bluuebunny/crossref_metadata_2025_split huggingface dataset:
Highly Enantioselective Hydrolysis of (R,S)‐Phenylalanine Isopropyl Ester by Subtilisin Carlsberg. Continuous Synthesis of (S) ‐Phenylalanine in a Hollow Fiber/Liquid Membrane Reactor
Authors: Ricks Edith E. , Estrada‐Valdés Maria C. , Mclean Thomas L. , Iacobucci Guillermo A. 
Published: March 2005

AbstractThe resolution of racemic phenylalanine esters with esterases has been reexamined in relation to the development of a continuous process based on the use of hollow fiber/liquid membrane (SLM) reactors. The requirement for high enantioselectivity was met by phenylalanine isopropyl ester, whose (R)‐enantiomer was found refractory to the action of subtilisin Carlsberg, in water, pH 7.5, at 25 °C. The continuous feeding of (R, S)‐Phe‐O‐iPr‐HCl at 2 mm/h to 0.7 g/L subtilisin resulted in steady‐state reaction conditions that gave (S)‐Phe‐OH of 95% enantiomeric excess (ee) at the theoretical rate. The unreacted (R)‐Phe‐O‐iPr, which permeated out continuously, had 80% ee and was racemized quantitatively by heating with a salicylaldehyde catalyst in refluxing toluene. Doubling the feeding rate (4 mm/h) created excessive accumulation of (R)‐Phe‐O‐iPr in the reactor, which inhibited the enzyme through nonproductive binding, lowering the (S)‐Phe‐OH resolution rate to 68% of the theoretical rate. Similar experiments with (R, S)‐Phe‐OMe‐HCl gave (S)‐Phe‐OH of 9–48% ee, due to the enzymatic hydrolysis of (R)‐Phe‐OMe experienced during the runs. When α‐chymotrypsin was used for the continuous hydrolysis of (R, S)‐Phe‐O‐iPr, the isolated (S)‐Phe‐OH showed 67% ee, indicating that substantial hydrolysis of the (‐R)‐ester had occurred.
Activity Regeneration in Continuous Clostridium acetobutylicum Bioconversions of Glucose
Authors: Reardon Kenneth F. , Baily James E. 
Published: March 2005

AbstractThe regeneration of product‐forming activity by periodic nutrient feeding was studied in a continuous immobilized Clostridium acetobutylicum reactor system. Activity levels were increased by feeding ammonia and vitamins during selected intervals of the experiments; these compounds were not otherwise present in the medium. In contrast with experiments utilizing rich, complex media, regeneration with a defined medium resulted in increased rates of product formation far in excess of rates of concomitant cell growth. During one nutrient feeding phase, the increase in the butyric acid production rate was five times that of the cell growth‐rate increase, while the analogous ratio for butanol was 18. A hypothesis explaining the patterns of substrate consumption and product formation during the regeneration and deactivation periods has been developed: decreasing levels of exogenous ammonia induce the high‐affinity ammonia uptake system, causing the adenylate energy charge of the cells to increase; this in turn leads to the repression of glucose carbon flow through the EMP pathway and to the use of the acid‐forming catabolic reactions for more efficient production of ATP. Pulsewise addition of three different vitamins during one regeneration phase yielded interesting results, including a 50 % increase in butyric acid production rate that appeared to be connected with the addition of biotin. Finally, enumeration of the different morphological types of immobilized cells provided the first measurements of population dynamics in an immobilized cell system. Changes in the numbers of each cell type are consistent with the sporulation and germination processes of C. acetobutylicum observed with suspended cells.
Rheological Properties of Plant Cell Suspensions
Authors: Ballica Rabia , Ryu Dewey D. Y. , Powell Robert L. , Owen Derek 
Published: March 2005

AbstractPacked cell and concentrated cell suspension cultures of Datura stramonium were exposed to oscillatory and steady shear on a Weissenberg rheogoniometer with a modified parallel plate geometry and to steady shear using other rotational viscometers. The viscoelastic properties of the cell suspensions were measured over a frequency range from 0.01 to 4 rad/s at low and high amplitudes. The applied strains were in the range of 1%‐36%. Increasing the strain caused cell damage (e.g., approximately 20% at a strain of 36% during 3 h of shearing). Cell cultures exhibited both viscoelastic and shear thinning behavior. The concentrated cell suspension cultures showed much lower dynamic viscosities and elastic moduli than the packed cell cultures. From the measured viscoelastic properties, yield stresses were calculated. The maximum calculated average yield stress was 4 × 104 dyn/cm2 for the packed cells and 325 dyn/cm2 for the concentrated cell suspension having a cell volume fraction (PCV, packed cell volume) of 0.9 mL/mL. From the steady shear experiments, yield stresses were also determined. The maximum calculated yield stress was 360 dyn/cm2 for a concentrated cell suspension (PCV = 0.9 mL/mL) and 95 dyn/cm2 for the less concentrated cell suspension (PCV = 0.6 mL/mL). It was confirmed that D. stramonium cell suspension cultures at high cell concentrations exhibit yield behavior which can be determined by either oscillatory or steady shear. The yield stress values determined in this study will be of some value to the bioprocess engineering of plant cells for high density cell cultivation and product recovery. In particular, these values will be important to determine bioreactor operating conditions such as agitation and aeration rates.
Mechanical Properties of Hydrocolloid Gels Filled with Internally Produced CO2 Gas Bubbles
Authors: Nussinovitch A. , Velez‐Silvestre R. , Peleg M. 
Published: March 2005

AbstractAgar (2%), alginate (1% algin), and κ‐carrageenan (1.5%) gel specimens were prepared from mother solutions that contained 0–2.5 % sodium bicarbonate (agar and carrageenan) or calcium carbonate (alginate). Upon immersion in a citric acid bath (0–2%), the carbonate reacted with the diffusing acid to produce numerous carbon dioxide bubbles. The compressive strength and deformability of the gas‐filled gels so produced were determined using a Universal testing machine and compared with those of pure gels and gels containing the carbonate but not subjected to the process after various immersion times. While the agar and alginate gels retained considerable mechanical integrity even after several hours, the carrageenan gels disintegrated after about 2–5 h. Under similar conditions, the number of bubbles produced in the agar gels was about twice that in the alginate gels, an observation that cannot be explained solely by stoichiometric considerations.
Recombinant β‐Galactosidase Production in Serum‐Free Medium by Insect Cells in a 14‐L Airlift Bioreactor
Authors: King G. A. , Daugulis A. J. , Faulkner P. , Goosen M. F. A. 
Published: March 2005

AbstractSpodoptera frugiperda (Sf9) insect cells were successfully cultured in serum‐free medium in a 14‐L airlift bioreactor. Cell densities as high as 1 × 107 cells/mL were achieved with specific growth rates of approximately 0.0286 h−1 (doubling time of 24 h). This system was also used to demonstrate the expression of a reported gene, β‐galactosidase (β‐gal), when cells were infected with a recombinant baculovirus. Approximately 0.33 mg of β‐gal/mL (i.e., 104 000 units/mL) of medium were obtained at the 14‐L scale, while about 0.95 mg of β‐gal/mL (i.e., 285 000 units/mL) of medium were obtained in small‐scale shaker flasks. The difference was attributed to a suboptimal infection in the large scale. Specific oxygen consumption rates decreased from 5.58 × 10−17 mol O2cell·s in early exponential growth to 3.13 × 10−17 mol (O2cell·s at 3 days post‐infection.
STM and AFM in Biotechnology
Authors: Haggerty Lorie , Lenhoff Abraham M. 
Published: March 2005

AbstractScanning tunneling microscopy (STM) and atomic force microscopy (AFM) are capable of providing atomic‐scale images, as well as insights into functional characteristics, of surfaces. Biological materials can be studied by depositing them on appropriate substrates, and samples have been imaged with varying degrees of resolution. In this review, the principles of STM and AFM are summarized, and practical aspects of implementing them for studies relevant to biotechnology are discussed, with the emphasis on investigations of biological macromolecules such as DNA and proteins. Items covered include samples, surfaces, medium, tips, and typical operating conditions. Finally, prospects are discussed for potential future applications to biotechnology.
The Monod Constant for Growth for Candida utilis on Ammonium in Continuous and Batch Cultures
Authors: Cooney Michael J. , Mcdonald Karen A. 
Published: March 2005

AbstractMonod constants for growth on ammonium were determined for Candida utilis under different growth conditions. Results indicate that values for Ks differed in batch and in continuous cultures. The addition of the vitamin thiamine, however, had no effect on Ks. By contrast, the maximum specific growth rate did not vary between batch and continuous cultures but was affected by the addition of the vitamin thiamine.
Nonisotropic Scattering Model for Estimation of Light Absorption Rates in a Suspension Culture of Coffea arabica Cells
Authors: Kurata Hiroyuki , Furusaki Shintaro 
Published: March 2005

AbstractA nonisotropic scattering model is proposed to estimate the light absorption rates in a heterogeneous photoreactor containing plant cells of Coffea arabica and is compared with an isotropic scattering model. In order to determine the directions of noniso‐tropically scattered light, optical theorems (geometrical optics and wave optics) are applied to light scattering caused by a homogeneous and perfect sphere. Two parameters, relative refractive index and attenuation coefficient, are used to characterize the ideal sphere. The primary feature of this model is that various phenomena of light scattering and absorption can be simulated by the combination of the two parameters. A measuring system composed of parallel plates was developed for evaluating the model. The nonisotropic scattering model was successfully applied to the heterogeneous reactor with the cultured plant cells.
Comparison of Metabolism of Hybridoma Cells Cultured in Media Supplemented with Whey or Fetal Calf Serum
Authors: Legrand C. , Capiaumont J. , Belleville F. , Nabet P. 
Published: March 2005

AbstractThe effects of industrial whey on cell physiology were studied in a repeated fed‐harvest mode using free‐suspended murine hybridoma cells. After several days of culture in medium containing 9% whey and 1% fetal calf serum (FCS), cell growth and viability, carbohydrate, amino acid, energy metabolism, and antibody production rates were investigated. Differences were found between cells cultured in whey medium and those cultured in conventional FCS medium. The cell growth obtained in medium supplemented with fresh whey was similar to that obtained in FCS medium. The viability showed an increase for hybridoma cells cultured in whey medium. Glucose consumption rates were similar, whereas the lactate production rate was higher in whey medium. The metabolic uptake rates of glutamine and ammonia increased in whey medium. More alanine, glutamate, glycine, and proline were produced; their production partly came from glutamine and lactate. The consumption rates of branched amino acids changed little; their utilization was higher in whey medium. Finally, antibody productivity was increased about 20% for cells cultured in medium containing whey.
Development of Scale‐Down Techniques for Investigation of Recombinant Escherichia coli Fermentations: Acid Metabolites in Shake Flasks and Stirred Bioreactors
Authors: Dahlgren Mary Ellen , Powell Alan L. , Greasham Randolph L. , George Hugh A. 
Published: March 2005

AbstractWe have developed shake‐flask screening conditions that are predictive of specific expression of the chimeric toxin, TGFα‐PE40, by recombinant Escherichia coli JM109 in stirred bioreactors. When a nutrient‐rich stirred bioreactor medium was used in shake flasks, neither the extent of growth nor the specific level of recombinant protein expression duplicated the performance in stirred bioreactor fermentations. Incomplete oxidation of glucose and concomitant accumulation of organic acid metabolites, as well as oxygen limitation and lack of pH control, were examined as contributors to the poorer performance in the flask. The medium buffering capacity, initial glucose level, and flask aeration were evaluated to establish the limits of “scale‐down” conditions for expression both in a complex nutrient medium (M101) similar to that used in stirred bioreactors and in a defined (FM) medium. Acid metabolites and ethanol were measured as indicators of carbon flow from glucose as well as indirect indicators of oxygen limitation. For the complex M101 medium, optimal shake‐flask performance in 250‐mL, nonbaffled flasks at 37°C occurred with 0.3X medium strength, supplementation with 0.3 M HEPES buffer (pH 7.5), and 10 mL of medium per flask. Cultures grown under these conditions produced a maximum density of 3.6 g of dry cell weight/L (as estimated by absorbance measurements at 600 nm) and maintained a pH near neutrality. Additionally, metabolite markers of anaerobic or microaerobic conditions, such as ethanol, lactate, and pyruvate, were not detected, and specific expression of TGFα‐PE40 was comparable to stirred bioreactors induced for expression at various biomass levels. When culture parameters were controlled within these limits, similar results were also observed in the defined FM medium.
Synthesis of β‐Ionone in an Aldehyde/Xanthine Oxidase/β‐Carotene System Involving Free Radical Formation
Authors: Bosser Aurélie , Belin Jean Marc 
Published: March 2005

AbstractThe enzymic cooxidation of β‐carotene (BC) by xanthine oxidase (XO) in aqueous solutions leads to β‐ionone (BI) and derivatives: epoxy‐β‐ionone (EPBI), dihydroactinidiolide, β‐cyclocitral, pseudoionone, etc. We demonstrate, in this article, that this is due to free radical (R⋅) formation during the first hour of stirring and the use of aldehydes as substrate. The bleaching of BC does not occur when the common substrate of XO, xanthine, is used; this proves that the superoxide anion O2⋅‐ alone is not active on BC. BI formation in this case is not observed.
Effect of Glucose on the Expression Parameters of Recombinant Protein in Escherichia coli during Batch Growth in Complex Medium
Authors: Li Xiaoli , Taylor Kenneth B. 
Published: March 2005

AbstractThe expression parameters were determined at different phases of batch growth of Escherichia coli JM101/pYEJ001 in complex medium and at different conditions of glucose addition. Thus the plasmid content, the RNA content, the RNA synthesis rate, the specific recombinant mRNA content, the specific recombinant mRNA synthesis rate, the recombinant chloramphenicol acetyltransferase content, and the overall protein synthesis rate were determined during growth with no glucose, with initial glucose, with glucose feeding during stationary phase, and with initial glucose plus glucose feeding during stationary phase. The results show that the specific rate of total RNA synthesis was enhanced in the presence of glucose at both exponential and stationary phases, while recombinant mRNA synthesis was enhanced only at stationary phase by glucose feeding. However, the steady‐state level of the recombinant mRNA was not changed under the same conditions. In addition, although the overall protein synthesis rate at exponential phase was enhanced in the presence of glucose, the specific recombinant protein level was unaffected. The specific synthesis rate of recombinant mRNA varied inversely with the plasmid content during exponential and stationary phases. Furthermore, changes in the specific activity of the recombinant protein were not correlated with either the changes in the specific synthesis rate of the recombinant mRNA or the overall protein synthesis rate. Therefore, the specific activity of the recombinant protein is not universally limited by its gene transcription rate or the overall protein synthesis capacity.
Carbon Flux Distributions at the Pyruvate Branch Point in Corynebacterium glutamicum during Lysine Overproduction
Authors: Vallino Joseph J. , Stephanopoulos Gregory 
Published: March 2005

AbstractIn order to achieve the theoretical maximum yield of lysine from glucose (75% molar), flux partitioning at principal branch points (glucose 6‐phosphate, phosphoenolpyruvate, and pyruvate) in the primary metabolic network of Corynebacterium glutamicum must be significantly altered from that observed during balanced growth, so that lysine precursors are synthesized in optimal stoichiometric ratios. In this article, we employ flux analysis, which provides a snapshot of metabolic flux distributions from extracellular measurements, in conjunction with two metabolic perturbations of the pyruvate dehydrogenase complex (PDC) to examine the possibility that flux partitioning at the pyruvate (Pyr) branch point (i. e., pyruvate availability) limits lysine yield. The two perturbation experiments involved (1) isolation and flux analysis of a PDC‐attenuated mutant of C. glutamicum and (2) flux analysis following the addition of fluoropyruvate (FP) to a standard fermentation during the initial period of lysine overproduction. No significant alteration in flux partition was observed at any of the three principal nodes in the PDC‐attenuated strain. However, the PDC mutation did cause a 70% uniform decrease in carbon flow throughout the network, which indicates that the lysine yield is not limited solely by a weakly rigid Pyr branch point. The addition of FP did not affect the lysine synthesis rate, but did temporarily redirect carbon flow away from the TCA cycle toward pyruvate excretion, which further confirms that the lysine yield is not pyruvate‐limited and that the Pyr branch point is not weakly rigid.
Microbial Oxidation of Hydrogen Sulfide in a Pilot‐Scale Bubble Column
Authors: Sublette Kerry L. , Hesketh Robert P. , Hasan Saleem 
Published: March 2005

AbstractThiobacillus denitrifians is a chemoautotroph and a facultatively anaerobic bacterium that can use reduced sulfur compounds as energy sources with oxidation to sulfate. Under anoxic conditions, nitrate may serve as a terminal electron acceptor with reduction to elemental nitrogen. T. denitrificans has previously been shown to remove hydrogen sulfide from sour gases under both aerobic and anoxic conditions. Hydrogen sulfide concentrations were reduced from 10 000 ppmv to undetectable levels with 1–2 s of gas‐liquid contact time in bench‐scale fermentors. The oxidation product, sulfate, accumulated in the liquid phase of the culture medium. Flocculated T. denitrificans has now been used to remove hydrogen sulfide (up to 1500 ppmv) from a sour gas in a 0.5‐m3 pilot‐scale bubble column. The bubble column was operated for 7 weeks under aerobic conditions, with gas feeds of air and hydrogen sulfide in nitrogen. Up to 97% removal of hydrogen sulfide was observed with complete oxidation to sulfate, which accumulated in the reactor medium. Limitations in H2S removal were due to mass transfer and not the microbiology of the system. More complete H2S removal can be accomplished by increasing gas‐liquid contact in a single reactor or using two bubble columns in series.
Block Methacrylic Polyampholytes as Protein Displacers in Ion‐Exchange Chromatography
Authors: Patrickios C. S. , Gadam S. D. , Cramer S. M. , Hertler W. R. , Hatton T. A. 
Published: March 2005

AbstractLow molecular weight methacrylic triblock copolymers bearing a block of negatively charged (adsorbing) residues, a sequence of positively charged (repelling) units, and a middle neutral hydrophobic block were utilized successfully in anion‐exchange displacement chromatography for the resolution of a crude mixture of/β‐lactoglobulins into the A and B isoforms. Owing to their low molecular mass and ampholytic nature, these novel displacers possess the attractive process‐scale features of easy column regeneration and polymer recycling.
Evaluation of the Cd, Cu, and Zn Biosorption in Two‐Metal Systems Using an Algal Biosorbent
Authors: De Carvalho R. P. , Chong K.‐H. , Volesky B. 
Published: March 2005

AbstractThe equilibrium sorption capacity of the formaldehyde‐cross‐linked brown seaweed Ascophyllum nodosum biomass was studied using two‐metal systems comprising either (Cu + Zn), (Cu + Cd), or (Zn + Cd). Three‐dimensional sorption isotherm surfaces were used in the evaluation of sorption performance, replacing simple isotherm curves. While each of the metals tested can inhibit the sorption of the others, at low total residual metal concentrations the total metal sorption uptake increases. At higher total metal concentrations, the total metal sorption uptake either remains constant, or there may be a slight decrease compared to single‐metal uptakes. This is an indication of a mutual interference in the sorption uptake of the two metals tested in each system.
Biomass Estimation in Plant Cell Cultures: A Neural Network Approach
Authors: Albiol Joan , Campmajó Carles , Casas Carles , Poch Manel 
Published: March 2005

AbstractThe special characteristics of plant cell cultures make it difficult to use conventional analytical techniques for on‐line biomass monitoring. Meanwhile, promising results have been obtained using mathematical models and recursive estimation algorithms. However, in this case, additional experimental effort is necessary to obtain a reasonable description of the process. Recently, techniques using more empirical approaches have been proposed to describe complex processes, minimizing the experimental work needed for their application. In this paper, we report on the use of artificial neural networks to monitor biomass evolution in plant cell cultures. The results obtained with a three‐layered network are presented. Method requirements and capabilities are compared with the method based on the extended Kalman filter used in previous work.
Cell Damage and Oxygen Mass Transfer during Cultivation of Nicotiana tabacum in a Stirred‐Tank Bioreactor
Authors: Ho Chung ‐Han , Henderson Kelley A. , Rorrer Gregory L. 
Published: March 2005

AbstractCell damage and oxygen mass transfer were investigated during the batch culture of Nicotiana tabacum (tobacco) cells in a 3‐L Applikon stirred‐tank bioreactor equipped with a marine‐blade impeller. Total cell density, % culture viability, viable cell density, and kLa in the culture were measured over a 24‐day cultivation period at impeller speeds of 100–325 rpm under a fixed air flow of 0.43 wm and a temperature of 27 °C. The maximum total biomass density decreased from 11.8 g of dry cell weight (DCW)/L at 100 rpm to 8.6 g of DCW/L at 325 rpm. The net specific biomass production rate of viable cells (μv) during the exponential phase and the viable cell death rate (kd) during the stationary phase were estimated from semilog plots of viable cell density vs cultivation time. Values for μv decreased from 0.175 to 0.077 day−1 whereas values for kd increased from 0.042 to 0.109 day−1 with increasing impeller tip speed (from 24 to 77 cm/s), clearly showing that increasing agitation intensity increased the rate of cell damage. The effect was most pronounced at tip speeds of 24–60 cm/s. With respect to oxygen mass transfer, culture kLa values were about 10–20% higher than their corresponding initial kLa° values, but as the cell density increased, the values for kLa ultimately decreased. Despite the reduction in oxygen mass transfer rates, at impeller speeds of 100–325 rpm and an aeration rate of 0.43 wm, oxygen starvation was not observed. Therefore, the reduction in biomass productivity can be attributed to cell damage by hydrodynamic forces and not by inadequate oxygen mass transfer.