β‐lactamase activity, in situ application of EDTA was found to be an efficient way of releasing the recombinant protein without sacrificing its productivity. The experimental results demonstrate that the presence of EDTA and PEA can substantially reduce the growth rate differential between plasmid‐free and plasmid‐bearing cells, suggesting possible improvement of plasmid stability by application of these cell membrence‐permeabilizing agents on a periodic basis.
Lethal events during gas sparging in animal cell culture
Authors: Jöbses I. , Martens D. , Tramper J. 
Published: December 2004

AbstractThe lethal effects of gas sparging on hybridoma cells obtained from a chemostat culture were examined in a bubble column. Experiments were performed to identify and quantify the main hazardous event: bubble formation, bubble rising, or bubble breakup. The results indicate that bubble breakup is the main cause of cell death. The protective activity of the surfactant Pluronic F68 against sparging seems to result from a direct interaction with the cells rather than influencing bubble‐liquid interface properties.
Development of pure culture biofilms of P. putida on solid supports
Authors: Shrove Gina S. , Olsen Ronald H. , Vogel Timothy M. 
Published: December 2004

AbstractPseudomonas putida biofilms were developed on and biofilm accumulation rate data were obtained for the following two classes of support materials: charged surfaces and noncharged hydrophobic and hydrophilic surfaces. The effects of surface roughness and porosity on the rate of microbial attachment were also examined.Materials bearing a net positive or negative surface charge supported the greatest biofilm accumulation and the highest biofilm accumulation rate. Uncharged hydrophobic materials achieved the next greatest biofilm accumulation, averaging approximately 50% of the total biomass which was accumulated on the charged surface materials after 16 days. Uncharged hydrophilic materials supported very little biofilm development. In general, biofilm accumulation increased with decreased surface roughness. The effect of pore size on biofilm accumulation was not conclusive.The biofilm accumulation kinetics showed an exponential accumulation rate for the charged surfaces and an approximately linear accumulation rate for the hydrophobic materials. This difference in accumulation kinetics is consistent with proposed differences in the physicochemical mechanism governing attachment to these two types of surface materials.
Analysis of mass transfer for immobilized cells in an extractive lactic acid fermentation
Authors: Yabannavar V. M. , Wang D. I. C. 
Published: December 2004

AbstractThe use of immobilization in extractive lactic acid fermentation by Lactobacillus delbrueckii is preferred. In this article, the mathematical simulations to examine the influences of substrate and product transport were performed to assess the overall performance. The simulations showed that transport of the substrate in k‐carrageenan beads was not a rate limiting factor. However, the model observed significant buildup of inhibitory product in large beads. The model was validated through comparisons with the experimental results. Finally, the model was used to predict the performance of the extractive fermentation under different operating strategies.
Promotion of oxygen transfer in three‐phase fluidized‐bed bioreactors by floating bubble breakers
Authors: Kang Y. , Fan L. T. , Min B. T. , Kim S. D. 
Published: December 2004

AbstractThe objective of the present study was to investigate a method to enhance the volumetric rate of oxygen transfer in three‐phase fluidized‐bed bioreactors. The rates of oxygen transfer from air bubbles to viscous liquid media were promoted by floating bubble breakers in three‐phase fluidized beds operated in the bubble coalescing regime. The liquid‐phase volumetric oxygen transfer coefficient has been recovered by fitting the axial dispersion model to the resultant data, and its dependence on the experimental variables, such as the gas and liquid flow rates, particle size, concentration of bubble breakers, and liquid viscosity, has been examined. The results indicate that the liquid‐phase volumetric oxygen transfer coefficient can be enhanced up to 20‐25%. The coefficient exhibits a maximum with respect to the volume ratio of the floating bubble breakers to the fluidized solid particles; it increases with increases in the gas and liquid flow rates and size of fluidized particles, while it decreases with an increase in the liquid viscosity. An expression has been developed to correlate the liquid‐phase volumetric oxygen transfer coefficient with the experimental variables.
Photosynthetic activity of plant cells solubilized in water‐in‐oil microemulsions
Authors: Hochkoeppler A. , Luisi P. L. 
Published: December 2004

AbstractWith the aim of possibly extending plant microbiology and photosynthesis beyond the usual applicability in aqueous solution, we investigated the solubilization of plant cells inorganic media with the help of water‐in‐oil microemulsions. Cells isolated from leaves of Rumex obtusifolius were solubilized in a water/2‐ethyl‐hexyl‐sodiumsulfosuccinate/isooctane system, containing 20% water (v:v) and 240 mM surfactant, and the oxygen evolution/consumption was measured polarographically. Although no oxygen evolution was detectable in the organic medium, the cells were able to carry out photosynthetic oxygen consumption at the expense of ascorbate. To a lesser extent, photosynthetic oxygen consumption was measured using N, N, N′, N′‐tetramethyl‐p‐phenylenediamine as electron donor. The rate of ascorbate photooxidation was linearly related to the concentration of cells.
Kinetics of loss of a recombinant plasmid in Bacillus subtilis
Authors: Shoham Yuval , Demain Arnold L. 
Published: December 2004

AbstractRecombinant plasmid pCEDS is structurally unstable in Bacillus subtilis cultures. We have previously shown that stability can be independently increased by changing from a complex medium supporting high growth rates to a chemically‐defined medium supporting a lower growth rate and removal of a 4.77‐kb EcoRI fragment from pCED3 to give plasmid YS1. Further stabilization was achieved by combining the two approaches. In the present work, we show that the stabilization of the plasmid‐encoded LacZ+ phenotype can be explained solely by the effect on the growth rate ratio between cells containing modified and parental plasmids. By using modified stability experiments (where a single cell rather than a suspended colony was used to initiate growth), independent growth rate measurements, and a simple mathematical model, we can describe the kinetics of the loss of the LacZ+ phenotype in terms of two variables, α and p (where α is the ratio of growth rates between modified and parental cells, and p is the probability of obtaining modified cells from parental cells). Under the conditions tested, the average values of α were 1.52 for cultures growing in complex medium, 1.28 for cultures growing in defined medium, and 1.18 for cultures containing the modified plasmid pYS1 growing in complex medium. The calculated p values ranged between 10−8 and 10−10 under all conditions. Plasmid (pYS137) was used to directly estimate plasmid deletion rates in B. subtilis and it showed a rate between 5 × 10−8 and 1.1 × 10−9 deletions/cell/generation. In contrast to B. subtilis, there were no detectable differences in growth rates between Escherichia coli strains harboring plasmid pCEDS and plasmid‐free cells. These results explain the observed stability of pCEDS in E. coli cultures and indicate that readily detected instability in B. subtilis cultures can be the result of rare deletion events.
Oxygen transfer in slurry bioreactors
Authors: Kawase Yoshinori , Moo‐Young Murray 
Published: December 2004

AbstractThe oxygen transfer in bioreactors with slurries having a yield stress was investigated. The volumetric mass transfer coefficients in a 40‐L bubble column with simulated fermentation broths, the Theological properties of which were represented by the Casson model, were measured. Experimental data were compared with a theoretical correlation developed on the basis of a combination of Higbie's penetration theory and Kolmogoroff's theory of isotropic turbulence. Comparisons between the proposed correlation and data for the simulated broths show good agreement. The mass transfer data for actual mycelial fermentation broths reported previously by the authors were re‐examined. Their Theological data was correlated by the Bingham plastic model. The oxygen transfer rate data in the mycelial fermentation broths fit the predictions of the proposed theoretical correlation.