bluuebunny/crossref_metadata_2025_split huggingface dataset:
Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid
Authors: Ryan Wen , Parulekar Satish J. 
Published: December 2004

AbstractEscherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid‐free cells were conducted in a well‐controlled bio‐reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid‐bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid‐free and plasmid‐bearing cells resulting from the extra metabolic burden on the plasmid‐bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi‐steady state representative of plasmid‐bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid‐free cells near the end of the quasi‐steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid‐bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid‐free cells. In continuous cultures, plasmid copy number during the quasi‐steady states increased with decreasing dilution rate from 50 (at 0.409 h−1) to 941 (at 0.233 h−1). Production of the plasmid‐encoded protein (β‐lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of β‐lactamase was observed at both low and high dilution rates.
A parametric study ot protease production in batch and fed‐batch cultures of Bacillus firmus
Authors: Moon Seung‐Hyeon , Parulekar Satish J. 
Published: December 2004

AbstractProteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen‐free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed‐batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design of bioreactors for production of protease and bulk chemicals by this bacterium.
The effect of an applied potential on the activity of carbonic anhydrase immobilized on graphite rods
Authors: Mclachlan K. L. , Crumbliss A. L. 
Published: December 2004

AbstractThe effect of both a positive and a negative applied potential on the p‐NPA hydrolysis activity of bovine carbonic anhydrase (BCA) immobilized on graphite rods has been investigated. Background experiments show that the pH‐activity profile for BCA free in solution is not affected by either a negative or a positive potential applied to graphite rods placed in the same solution. However, the activity of BCA immobilized by covalent attachment to a graphite rod is influenced by a potential externally applied to the graphite rod. An overall increase in activity (as determined by the initial rate of the p‐NPA hydrolysis reaction) is observed in the presence of a ‐0.2 V (Ag/AgCl) applied potential, while decreased activity is evident at +0.6 V (Ag/AgCl). This is indicative of an electrolyte anion effect rather than a local pH effect. In the presence of the specific anion inhibitors Cl− and SCN−, the relative BCA activity increases at ‐0.2 V (Ag/AgCl) and decreases at +0.6 V (Ag/AgCl) are consistent with the different BCA inhibition constants for Cl− and SCN−. Accelerated loss of immobilized BCA activity also accompanies the application of the external potentials, particularly at +0.6 V (Ag/AgCl). Results described here represent an early example of potentiostatic control of nonredox enzyme activity. Several possible mechanisms are discussed including specific anion inhibition, enzyme surface charge/charged support material interactions, and charged product inhibition. It is likely that a combination of such mechanisms is operational in this system. The implications of external potentials affecting the activity of immobilized enzymes in the design of stable immobilized enzyme electrodes are also discussed.
Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions
Authors: Ju Lu‐Kwang , Lee Jaw F. , Armiger William B. 
Published: December 2004

AbstractThe effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO2TM emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO2 emulsions with very fine emulsion particles (< 0.2 μm) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration‐based oxygen diffusion coefficients has also been discussed.
Fractal dimensions and porosities of Zoogloea ramigera and Saccharomyces cerevisae aggregates
Authors: Logan Bruce E. , Wilkinson Daniel B. 
Published: December 2004

AbstractThe fractal nature microbial aggregates is a function of the type of microorganism and mixing conditions used to develop aggregates. We determined fractal dimensions from length‐projected area (D2) and length‐number scaling (D3) relationships. Aggregates of Zoogloea ramigera developed in rotating test tubes were both surface and mass fractals, with fractal dimensions of D2 = 1.69 ± 0.11 and D3= 1.79 ± 0.28 (±standard deviation), respectively. When we grew this bacteria in a bench‐top fermentor, aggregates maintained their surface fractal characteristics (D2 = 1.78 ± 0.11) but lost their mass fractal characteristics (D3 = 2.99 ± 0.36). Yeast aggregates (Saccharomyces cerevisae) grown in rotating tests tubes had higher average fractal dimensions than bacterial aggregates grown under physically identical conditions, and were also considered fractal (D2 = 1.92 ± 0.08 and D3 = 2.66 ± 0.34). Aggregates porosity can be expressed in term of a fractal dimensions, but average porosities are higher than expected. The porosities of yeast aggregates (0.9250–0.9966) were similar to porosities of bacterial aggregates (0.9250‐0.9966) cultured under the same physical conditions, although bacterial aggregates developed in the reactor had higher average porosities (0.9857‐0.9980). These results suggest that that scaling relationships based on fractal geometry may be more useful than equations derived from Euclidean geometry for quantifying the effects of different fluid mechanical environments on aggregates morphology and characteristics such as density, porosity, and projected surface area.
Use of microcalorimetric monitoring in establishing continuous energy balances and in continuous determinations of substrate and product concentrations of batch‐grown Saccharomyces cerevisiae
Authors: Larsson Christer , Blomberg Anders , Gustafson Lena 
Published: December 2004

AbstractEnergy balance calculations were performed for different physiological states during batch growth of Saccharomyces cerevisiae with glucose as carbon and energy source. For the different physiological states, energy recoveries close to one were obtained, which permitted a continuous control that the constantly changing growth process was quantified accurately. During the respiro‐fermantative phase of growth, during which glucose served as the carbon and energy source, a low‐heat‐yield value (ΔQx) of −8.6 kJ/g dry biomass formed was obtained. This low‐heat‐yield value was due to the mainly fermentative metabolism during the middle of this phase of growth. After a transition phase, the ethanol produced during the respiro‐fermentative growth was respired. During this respiratory phase, the heat yield values increased markedly, resulting in a lowest value of −42.7 kJ/g. The low‐heat‐yield values of the respiro‐fermentative growth is not a reflection of the most efficient metabolism of S. cerevisiae. On the contrary, during the middle of this phase, 74% of the energy input was dissipated as ethanol, 6% was dissipated as heat, and the energy conserved as biomass was just 13%, while during the early respiratory phase, 69% of the energy input was dissipated as heat, and 22% of the energy input was conserved as biomass. By mathematical modeling and direct monitoring on‐line of the rate of heat production, continuous calculations of (1) glucose consumption, and (3) biomass production were performed, and were shown to correlate closely with measured values for the continuously changing growth process.
Cybernetic modeling of iron‐limited growth and siderophore production
Authors: Alexander Matthew L. , Ramkrishna D. 
Published: December 2004

AbstractThe cybernetic modeling framework developed by Ramkrishna and co‐workers has been applied to a case of bacterial metabolite production, namely the production of siderophores (iron‐chelating agents) associated with iron‐limiting fermentation conditions. Experimental growth data showed that, even though final biomass levels were controlled by exhaustion of the carbon source, iron‐limiting conditions also affected the biomass yield. A structured model which includes the process of an iron‐limiting energy resource production was able to quantitatively account for this apparent dual‐substrate limitation over a wide range of batch and continuous operating conditions. The experiments data also showed quite large difference in iron uptake over the wide range of operating condition and iron levels investigated. The inclusion in the model of the processes of low and high (siderophore‐mediated) affinity iron transport, and siderophore production led to simulation results that were in good quantitative agreement with the siderophore, medium and cell iron levels, in both batch and steady‐state continuous culture operating conditions.
Production of monoclonal antibody using free‐suspended and immobilized hybridoma cells: Effect of serum
Authors: Lee Gyun Min , Varma Amit , Palsson Bernhard O. 
Published: December 2004

AbstractThe effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free‐suspended and immobilized S3H5/γ2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 ± 0.12 day−1 (±standard deviation), while that in medium containing 1% serum was approximately 0.73 ± 0.12 day−1. The specific MAb productivity was almost constant at 3.69 ± 0.57 μg/106 cells/day irrespective of serum concentration reached a maximum at ca. 1.8 × 106 cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free‐suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.
Use of a lipase immobilized in a membrane reactor to hydrolyze the glycerides of butteroil
Authors: Malcata F. Xavier , Hill Charles G. , Amundson Clyde H. 
Published: December 2004

AbstractA lipase from A spergillus niger, immobilized by adsorption on a microporous, polypropylene flat‐sheet membrane, was used to effect the continous hydrolysis of the glycerides of melted butterfat at 35°C. For the reaction conditions used in this research, a pseudo‐zero order rate expression can be used to model the kinetics of the overall hydrolysis of butterfat. Multiresponse nonlinear regression methods were employed to determine the kinetic parameters of a multisubstrate rate expression derived fro ma mechanism based on the general Michaëlis–Menten approach. For the multiresponse data taken at pH 7.0, the dependence of the maximum rate of release of each fatty acid residue of butterfat on its carbon chain length is accurately described by a skewed, bell‐shaped (or Γ‐type) distribution. Data taken at five different pH values were fit assuming a Dixon–Webb diprotic model for the pH dependence of the reaction rate. The thermal deactivation of the immobilized lipase obeyed first‐order kinetics with a half‐life of 19.9 days at 35°C. The multisubstrate model is useful for the prediction of the free fatty acid profile of lipolyzed butterfat, whereas the lumped‐substrate model provides an estimate of the overall degree of hydrolysis as a function of the reactor space time.
Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture
Authors: Robinson David K. , Memmert Klaus W. 
Published: December 2004

AbstractA clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady‐state, continuous culture at dilution rates ranging from 0.21 to 1.04 day−1. The steady‐state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day−1. Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.
Expression of recombinant protein A from the lac promoter in Escherichia coli JM83 is not subject to catabolite repression when grown under specific conditions of continuous culture
Authors: Warnes Alan , Stephenson John R. , Fooks Anthony R. , Melling Jack , Brown Michael R. W. 
Published: December 2004

AbstractAlthough widely used in experimental and industrial situations, genetically engineered plasmids containing the lac promoter from Escherichia coli are subject to catabolite repression when grown in glucose‐containing media. Several methods of overcoming this problem have been investigated by studying the expression of the protein A gene from Staphylococcus aureus under the control of the Escherichia coli lac promoter. When glycerol is used as a sole carbon source, the plasmid is unstable and is rapidly lost from the culture. When the bacteria are grown in chemostats under glucose limitation, the plasmid is maintained, even at high dilution rates, and the expression of protein A is similar to that observed when glycerol was used. The balance between metabolic load and protein A expression seems to be maintained by reducing the gene dose to a tolerable level. Depending on the metabolic conditions prevailing in the culture, this is achieved, either by reducing the copy number of the plasmid or in extreme cases by removing the plasmid altogether.
Observing protein synthesis, export, and tryptophan incorporation by front‐surface fluorescence
Authors: Lipton A. J. , Domach M. M. 
Published: December 2004

AbstractFront‐surface detection of emission from fluorophores in the presence and absence of light‐scattering particles was contrasted to right‐angle and wave‐guide detection. We found that front‐surface detection was the least prone to the reabsorption, inner‐filtering, and scattering effects that can plague fluorescent measurements. Front‐surface detection was thus used to assess the use of protein and ANS fluorescence as a means of monitoring events in bacterial fermentations. Protein fluorescence appeared to track well changes in optical density during balanced growth. However, during the lag associated with diauxic growth and after exposure to ampicillin, protein fluorescence became decoupled from cellular growth in a manner consistent with prior observations and the known effect of ampicillin on cells. ANS proved to be nontoxic and capable of reporting the occurrence of protein release from cells. The spectral shifts of tryptophan indicated that the incorporation of tryptophan into cellular protein can be monitored.
Bioleaching of zinc sulfide concentrate by Thiobacillus ferrooxidans
Authors: Konishi Yasuhiro , Kubo Hideaki , Asai Satoru 
Published: December 2004

AbstractThe kinetics of the bioleaching of ZnS concentrate by Thiobacillus ferrooxidans was studied in a well‐mixed batch reactor. Experimental studies were made at 30°C and pH 2.2 on adsorption of the bacteria to the mineral, ferric iron leaching, and bacterial leaching. The adsorption rate of the bacteria was fairly rapid in comparison with the bioleaching rate, indicating that the bacterial adsorption is at equilibrium during the leaching process. The adsorption equilibrium data were correlated by the Langmuir isotherm, which is a useful means for predicting the number of bacteria adsorbed on the mineral surface. The rate of chemical leaching varied with the concentration of ferric iron, and the first‐order reaction rate constant was determined. Bioleaching in an iron‐containing medium was found to take place by both direct bacterial attack on the sulfide mineral and indirect attack via ferric iron. In this case, the ferric iron was formed from the reaction product (ferrous iron) through the biological oxidation reaction. To develop rate expressions for the kinetics of bacterial growth and zinc leaching, the two bacterial actions were considered. The key parameters appearing in the rate equations, the growth yield and specific growth rate of adsorbed bacteria, were evaluated by curve fitting using the experimental data. This kinetic model allowed us to predict the liquid‐phase concentrations of the leached zinc and free cells during the batch bioleaching process.
Degradation of microcrystalline cellulose: Synergism between different endoglucanases of Cellulomonas sp. ATCC 21399
Authors: Poulsen O. M. , Petersen L. W. 
Published: December 2004

AbstractThree immunologically and enzymatically distinct endoglucanases of Cellulomonas sp. ATCC 21399 were purified previously. Endoglucanase A and endoglucanase B acted synergistically on microcrystalline cellulose (Avicel), whereas no synergistic action was observed between endoglucanase B or endoglucanase C. Only endoglucanase A was capable of hydrolyzing Avicel when acting alone and this enzyme resulted in “short fiber formation” when acting on Avicel. The end product of hydrolysis of acid swollen Avicel produced by the three endoglucanases was in all cases dominated by cellobiose and showed lower content of glucose and cellotriose. Higher cellodextrins appeared as transient end products. The results indicate that the function of endoglucanase A in the cellulase system of Cellulomonas might be very similar to the function of the cellobiohydrolases of Trichoderma reesei.
Hydrolysis of penicillin G by combination of immobilized penicillin acylase and electrodialysis
Authors: Ishimura Fumihiro , Suga Ken‐Ichi 
Published: December 2004

AbstractPhenylacetic acid, as inhibitory product, was formed from a hydrolysis of penicillin G by immobilized penicillin acylase. In this article, electrodialysis was applied to remove phenylacetic acid continuously from the reaction mixture and to enhance an efficiency of the reaction. When 268 and 537 mM of penicillin G solution were used as the substrate, the concentration of phenylacetic acid in the reaction mixture could be maintained at less than 81 and 126 mM, respectively, and eventually, 86% and 88% of phenylacetic acid produced were removed from the reaction mixture at the end of the hydrolysis, respectively. Times required to reach 96% and 94.8% conversion from 268 and 537 mM of initial penicillin G could be reduced to 65% and 64% respectively, by means of electrodialysis; while 3.0% and 4.3% of initial penicillin G of 268 and 537 mM were permeated out of the reaction chamber during the hydrolysis, respectively. However, a loss of penicillin G by permeation could be reduced from 4.3% to 3.4% by a repeated addition of penicillin G.