bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protection mechanisms of freely suspended animal cells (CRL 8018) from fluid‐mechanical injury. Viscometric and bioreactor studies using serum, pluronic F68 and polyethylene glycol
Authors: Michaels James D. , Petersen Jonathan F. , Mclntire Larry V. , Papoutsakis Eleftherios T. 
Published: December 2004

AbstractWe use bioreactor and viscometric studies to examine the mechanism by which three additives, fetal bovine serum (FBS), pluronic F68, and polyethylene glycol (PEG), protect the freely suspend CRL‐8018 cells from damage due to interactions with bubbles in agitated bioreactors. In bioreactor studies, the protective effect of an addictive could be due to either changes in the ability of the cell resist shear (biological mechanism) or to changes in the medium properties that effect the level or frequency of forces experienced by the cells (physical mechanism). Bioreactor studies show that protection by all three addictives occurs whether the cells are grown in the presence of the addictives (long exposure) or the addictives are added to medium after the cells were exposed to detrimental agitation intensity (short exposure). In the viscometric studies, exposure of cells to laminar shear in the absence of gas‐liquid interfaces assesses only the ability of the cells to resist a constant level of shear in a medium with or without an additive. Viscometric studies show that prolonged exposure to FBS makes the cells more shera tolerant, but that short (30–120 min) exposure to FBS does not affect their shear tolerance. We thus conclude that the protective effect of FBS in bioreactors id of both physical and biological nature. The biological contribution is metabolic in nature rather than fast acting. Viscometric studies show that either long or short exposure of the cells to either F68 or PEG does not make the cells more shear tolerant. WE therefore conclude that the protective effect of F68 and PEG does not make the cells more shear tolerant. We therefore conclude that the protective effect of F68 and PEG in bioreactors is physical in nature.
Kinetics, mechanism, and time course analysis of lipase‐catalyzed hydrolysis of high concentration olive oil in AOT–isooctane reversed micelles
Authors: Tsai Shau‐Wei , Chiang Chen‐Li 
Published: December 2004

AbstractCandida rugosa lipase has been used to investigate the hydrolysis of high concentration olive oil in the AOT–isooctane reversed micellar system at Wo = 10, pH 7.1, and 37°C. Results from this work show the hydrolytic reaction obeys Michaelis–Menten kinetics up to the initial substrate concentration of 1.37M, with turnover number kcat and Michaelis constant KM of 67.1 μmol/min mg enzyme and 0.717M, respectively. A competitive inhibition by the main product, oleic acid, has been found with a dissociation constant KI for the complex EP* of 0.089M. The rate equation was further analyzed in the time course reaction and was found in agreement with the experimental results for lower substrate concentrations, up to 0.341M. Large deviation occurred at high substrate concentrations, which may be due to the effects of large consumption of water on kinetics, on the formation of glycerol, and on the deactivation of lipase in the hydrolysis reaction as well.
Process development with nitrogenase‐producing Escherichia coli C‐M74 (pUS1) CellS
Authors: Ahlmann N. , Schügerl K. 
Published: December 2004

AbstractA process for the continuous fermentation of the genetically modified, nitrogenase‐producing Escherichia coli C‐M74 (pUS1)‐strain has been developed. This strain, which is able to fix molecular nitrogen, has the nifgenes of the bacterium Klebsiella pneumoniae. Cell growth and nitrogenase activity of the enzyme have been optimized both in batch and continuous fermentations. For the fermentations, trial runs were performed by cultivating the E. coli cells in 50‐ml culture bottles. The medium composition was varied in order to provide high biomass production and nitrogenase activity. For an effective fermentation control, an on‐line analysis was built up for the substrates ammonium and glucose. Other medium components such as ampicillin, citric acid, acetic acid, nitrogenase activity, and protein were measured by using different off‐line methods. Modern optical methods like in‐line microfluorometry for monitoring the culture fluorescence and laser flow cytometry for the estimation of DNA and protein content were also employed. Plasmid stability was also determined.
The Effect of sorbitol on acid phosphatase deactivation
Authors: Gianfreda Liliana , Toscano Giuseppe , Pirozzi Domenico , Greco Guido 
Published: December 2004

AbstractAcid phosphatase thermal deactivation follows a complex path: an initial decay toward an equilibrium distribution of at least two intermediate structures, mutually at the equilibrium, followed by a final breakdown toward a completely inactive enzyme configuration. The results obtained in the presence of sorbitol have been compared to those produced in the course of purely thermal deactivation of the native enzyme. For any sobitol concentration, an equivalent temperature is calculated that results in exactly the same activity‐versus‐time profile. This suggests enzyme deactivation to be controlled by a single, unchanging step. Immobilized enzyme runs have been performed, as well, by entrapping acid phosphates within a polymeric network formed onto the upstream surface of an ultrafiltration membrane. The stabilizing effect of entrapment cumulates with that produced by sorbitol. In this case, however, an equivalent temperature cannot be determined, thus indicating that a different deactivation mechanism is followed.
Steroid bioconversion in a microemulsion system
Authors: Smolders A. J. J. , Pinheiro H. M. , Noronha P. , Cabral J. M. S. 
Published: December 2004

AbstractThe Δ1,2‐dehydrogenation of high concentrations of the steroid ‐methyl‐Reichstein's compound S‐21‐acetate (16MRSA) in a microemulsion system was studied using heat‐dried and thawed Arthrobacter simplex cells as biocatalyst. The microemulsion system consists of an organic phase [75−95% (v/v)] with steroid (1−60 g/Ltot), an aqueous phase [5−25% (v/v)] containing the cells (5−30 g/Ltot), and a neutral surfactant (5−20 g/L organic solvent). Benzene derivatives, which solubilize 16MRSA up to 94 g/L, and phospholipids were used as organic solvents and surfactants, respectively, and menadione was added as an external electron acceptor. Factors affecting the dehydrogenation rate in the microemulsion system were studied. The influences of the 16MRSA and the menadione concentration on the dehydrogenation rate were described by Michaelis‐Menten kinetics, apparent V′max and K′m values of 2.06 g/g dry weight h and 18.9 g/L for 16MRSA and 4.97 g/g dry weight h and 1.91 g/L for menadione being obtained. Optimal menadione concentration was dependent on the steroid concentration was dependent on the steroid concentration used. The reaction was strongly inhibited by high product concentrations. Much higher activities were obtained with the thawed cells than with the dried cells, conversions of 98% being reached within 14−16 h. for 16MRSA and cell dry weight concentrations of 40 and 10 g/L, respectively. Activity retention in a batch stirred tank reactor remained constant during the first 16−24 h of operation and then decreased, depending on the stirring rate; 22 to 65% of the initial reaction rate was obtained after 48 h at stirring rates of 650 and 2000 rpm, respectively.
Enzymatic preparation of organometallic compounds with second‐order Optical nonlinearities
Authors: Yamazaki Yoshimitsu , Hosono Kuniaki , Matsuda Hiro , Minami Nobutsugu , Asai Michihiko , Nakanishi Hachiro 
Published: December 2004

AbstractTwelve species of optically active metallocene derivatives having a 4‐nitrophenyl group were prepared with horse liver alcohol dehydrogenase‐ or lipase‐catalyzed optical resolution as the key step. The second harmonic generation (SHG) efficiently of these products was measured by the power method using the fundamental light from the Nd:YAG laser. (−)‐1‐ (4‐Nitrophenylthio)ethylferrocene (9), (+)‐1‐(4‐nitrophenylthio)ethylruthenocene (16), (+)‐1‐(4‐nitrophenylthio)ethylosmocene (19), (+) ‐1‐(5‐nitro‐2‐pyridylthio)ethylruthenocene (21), and (+) ‐1‐[(4‐nitrophenylhydrazono)methyl] ‐2‐methylferrocene (12) showed SHG signals. The highest SHG efficiency was found with (+) ‐16, being 27 times more intense than the commonly used urea standard.
Protein extraction by reverse micelles: A study of the factors affecting the forward and backward transfer of α‐chymotrypsin and its activity
Authors: Marcozzi Giordana , Correa Nancy , Luisi Pier Luigi , Caselli Maurizio 
Published: December 2004

Abstractα‐chymotrypsin is taken as a model protein to investigate three aspects of the protein extraction by reverse micelles: (1) the comparison between the two forward transfer techniques, i.e., the liquid‐liquid and the solid state‐liquid transfer; (2)the back‐transfer, i.e., the capability of the protein to be recovered from the micellar solution; and (3) the maintainance of the enzyme activity at the end of the extraction cycle. Concerning the forward transfer from the liquid phase, we study first the effect of salt initially present in the aqueous phase on the equilibrium concentration of the extracted species; further, we study the forward protein extraction from the solid state, and the effect of pH, salt, and protein concentration on the transfer efficiency. Concerning the back transfer, we find the somewhat surprising result, that the percentage of protein back‐extraction depends on the type and concentration of salt used for the forward transfer. Preliminary data concerning an alternative method for the back‐transfer using silica gel to liberate the protein from the micellar environment, are presented. Finally, it is found that the enzyme activity depends again on the type and concentration of salt used for the forward transfer.
Characteristics of batch culture of a recombinant bacillus brevis excreting a foreign esterase
Authors: Tulin Edgardo E. , Takagi Hiroaki , Ueda Shunsaku , Yamagata Hideo , Yamane Tsuneo , Udaka Shigezo 
Published: December 2004

AbstractThe productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD315 harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35°C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%‐92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12‐h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.
The effects of plasmid content, transcription efficiency, and translation efficiency on the productivity of a cloned gene protein in Escherichia coli
Authors: Kim Jeong‐Yoon , Ryu Dewey D. Y. 
Published: December 2004

AbstractIn order to investigate how plasmid content, transcription efficiency, and translation efficiency affect the productivity of a cloned gene protein, a new vector (pPLc‐RP4.5) was constructed. The vector has PL promoter and lacZ as a structure gene 4.5S RNA gene between PL promoter and lacZ gene. We took advantage of the characteristic that the 4.5S RNA is accumulated inside E. coli cells and can be quantitatively measured. A two‐stage continuous culture system in combination with a temperature‐sensitive gene switching system was used to study the performance of the recombinant fermentation. It was found that the plasmid content as varied by the dilution rate in the production stage showed a different pattern from that in the growth stage. The result showed that promoter strength had a greater influence on the overall gene expression efficiency of a cloned gene than the plasmid content, and the overall gene expression efficiency was largely dependent upon translation efficiency when a multicopy plasmid (pBR322 derivative and rop‐) and a strong promoter (PL) were used to express a heterologous protein in E. coli.
Extractive bioconversions in aqueous two‐phase systems: Enzymatic hydrolysis of casein proteins
Authors: Mukataka S. , Haynes C. A. , Prausnitz J. M. , Blanch H. W. 
Published: December 2004

AbstractPartition coefficients in poly(ethylene glycol)/dextran aqueous two‐phase systems are reported for mixed‐casein and its components, α, β and κ casein. Rates of casein proteolysis by α‐chymotrypsin and by trypsin are reported in single‐phase and aqueous two‐phase reactor systems. The advantages resulting from selective partitioning of substrates, enzymes, and products are examined in terms of relative volumetric reaction rates.
Optimization of the production of full‐length rCD4 in baculovirus‐infected Sf9 cells
Authors: Lazarte Jaime E. , Tosi Pierre‐Francois , Nicolau Claude 
Published: December 2004

AbstractThe baculovirus‐insect cell system is reliable in expressing a variety of recombinant proteins. A recombinant baculovirus encoding the full length human CD4 has been used to infect Spodoptera frugiperda 9 cells in 6‐L‐airlift fermentors. The procedured described in this report permitted a 6.5‐fold enhancement of rCD4 expression as compared to standard procedures previously published. The increase of rCD4 expression on the cell surface was achieved by using the following steps: (1) Optimal seeding density of 0.8 × 106 cells/mL used to multiply cells at a maximum exponential growth of 4.5 × 106; (2) high multiplicity of infection (MOI) of 580 PFU/cell; (3) addition of medium at time of infection. In addition to full‐length rCD4, a “short” rCD4 with largely deleted cytoplasmic sequence (last 31 C‐terminal amino acids) was also efficiently expressed.
Performance of mammalian cell culture bioreactor with a new impeller design
Authors: Shi Yuan , Ryu Dewey D. Y. , Park Sun Ho 
Published: December 2004

AbstractTo improve the oxygen transfer in a mammalian cell bioreactor, a new type of impeller consisting of a double‐screen concentric cylindrical cage impeller (annular cage impeller in short) was designed and its mass transfer rate evaluated. This new impeller design increases the specific screen area, and the convective mass transfer rate through the annular cage was significantly increased. The oxygen transfer rates with the new impeller and the commercially available cell‐lift impeller (CelliGen by New Brunswick Scientific Co.) were evaluated and their performance compared at various rates of aeration and agitation. The results showed that with the new impeller, the oxygen transfer rate was increased by 19% in water and 21% in cell‐free culture medium supplemented with 10% horse serum, the total hybridoma cell concentration was increased to 3.4 × 107 cells/mL, and the IgG1 subtype monoclonal antibody (MAb) product concentration was also increased to 512 mg/L in perfusion culture of murine hybridoma cell line 62′D3. These improvements in oxygen transfer rate, cell concentration, and MAb product concentration are all very significant. The mass transfer resistance in the cell‐lift impeller system was found to be mainly due to the surface area of the single‐screen cage impeller. The new annular cage impeller not only provided the increased surface area for convective oxygen transfer but also protected cells from hydrodynamic shear damage, thereby achieving a significant bioprocess improvement in terms of higher viable cell concentration, higher product concentration, and higher oxygen transfer rate in the mammalian cell bioreactor system.
Dependence on growth phase and temperature of the composition of a nitrogen‐fixing cyanobacterium
Authors: Fontes Agustín G. , Moreno José , Vargas M. Angeles , Rivas Joaquín 
Published: December 2004

AbstractThe effects of growth pahase and temperature on the molecular and elemental composition of Anabaena variabilis cells grown under nitrogen‐fixing conditions have been studied in batch cultures. Irrespective of the growth temperature, this cyanobacterium underwent a gradual increase in its protein and lipid contents in the transition from exponential to deceleration pahse that was accompanied by a parallel decrease in carbohydrates and nucleic acids. Also in response to this, transition increases in carbon, nitrogen, and hydrogen contents with a concomitant decline in oxygen content was a common pattern for all growth temperatures tested. Whereas temperature rise did not affect significantly the protein and carbohydrate contents in exponentially growing cells, for cells in the deceleration phase proteins declined and carbohydrates increased with increasing temperature. From growth and elemental composition data several bioenergetic parameters were derived for A. variabilis cells. Both aging of cultures and rise in temperature resulted in increases of both biomass degree of reduction and heat of combustion. Nevertheless, biomass energetic yield was only slightly affected by variations in growth temperature and the maintenance coefficient was virtually constant within the range of temperatures tested. © 1992 John Wiley & Sons, Inc.
Oxygen transfer in liquid‐impelled loop reactors using perfluorocarbon liquids
Authors: van Sonsbeek H. M. , De Blank H. , Tramper J. 
Published: December 2004

AbstractOxygen transfer in the liquid‐impelled loop reactor is described for a setup in which the perfluorochemical FC40 is aerated externally. Two sizes of reactors are investigated. The mass‐transfer coefficient k appears to be lower with a factor of about 0.6 compared to gas liquid systems. the specific exchange area in the present experimental setup is found to be favorable when compared with gas liquid bioreactors at the same superficial dispersed‐phase velocities. However, slow coalescence of the dispersed‐phase drops in the phase separation section limits the dispersed‐phase flow rate seriously. In Case this become crucial from the point of view of oxygen supply, special measures need to be found or alternatives such as combined sparging of air and solvent. © 1992 John Wiley & Sons, Inc.
Activity of mushroom polyphenol oxidase in organic medium
Authors: Burton Stephanie G. , Duncan John R. , Kaye Perry T. , Rose Peter D. 
Published: December 2004

AbstractA kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p‐cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p‐substituted phenolic substrates, yielding o‐quinone products. Michaelis–Menten kinetics were used to obtain apparent KM and Vmax values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o‐quinones were measured by a novel method: 1H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water‐sensitive, unstable o‐quinones. © 1993 John Wiley & Sons, Inc.
Construction and evaluation of a metal ion biosensor
Authors: Tescione Lia , Belfort Georges 
Published: December 2004

AbstractEscherichia coli, genetically engineered with a mercury(II)‐sensitive promoter and the lux genes from Vibrio fischeri, were used as microbial bioluminescent sensors for the detection of mercury. Evaluation of this genetic construction was carried out by determining the effects of various parameters on cell suspensions maintained at constant conditions in a small 100‐mL vessel. The strongest light intensities and quickest induction times occurred with cells in the midexponential growth phase maintained at 28°C, concentrated to 1 × 109 cells/mL, mixed at very fast speeds, and aerated at 2 vvm (volume of air per volume of culture per minute) during light measurement in the small vessel. The cells were sensitive to the mercuric ion in the range of 20 nM to 4 μM (4 to 800 ppb), and the total response time was on the order of 1 hour, depending on the above parameters. The cells exhibited great specificity for mercury. The cells had almost equal specificity for organic and inorganic forms of the mercuric ion and responded more weakly to the mercurous ion. A simple, inexpensive, durable miniature probe (3 mL) was constructed and operated using the optimum parameters found in the small vessel as a guide. The range of sensitivity to the mercuric ion detected in the probe was 10 nM to 4 μM when aeration was provided. © 1993 John Wiley & Sons, Inc.
Temperature control of growth and productivity in mutant Chinese hamster ovary cells synthesizing a recombinant protein
Authors: Jenkins Nigel , Hovey Alison 
Published: December 2004

AbstractThe use of a temperature switch to control the growth and productivity of temperature‐sensitive (ts) mutants was investigated to extend the productive life span of recombinant Chinese hamster ovary (CHO) cells in batch culture. Bromodeoxyuridine was used at 39°C to select mutagenized CHO‐K1 cells, which resulted in the isolation of 31 temperature‐sensitive mutants that were growth inhibited at 39°C. Two of these mutants were successfully transfected with the gene for tissue inhibitor of metalloproteinases (TIMP) using glutamine synthetase amplification, and a permanent recombinant cell line established (5G1‐B1) that maintains the ts phenotype.Continuous exposure to the nonpermissive temperature (npt) of 39°C led to a rapid decline in cell viability. However, a temperature regime using alternating incubations at 34°C and 39°C arrested the 5G1‐B1 cells while retaining a high cell viability for up to 170 h in culture. The specific production rate of the growth‐arrested cells was 3–4 times that of control cultures maintained at a constant 34°C over the crucial 72–130‐h period of culture, which resulted in a 35% increase in the maximum product yield. Glucose uptake and lactate production both decreased in arrested cells. Flow cytometric analysis indicated that 5G1‐B1 cells arrested in the G1 or G0 phase of the cell cycle, and no major structural damage was caused to these cells by the alternating temperature regime.These results demonstrate that growth‐arrested ts CHO cells have increased productivity compared to growing cultures and maintain viability for longer periods. The system offers the prospect of enhancing the productivity of recombinant mammalian cells grown in simple batch fermentors. © 1993 John Wiley & Sons, Inc.