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  • Inorganic Chemistry  (693)
  • Biochemistry and Biotechnology  (603)
  • 1995-1999
  • 1990-1994  (1,296)
  • 1950-1954
  • 1930-1934
  • 1990  (1,296)
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  • 1995-1999
  • 1990-1994  (1,296)
  • 1950-1954
  • 1930-1934
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  • 1
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A thermodynamic model was used in this study to predict the adhesion of Phanerochaete chrysosporium INA-12 as conidiospores or mycelium to various solid carriers. Theoretical predictions were closely reflected by experimental results. Amount of immobilized mycelium was higher for hydrophobic (polypropylene and polyurethane) than for hydrophilic carrier (stainless steel and grey). Lignin peroxidase production was stimulated in the same way. However, better results were obtained with polyurethane than with polypropylene and with grey than with stainless steel. These results were attributed to roughness effects of solid surfaces. Surface morphology characterization showed that the surface roughness parameter RA was higher for polyurethane and grey as compared to polypropylene and stainless steel, respectively. On the other hand, polyurethane is not simply rugous; it has an intraparticle porosity as well as a higher total surface area as compared to polypropylene.
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  • 2
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    Biotechnology and Bioengineering 35 (1990), S. 630-645 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Simultaneous wet milling and enzymatic hydrolysis of newsprint in a ball mill reactor (BMR) and in an attrition mill reactor (AMR) were studied. The effect of milling on the enzyme deactivation was also investigated. Variables studied included milling media size, material, and the presence or absence of an air-liquid interface in the reactor. At a reaction temperature of 45°C and a pH of 4.5 and in the presence of an air-liquid interface, the amount of reducing sugars produced by hydrolysis using a BMR was typically 5 to 15%less than the amount produced using an AMR. Without an air-liquid interface there was no significant difference. The effect of an air-;liquid interface on enzyme deactivation without any substrate was also studied. Enzyme activity after six hours of milling ranged from 1 to 85% ofthe starting activity. In both the AMR and the BMR, the extent of enzyme deactivation was 20 to 50% greater with an air-liquid interface present than with the air-liquid interface absent.
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  • 3
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    Biotechnology and Bioengineering 35 (1990), S. 691-701 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Predictions may be made for the influence of solvent choice on the equilibrium position of biocatalyzed reactions, based on data for the liquid-liquid distribution of the reactants. The most reliable predictions are probably for dilute systems, based on partition coefficients or correlations derived from them. The effective equilibrium constant for esterification reactions is predicted to alter by more than four orders of magnitude on changing between different water-immiscible solvents. The equilibrium constant correlates well with the solubility of water in the solvent, and is most favorable for synthesis in the least polar solvents (aliphatic hydrocarbons). Similar effects seem to apply for other reactions, including oxidation of alcohols and hydrolysis of chlorides. Predictions can be made for nondilute systems using the UNIFAC system of group contributions, but the reliability of these is more questionable.
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  • 4
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    Biotechnology and Bioengineering 35 (1990), S. 702-711 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The scaleup of the technique of plant cell surface immobilization was performed successfully in specifically designed laboratory size bioreactors. The immobilizing matrix was formed into a vertically wound spiral providing for a high immobilizing area-to-volume ratio (0.8-1.2 cm-1). A modified airlift and a mechanically stirred vessel delivered a best bioreactor performance characterized by low biomass frothing and highly efficient plant cell attachment and retention (≥96%). The growth of Catharanthus roseus cells investigated in these bioreactors was found not to be mass transfer limited. It required mild mixing and aeration levels (kLa ∼ 10-15 h-1). The biomass formation pattern of surface immobilized plant cells generally exhibited a linear growth phase followed by a stationary phase characterized by the presence of residual carbohydrates in the medium, contrary to suspension cultures. This behavior was found to depend on the plant cell type and/or line cultured, as well as on the inoculum age. The space restriction and unidirectional growth of the SIPC biofilm combined with the limited availability of essential intracellular nutrients rapidly accumulated from the medium by the stationary phase inoculated plant cells all likely contributed to the culture behavior.
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  • 5
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    Biotechnology and Bioengineering 35 (1990), S. 751-751 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 6
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    Biotechnology and Bioengineering 35 (1990), S. 820-830 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new kinetic model is developed which provides a simple quantitative description of the growth of mycelial organisms. The main concept of this model is that the fungal hypha can be regarded as a form of self-extending tubular reactor. Nutrients are absorbed along the length of the “reactor,” converted into cell envelope precursors and transported forward to the growth tip where they become consumed in the process of extension; Michaelis-Menten kinetics are assumed to operate at this point. The model is capable of predicting changes in the rates of hyphal extension, branching, and fragmentation during mycelial growth in submerged culture and consequently variations in the hyphal growth unit. Simulation studies using the model are presented and the results are evaluated with reference to data from an industrial-type fed-batch penicillin fermentation.
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  • 7
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    Biotechnology and Bioengineering 35 (1990) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 8
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    Biotechnology and Bioengineering 35 (1990), S. 850-851 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 9
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    Biotechnology and Bioengineering 35 (1990), S. 897-906 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model for the effects of NH4Cl addition to the growth medium on hybridoma cell intracellular pH (pHi) is presented which includes a detailed description of the properties of the Na+/H+ exchanger. The model is used to calculate the steady-state value of pHi as a function of the extracellular NH4Cl concentration, employing parameter values taken from the literature except for the cell permeability to NH4+ This parameter value, estimated to be 3.5 × 10-7 cm/s, is obtained by fitting simulation results to experimental data obtained previously for the steady-state value of pHi in the presence of 10mM NH4Cl. The model simulates well the major features of previously observed experimental pHi changes following NH4Cl addition: the transient characteristics, dependence on the NH4Cl concentration, and lack of dependence on the external pH value. The effect of parameter variations on model results is also investigated in additional simulations. The general modeling framework employed here should be useful for other types of mammalian cells.
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  • 10
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    Biotechnology and Bioengineering 35 (1990), S. 948-950 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 11
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    Biotechnology and Bioengineering 35 (1990), S. 955-965 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel process has been developed which uses reversed micelles to isolate denatured protein molecules from each other and allows them to refold individually. These reversed micelles are aqueous phase droplets stabilized by the surfactant AOT and suspended in isooctane. By adjusting conditions such that only one protein molecule is present per reversed micelle, it was possible to achieve independent folding without encountering the problem of aggregation due to interactions with neighboring molecules. The feasibility of this process was demonstrated using bovine pancreatic ribonuclease A as a model system. It was shown that denatured and reduced ribonuclease can be transferred from a buffered solution containing guanidine hydrochloride into reversed micelles to a greater extent than native enzyme under the same conditions. The denaturant concentration can then be significantly reduced in the reversed micellar phase, while retaining most of the protein, by means of extractive contacting stages with a denaturant-free aqueous solution. Denatured and reduced ribonuclease will subsequently recover full activity inside reversed micelles within 24 h upon addition of a mixture of reduced and oxidized glutathione to reoxidize disulfide bonds. Extraction of this refolded enzyme from reversed micelles back into aqueous solution can be accomplished by contacting the reversed micelle phase with a high ionic strength (1.0M KCl) aqueous solution containing ethyl acetate.
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  • 12
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    Biotechnology and Bioengineering 35 (1990), S. 1024-1033 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model for an ideal chemostat in which one microbial population feeds on another and where Monod's model is used for the specific growth rates of both populations predicts a less stable behavior for the system than the one observed experimentally. Various factors have been proposed as being the reason for the increased stability of such systems. In this work, the effect of spatial heterogeneity on the dynamics of the microbial feeding interaction is studied. It is concluded that spatial heterogeneity has a stabilizing effect on the system. This effect combined with other factors could be the reason for the increased stability observed in systems where a microbial feeding interaction occurs.
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  • 13
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    Biotechnology and Bioengineering 35 (1990) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 14
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    Biotechnology and Bioengineering 35 (1990), S. 8-14 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A fast inferential, multivariable adaptive optimization algorithm based on a fast responding off-gas data, the carbon dioxide evolution rate (CER), has been developed and applied to a continuous baker's yeast culture to maximize the cellular productivity in simulation and experimental studies. In the simulation study the process was optimized based on CER measurements using readily available steady-state data on the ratio between the cellular productivity and the CER. It was shown that the algorithm is two to three times faster than the algorithm based on cell mass concentration measurements. In the experimental study the CER was maximized without any information on the relationship between the cellular productivity and the CER. It took about 40 h for the process to converge, while about 80 h was required when the optimization was based on cell mass measurements. The attained steady state was found to be different but fairly close to that obtained with cell measurements. Briefly discussed is a switching to the cell-mass-based algorithm at the final stage of the optimization to overcome a potential inaccuracy.
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  • 15
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    Biotechnology and Bioengineering 35 (1990), S. 540-546 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple model for prediction of liquid velocity in external-loop airlift bioreactors has been developed. Theoretical correlations for friction factor of gas-non-Newtonian two-phase flows and for liquid velocity in the riser were derived using the concept of an eddy diffusivity. The predictions of the proposed model were compared with the available experimental data for the friction factor and the liquid velocity in the riser of external-loop airlift contactors. Satisfactory agreement was obtained.
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  • 16
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    Biotechnology and Bioengineering 35 (1990), S. 547-558 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A kinetic model for anaerobic digestion of insoluble solid-state substrates was developed. Rate equations for cell growth and substrate consumption were derived based on the assumption that the microorganisms assimilate the substrate mainly at the point of contact where they grow. The model emphasizes effects of substrate particle size, organic loading, and cell concentration on the rates of cell growth and substrate utilization. Batch digestion of a stearic acid emulsion with a mean particle size of 2.0 μm and a biological sludge was conducted at 30 and 37°C to verify the proposed model. Agreement between the experimental and calculated results indicated the validity of the model for describing the microbial degradation of insoluble solid-state substrates. Further examinationof the model revealed that with low cell substrate affinity or at low cell concentration, it coincided with a Michaelis-Menten type kinetics in which the effect of particle size was taken into consideration.
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  • 17
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    Biotechnology and Bioengineering 35 (1990), S. 598-608 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experiments have been carried out on a laboratory-scale adsorber packed with 3.3 kg cornmeal. The effects of varying particle size, feed concentrations and flow rates, and regeneration conditions on the product quality and breakthrough times were investigated. It was found that 85% (w/w) vapor feed gave the best product, and product yields of up to 99.8% (w/w) alcohol were obtained at energy costs as low as 4.0 MJ kg-1. It is suggested how this figure could be improved.
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  • 18
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 19
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    Biotechnology and Bioengineering 35 (1990), S. 483-491 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Gas holdups and volumetric mass transfer coefficients were measured in a concentric tube airlift reactor designed for the microbial desulfurization of coal. The solutions studied were comprised of an acidified basal salts solution containing thirteen different weight percentages (0 to 40) of coal (74 μm Ohio #1) at three different temperatures (30, 50, and 72°C). Gas holdup ∊G decreased with solids loading for the entire range studied. An enhancement in the volumetric mass transfer coefficient KLa with respect to that in pure solution was observed from zero to approximately 5 wt % (solids volume fraction ∊s = 0.035), the maximum enhancement occurring at approximately 2 wt % (∊s = 0.014). At higher solids fractions, the mass transfer coefficient decreased with further solids additions. Gas holdups and the mass transfer coefficients increased with temperature over the studied range. The KLa and ∊G were correlated to three process variables separately and the separate correlations combined to yield generalized correlations for the mass transfer coefficient and gas holdup for this system. The correlations may be used for design, operation, and ost estimation of such systems.
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  • 20
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    Biotechnology and Bioengineering 35 (1990), S. 518-524 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Water-insoluble proteases were prepared by immobilizing papain and chymotrypsin onto the surface of polyacrolein microspheres with and without oligoglycines as spacer. The activity of immobilized proteases was found to be still high toward small ester substrates, but very low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases without spacer decreased gradually with the decreasing surface concentration of the immobilized proteases on the microspheres. On the contrary, the immobilized proteases with oligoglycine spacers gave an almost constant activity for the substrate hydrolysis within the surface concentration region studied and gave a much higher relative activity than those without any spacer. With the longer spacer, the immobilized enzymes showed a higher activity toward casein hydrolysis, whereas there was an optimum length for the spacer when hydrolysis was carried out toward the low-molecular-weight substrate. The thermal stability of the immobilized proteases was higher than that of the respective native proteases. The initial enzymatic activity of the immobilized proteases maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.
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  • 21
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    Biotechnology and Bioengineering 35 (1990) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 22
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    Biotechnology and Bioengineering 35 (1990), S. 809-819 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model describing growth of an outdoor algal (Spir-ulina platensis)culture was developed. The model can simulate biomass production, pH, growth rate, oxygen evolution, and CO2 fixation rate. It was calibrated and validated against experimental data obtained by a novel automatic data logger/controller instrumentation which can number most vital parameters of the culture including on line estimation of oxygen production rate (OPR). The importance of understanding light distribution through the pond and its effects on the photosynthesis and respiration processes are emphasized. A maximum yield of about 38g day-1 m-2 under optimal conditions is predicted. The present model can also be a useful tool for optimization of algal mass production sites.
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  • 23
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    Biotechnology and Bioengineering 35 (1990), S. 586-597 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two theoretical approaches were examined to quantify observed experimental oxygen transfer enhancements in aqueous/perfluorocarbon (pfc) fermentation systems. Steady-state macroscopic balance results indicated that enhancements in the presence of reaction were a function of the gas bubble-to-droplet diameter ratio for pfc-in-water dispersions, but primarily a function of only the droplet diameter for water-in-pfc dispersions. The problem of unsteady-state diffusion into a semi-infinite composite medium was employed to predict enhancements in the absence of reaction based on the relative permeabilities of the perfluorocarbon and water phases. Since potential enhancements exceeded actual enhancements in the presence of an oxygen consuming reaction for water-in-pfc dispersions, the use of less expensive, lower oxygenpermeable oil phases is recommended in practice.
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  • 24
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inactivation of α-chymotrypsin in aqueous solutions of alcohols and diols proceeds both reversibly and irreversibly. Reversible loss of the specific enzyme activity results from conformational changes (unfolding) of the enzyme detected by fluorescence spectroscopy. Multipoint covalent attachment to the matrix of polyacryl-amide gel by copolymerization method stabilizes α-chymotrypsin from denaturation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. Immobilization protects the enzyme also from irreversible inactivation by organic solvents resulting from bimolecular aggregation and autolysis.
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  • 25
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    Biotechnology and Bioengineering 36 (1990), S. 865-878 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant 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.
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  • 26
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    Biotechnology and Bioengineering 36 (1990), S. 911-920 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The role of the plasma membrane fluidity (PMF) on the shear sensitivity of HB-32 hybridomas to laminar fluid shear was investigated. Steady-state fluorescence anisotropy (rs) of the cationic fluorescent probe 1-[4-(trimethylamino) phenyl]-6-phenylhexa-1,3,5-triene, was used to evaluate the PMF of whole hybridoma cells. The PMF was manipulated by the addition of the anesthetic benzyl alcohol, by temperature changes and by cholesterol enrichment. The effect of these PMF modifying procedures on the shear sensitivity of HB-32 was assessed by exposing the cells to defined levels of laminar shear stress in a Couette flow device. Conditions that resulted in lower rs values (indicating higher PMF) yielded a more fragile cell. Batch cultivations supplemented with the shear protective agent Pluronic® F-68 showed higher values of rs compared to control experiments during various growth phases, suggesting that the protective mechanism of Pluronic F-68 relies on its ability to decrease the PMF through direct interaction with the plasma membrane. The protective mechanism of serum against turbulent fluid shear is also discussed from analysis of growth and death kinetics of agitated and static cultures at increasing serum levels. The results of this study show that the fluid state of the plasma membrane is important in determining the integrity of hybridomas when exposed to lethal shear levels. It is concluded that increasing membrane fluidity correlates with increasing shear sensitivity.
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  • 27
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    Biotechnology and Bioengineering 36 (1990), S. 965-969 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 28
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Production of various extracellular enzymes (the β-lactamases from Streptomyces albus G, Streptomyces cacaoi, Actinomadura R39, and the DD-carboxypeptidase from Streptomyces R61) by genetically engineered Streptomyces lividans TK24 in Lennox broth medium reached a maximum after 36 to 48 h. Subsequently, the enzyme activity drastically decreased probably due to an increased pH value and the production of an inactivator by Streptomyces lividans. Protease activity did not seem to play a major role. The increased pH and inactivator synthesis are related to amino acid catabolism and generally result in cellularlysis. The use of a medium where the catabolism of amino acids was made less likely by the presence of glucose and NH4Cl and by buffering at pH 7.4 considerably inproved the yield. Furthermore, the water activity of the medium seemed to be an important parameter for the production of extracellular proteins by genetically engineered Streptomyces. Better production was observed when the water activity was decreased to 0.96-0.98 by addition of sucrose.Under those conditions, the concentration of extracellular enzyme reached about 0.3 g (1 g in the best case)/L of culture supernantant.
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  • 29
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    Biotechnology and Bioengineering 35 (1990), S. 771-780 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Regulated secretion, i.e., the ability of certain specialized animal cells to store secretory proteins intracellularly and release them upon stimulation, may be used to realize production schemes that facilitate downstream processing of protein products. Mouse AtT-20 cells expressing recombinant human insulin and human growth hormone (hGH) were found to secrete the proteins at relatively low and constant rates when exposed to media with no secretion agonists: basal rates were 1.0-1.6 μU insulin-reiated peptides and 0.38 ng hGH/105 cells-h. When induced with 8 brorno-cyclic AMP (BrcAMP), the cells secreted recombinant proteins at initial rates 3.5-9-fold higher. A cycling secretion experiment was conducted with the insulin-producing cells in which the cells were exposed alternately to complete growth medium and to secretion medium with BrcAMP. During the first three cycles, the cells secreted immunoreactive insulin at the foregoing high induced rates when they were exposed to BrcAMP. The cells then started to detach from the culture surface, leading to a reduction of BrcAMP-induced secretion. Operational modifications that may result in improved system performance are discussed.
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  • 30
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Somatic embryos in liquid culture can serve as a mass cloning system in a plant propagation program. A quantitative formulation of embryo development obtained from cell suspension cultures is used to develop a segregated kinetic model. The model is based on standard classification schemes as previously developed by plant physiologists. Dependent variables include carbohydrate concentrations (sucrose, fructose, and glucose) and biomass apportioned among the inoculum (free single cells, cell clusters), normal developmental stages, and aberrant cell and embryo types. Good agreement between the model and experimental results is indicated and allows for a rigorous approach to media optimization and reactor scaleup for embryo formation.
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  • 31
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    Biotechnology and Bioengineering 35 (1990), S. 802-808 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model was developed and tested to simulate the generation and transfer of heat in solid substrate fermentation (SSF). The experimental studies were realized in a 1-L static bioreactor packed with cassava wet meal and inoculated with Aspergillus niger. A simplified pseudohomogeneous monodimensional dynamic model was used for the energy balance. Kinetic equations taking into account biomass formation (logistic), sugar consumption (with maintenance), and carbon dioxide formation were used. Model verification was achieved by comparison of calculated and experimental temperatures. Heat transfer was evaluated by the estimation of Biot and Peclet heat dimensionless numbers 5-10 and 2550-2750, respectively. It was shown that conduction through the fermentation fixed bed was the main heat transfer resistance. This model intends to reach a better understanding of transport phenomena in SSF, a fact which could be used to evaluate various alternatives for temperature control of SSF, i.e., changing air flow rates and increasing water content. Dimensionless numbers could be used as scale-up criteria of large fermentors, since in those ratios are described the operating conditions, geometry, and size of the bioreactor. It could lead to improved solid reactor systems. The model can be used as a basis for automatic control of SSF for the production of valuable metabolites in static fermentors.
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  • 32
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    Biotechnology and Bioengineering 35 (1990), S. 831-836 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Complex coacervation is a phenomenon of phase separation that may occur in a solution of positively and negatively charged polyions. The resulting two phases are distinguished by the total concentration of both polyions, with the concentrated phase often containing vesicular structures composed of the two polyelectrolytes. We have used this phenomenon in an attempt to-prepare a hemoglobin-based red blood cell analog. Hemoglobin-containing coacervate vesicles have been prepared from gelatin A and the polyanionic carbohydrates acacia, pectin, or dextranstilfate. Hemoglobin seems to be anchored into the vesicle walls through interaction of its polyanion binding site with the negatively charged residues on the carbohydrates. Oxygen binding by the immobilized HbA is reversible and cooperative, with p50 values at 20°C of 2.8, 6, and 24 mm Hg for the acacia- (pH 7.5), pectin- (pH 6.6), and dextransulfate-(pH 6.6) derived coacervates. Kinetic studies on CO binding show that the rate of CO uptake by the coacervates (t½ = 13-27 ms at 0.5 mM CO) is similar to that of human erythrocytes.The HbA-containing coacervates slowly dissolve in isotonic salt solutions (145 mM NaCl, pH 7.4), but they can be stabilized by treatment with glutaraldehyde. Oxygen binding by HbA incorporated into the stabilized coacervates derived from dextran sulfate is very similar to oxy gen binding by human red blood cells: p50 = 26 mm Hg and n = 1.89 at 37°C in isotonic salt. These results show how a novel approach, based on an old concept, has led to the preparation of immobilized HbA, with functional properties similar to those of intraerythrocytic HbA.
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  • 33
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    Biotechnology and Bioengineering 35 (1990), S. 861-869 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An extractive acetonobutylic fermentation process is developed by integrating bioproduction, Ultrafiltration, and distillation, providing simultaneous retention of biomass, selective removal of inhibitors from the permeate, as well as separation and purification of acetone-butanol-ethanol solvents. Successive batch fermentations were performed with normal pressure distillation (98°C), which permitted prolonging and enhancing (by a factor of 3) solvent production, with very few volume exchanges of medium (average dilution rate ws 0.002 h-1), and recovering on-line concentrated solvents. Different operating conditions were also tested in order to study the presence of extracellular autolytic enzymes as inhibition factors: It was shown that, (1) extracellular autolytic activity remains low during the larger part of fermentations, even without enzyme-inactivating thermotreatment in the distillation boiler, and (2) high-temperature distillation causes deleterious effects to the culture medium for long duration treatments. Progressive improvements of the process were achieved, first, by managing continuous runs, providing a minimum renewal of the culture medium and, mainly, by decreasing temperature and pressure of distilation. Solvent productivity then reached 2.6 g/L h for a 0.036 h-1 average dilution rate, corresponding to a feed concentration of 156 g/L glucose actually consumed.
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  • 34
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    Biotechnology and Bioengineering 35 (1990), S. 907-920 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A general model for aerobic yeast growth in batch culture is presented. It is based on the concept that the aerobic metabolism of all yeasts is determined by the relative sizes of the transport rate of sugar into the cell and the transport rate of respiratory intermediates into the mitochondrion. If the rate of sugar uptake rate exceeds the rate of transport of respiratory intermediates into the mitochondrion (as in Saccharomyces cerevisiae, S. uvarum, and S. pombe), the metabolism exhibits the features of ethanol excretion and limited specific oxygen uptake rate. If the rate of transport of respiratory intermediates into the mitochondrion is of the same order as the transport of sugar into the cell (as in Candida utilis), the metabolism is characterized by little or no ethanol excretion and a much higher specific oxygen uptake rate. Batch data from an extensive range of yeast and carbon sources is used to illustrate the use of this model. The ability of this model to fit such an extensive range of experimental data suggests that it can be used as a generalized model for aerobic yeast growth.
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  • 35
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    Biotechnology and Bioengineering 35 (1990), S. 1042-1050 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To study the influence of ammonium on an antibiotic cultivation, mass transfer measurements of ammonium through microporous hydrophobic membranes using different stripping methods were carried out and compared. The higher overall mass transfer coefficients for ammonium were obtained with an acid stripping solution compared to water, vacuum, or sweeping air. A hollow fiber module for in situ removal of ammonium during cultivation was designed and operated in an external bypass to a 10-L fermentor. Compared to a control fermentation, the cell dry mass could be increased 2.6 times and the antibiotic concentration 8 times, if the in situ ammonium removal was in operation.
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  • 36
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    Biotechnology and Bioengineering 35 (1990), S. 1067-1077 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of NH4Cl addition on batch hybridoma cell growth at different external pH values (pHe) were investigated in a bioreactor at constant pH and dissolved oxygen concentration. In agreement with measurements in flasks, changes in pHe over the range 6.8-7.6 had minor effects on growth. Addition of 3 mM NH4Cl had little effect on cell growth while 10 mM NH4Cl caused a substantial growth inhibition, Measurements of the effects of pHe and NH4Cl concentration on cell metabolism gave similar results for cells grown in flasks in an incubator and in the bioreactor. As pHe decreases, the integral cell yield on glucose increases. There is a correlation between the effects of pHe on glycolysis and previous measurements of its effects on intracellular pH (pHi). Increases in NH4Cl concentration were previously determined to decrease pHi and are shown here to decrease the integral cell yield on glucose. At all pHe values in the absence of NH4Cl, glutamine is depleted at the time the maximum cell density is reached. Both pHe decreases and NH4Cl concentration increases lead to decreases in the integral cell yield on glutamine. Changes in pHe and in the NH4Cl concentration that cause growth inhibition have no effect on the specific antibody production rate for cells grown in flasks in an incubator or in the bioreactor. Changes in the NH4Cl concentration have no effect on the quality of the antibody produced, to a first level of characterization.
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  • 37
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mass transfers of O2, CO2, and water vapor are among the key processes in the aerobic, autotrophic cultivation of moderate and extreme thermophiles. The dynamics and kinetics of these processes are, in addition to the obvious microbial kinetics, of crucial importance for the industrial desulfurization of high-pyritic coal by such thermophiles. To evaluate the role of the temperature on the gas mass transfer, kLa measurements have been used to supplement the existing published data. Oxygen mass transfer from gas (air) to liquid (5 mM H2SO4 in water) phase as a function of the temperature has been studied in a laboratory-scale fermentor. At 15, 30, 45, and 70°C, (kLa)o values (for oxygen) were determined under three different energy input conditions by the dynamic gassing in/out method. The (kLa)o was shown to increase under these conditions with increasing temperature, and straight lines were obtained when the logarithm of (kLa)o was plotted against the temperature. By multiplying the equilibrium concentration of O2 in water with (kLa)o maximal, O2 transfer capacities were calculated. It appeared that in finite of a decreased solubility of O2 at elevated temperature in mechanically mixed fermentors the calculated transfer capacities showed only minor changes for the range between 15 and 70°C. However, in an air-mixed fermentor the transfer capacity of O2 decreased slowly but steadily.Carbon dioxide mass transfer was predicted by calculations on the basis of the data for oxygen transfer. The maximal CO2 transfer capacity, calculated as the product of the equilibrium CO2 concentration times (kLa)c, decreased slowly as the temperature increased over the range 15-70°C under all three energy input conditions. Subsequent process design calculations showed that for aerobic, autotrophic cultures, CO2 limitation is more likely to occur than O2 limitation.
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  • 38
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    Biotechnology and Bioengineering 35 (1990), S. 1161-1163 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 39
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    Biotechnology and Bioengineering 35 (1990), S. 1169-1173 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 40
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    Biotechnology and Bioengineering 36 (1990), S. 275-287 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inhibition of Trichoderma reesei cellulase by sugars (glucose, δ-gluconolactone, and cellobiose) and solvents (ethanol, butanol, and acetone) was studied using cellulose azure. Glucose, cellobiose, ethanol, and butanol were noncompetitive inhibitors, δ-gluconolactone was a mixed inhibitor, and acetone was a noncompetitive activator. Converting cellobiose to glucose reduces the effective inhibitor binding constant by 6 times and converting cellobiose to ethanol reduces it by 16 times.
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  • 41
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    Biotechnology and Bioengineering 36 (1990), S. 617-622 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lipase from Pseudomonas fluorescens biotype I was immobilized by adsorption of anion exchange resin using glutaraldehyde to enhance the adsorption. The activity yield of the immobilized lipase was very low (below 1%) when lipase activity was measured using emulsion substrate. The activity yield was 10-70% when lipase activity was measured using non-emulsion substrate. Countercurrent reactors for hydrolysis of oil using non-emulsion substrate were studied. A fluidized bed reactor was found to be superior to a fixed bed one since in a fixed bed reactor the separation rate of the two layers was slow and the flow rate of the reactor had to be slower than the separation rate. A fluidized bed reactor system equipped with settling compartments and stirring compartments was devised. Continuous lipolysis at 60 °C and continuous separation of oily product and water soluble product were performed. After continuous operation for more than 3 months, 70% of the initial activity of the immobilized lipase was observed at the end of the reaction.
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  • 42
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    Biotechnology and Bioengineering 36 (1990), S. 636-641 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel viscous sensor utilizing AT-cut quartz crystal to monitor the viscosity of fermentation broth was developed. The sensor system was constructed from the piezoelectric quartz crystal fixed to the cell, exposing only one side of the quartz crystal electrode, an oscillating circuit, a peak level meter, and a personal computer. In order to investigate the characteristics of the sensor system, a sensor signal relating to the resonant resistance of the quartz crystal was measured using dextran solutions with different molecular weights. The linear relationship was obtained between the sensor signal and the (ρη)1/2 of the liquid, where ρ and η are the density and viscosity, respectively. The sensor signal was dependent not only on the viscosity of the liquid but also on the molecular weight of dextran, because dextran solution shows a non-Newtonian property. The sensor system was applied for the on-line monitoring of the viscosity in dextran fermentation. A good correlation was observed between the sensor signal and the viscosity value measured with a rotational viscometer for the fermentation broth. Little bubbling effect and agitation of the sensor signal were observed, showing that this system can be utilized for viscosity monitoring in a bioprocess.
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  • 43
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    Biotechnology and Bioengineering 36 (1990), S. 377-386 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In Part I of this series,1 we derived a model and made simulations for a multistage fluidized bed reactor (MFBR). It was concluded that the MFBR can be an attractive alternative for a fixed bed reactor when operated with a deactivating biocatalyst. In Part II of this series, the design of a laboratory-scale MFBR and its evaluation to investigate the practical feasibility of this reactor type, will be described. Experiments with a duration as long as 10 days were carried out successfully using immobilized glucose isomerase as a model reaction system. The results predicted by the model are in good agreement with the measured glucose concentration and biocatalyst activity gradients, indicating perfect mixing of the particles in the reactor compartments.The diameters of the biocatalyst particles used in the experiments showed a large spread, with the largest being 1.7 times the smallest. Therefore, an additional check was carried out, to make sure that the particles were not segregating according to size. Particles withdrawn from the reactor compartments were investigated using an image analyzer. Histograms of particle size distribution do not indicate segregation and it is concluded that the particles used have been mixed completely within the compartments. As a result, transport of biocatalyst is nearly plug flow.
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  • 44
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    Biotechnology and Bioengineering 36 (1990), S. 402-410 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gram-positive bacteria, Acinetobacter calcoaceticus, is capable of accumulating biopolymer in the carrier matrix of an immobilized cell system. Several possible mechanisms for the biopolymer accumulation are evaluated. It appears that direct solid surface polymer adsorption and polymer diffusion limitation within the pore space are minor factors in biopolymer accumulation. Calculations demonstrate that the cell bound polymer to dry cell weight ratio is much higher for immobilized cells than for free cells. The higher cell-bound polymer to dry-cell-weight ratio for immobilized cells as well as the accumulation of the immobilized cells in the Celite matrix are believed to be the main factors for biopolymer accumulation in the Carrier matrix. Further studies reveal that the cell-bound polymer to dry-cell-weight ratio is strongly affected by shear forces. At zero shear stress, such as would be present in the carrier matrix, cell bound polymer to dry cell weight ratio can be as high as 1.6. As the shear stress increases, this ratio decreases. When shear stress increases above 5 dyn/cm2, a level equivalent to the shear experienced by free cells in a stirred tank fermentation, cell-bound polymer decreases to less than 20% of dry cell weight. A macroscopic model is developed to describe the effect of shear stress on the cell-bound polymer to dry-cell-weight ratio.
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  • 45
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    Biotechnology and Bioengineering 36 (1990), S. 437-445 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The problem of dynamically modeling a chemostat is addressed. Using the results of continuous culture experiments for the growth of a strain of Saccharomyces cerevisiae on a glucose-limited medium, a general approach to developing dynamic models is discussed. The approach to develop and verify the model involves three different types of experiments: steady-state, dynamic step response, and feedback identification.
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  • 46
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    Biotechnology and Bioengineering 36 (1990), S. 493-505 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: As part of the process optimization of a two-stage continuous culture system, the effect of growth rate μ2app on the performance of the second stage (production stage) was studied in a recombinant Escherichia coli K12 (ΔH1Δtrp/pPLc23trpA1). Important parameters considered were specific gene expression rate, plasmid content, and plasmid stability, all of which were closely related to the cell growth rate and the production rate of the cloned gene product (trpα). When operating conditions were maintained constant (T1 = 35°C, D1 = 0.9 h-1, T2 = 40°C, and D2 = 0.7 h-1) and μ2app was varied, plasmid content in the second stage showed its maximum at μ2app = 0.4 h-1 and decreased thereafter. Specific gene expression rate linearly increased with increasing μ2app, while plasmid stability decreased. Optimum cell growth rate giving the maximum value in overall productivity was observed at around μ2app = 0.4 h-1. The contribution or role of the three parameters, specific gene expression rate, plasmid content, and plasmid stability in exhibiting the maximum productivity at the optimal μ2app is discussed.
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  • 47
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    Biotechnology and Bioengineering 36 (1990), S. 539-545 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 48
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    Biotechnology and Bioengineering 36 (1990), S. 879-886 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A hollow-fiber membrane reactor was designed and constructed to allow perfusion of entrapped, dense Escherichia coli cells with nutrient medium during examination of cell metabolism using nuclear magnetic resonance (NMR) spectroscopy. Phosphorus-31 NMR spectra of the perfused cells included peaks for nucleoside di- and triphosphates, sugar phosphates, and pH-sensitive peaks for inorganic phosphate. The observed intensity of the lumenal inorganic phosphate peak was found to depend on flow rate, ruling out the use of this peak as a concentration reference. Absolute intracellular pH values obtained from NMR measurements were found to be accurate to 0.2 pH units due to uncertainties in intracellular ionic concentrations. Relative pH values, however, were found to be sensitive to cell energetic status. The response of E. coli intracellular pH following a shift to carbon starvation medium was monitored with a resolution of 3 min. Use of a hollow-fiber reactor for cell containment and perfusion during NMR spectroscopy enables metabolic experiments of longer duration and of greater variety than is possible using standard, nonperfused sample tubes.
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  • 49
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Thiosphaera pantotropha is capable of aerobic heterotrophic nitrification and both aerobic and anaerobic denitrification. These phenomena have been studied in acetate-limited aerobic and anaerobic continuous cultures supplied with ammonia and nitrate. The internal reaction rates were defined, based on biochemical knowledge. The observable external conversion rates are related through a linear equation on the basis of the specified internal reaction rates. The linear equation is a Pirt relation extended for microbial systems with multiple electron donors (acetate and ammonia) and electron acceptors (oxygen and nitrate). The coefficients in this equation were estimated from the continuous culture measurements, and are composed of parameters involved in ATP production and consumption by the microorganism. It is shown that with realistic values for these parameters, the metabolically structured model describes the aerobic as well as the anaerobic experiments.
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  • 50
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    Biotechnology and Bioengineering 36 (1990), S. 960-964 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 51
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mathematical 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.
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  • 52
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    Biotechnology and Bioengineering 36 (1990), S. 1070-1082 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method is presented for the estimation of the standard Gibbs energies of formation of biochemical compounds (and hence the Gibbs energies and equilibrium constants of biochemical reactions) from the contributions of groups. The method employs a large set of groups and special corrections. The contributions were estimated via multiple linear regression, using screened and weighted literature data. For most of the data employed, the error is less than 2 kcal/mol. The method provides a useful first approximation to Gibbs energies and equilibrium constants in biochemical systems.
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  • 53
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    Biotechnology and Bioengineering 36 (1990), S. 1090-1096 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cibacron-blue-dextran conjugates have been produced with a range of ligand loadings using a dextran preparation of average molecular weight of 2 × 106. The equilibrium binding capacity of these ligand conjugates for lysozyme was determined using a gel permeation procedure to separate bound from free protein. The results obtained give clear evidence for at least two types of binding showing a marked difference in affinity. For the higher-affinity interaction the half-saturation constant decreases with increasing ligand loading. The number of dye molecules participating in binding is proportional to loading up to 154 mol dye/mol dextran but is reduced at the highest loading used (315 mol dye/mol dextran). This may be due to steric interference or to dye stacking reducing the number of dye molecules available for binding.
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  • 54
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    Biotechnology and Bioengineering 36 (1990), S. 1110-1118 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: n-Hexadecane was added to fermentation media to increase the medium oxygen solubilities, thus enhancing oxygen transfer rates in penicillin fermentations. For shake flask fermentations, cells were found to grow faster in the flasks with n-hexadecane than those without. The addition of n-hexadecane to penicillin fermentations was shown to significantly increase cell growth and penicillin production and reduce formation of mycelial pellets. The result was attributed to the enhancement of oxygen transfer in mycelial fermentations due to the higher oxygen solubilities of fermentation media achieved by adding n-hexadecane.
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  • 55
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    Biotechnology and Bioengineering 36 (1990), S. 1151-1154 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 56
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    Biotechnology and Bioengineering 36 (1990), S. 854-864 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Even when microorganisms are grown in highly agitated fermentors, calculation predicts a mismatch between the microscale of the turbulence (where the smallest eddy is typically 50-300 μm diameter) and the cellular dimensions (1-5 μm). The cell thus spends substantial portions of time in an apparently stagnant eddy, depleted of nutrients. The local fluid microscales were measured in a laboratory fermenter to confirm this. Using S. cerevisiae in continuous culture, it is shown that the local microscales influence cell metabolism dramatically. The issues addressed in this study are thus micromixing and microsegregation of reactants and how they influence cell yield.
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  • 57
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    Biotechnology and Bioengineering 36 (1990), S. 849-853 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To attain both high productivity and efficient recovery of ethanol from broth, a membrane bioreactor consisting of a jar fermentor and a pervaporation system was applied to the direct production of ethanol from uncooked starch with a thermophilic anaerobic bacterium, Clostridium thermohydrosulfuricum. From four types of ethanol-selective membranes tested, microporous polytetrafluoroethylene (PTFE) membrane, the pores of which are impregnated with silicone rubber, was chosen for its large flux, high ethanol selectivity, and high stability. During fed-batch fermentation with pervaporation in the membrane bioreactor, ethanol was continuously extracted and concentrated in two traps with concentrations at 5.6%-6.2% (w/w) in trap 1 (20 °C) and 27%-32% (w/w) in trap 2 (liquid N2), while the ethanol concentration in the broth was maintained at 0.85-0.9% (w/w). Due to the low ethanol concentration in the broth, and the immobilization of bacterial cells by the membrane, the number of viable cells, and, eventually, the ethanol productivity, increased in the membrane bioreactor.
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  • 58
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    Biotechnology and Bioengineering 36 (1990), S. 902-910 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The heterogeneous nature of hollow fiber reactors for cell cultivation requires special considerations for proper design and operation. Downstream concentration of high-molecular-weight proteins has been measured in the shell side of ultrafiltration hollow fiber bioreactors. This distribution resulted from shell-side convective fluxes which caused a concentration polarization of proteins retained by the ultrafiltration membranes (nominal 3 × 104 D cutoff). Measurements of the axial hybridoma cell distribution also revealed a downstream concentration of viable cells during the first month of perfusion operation. This is believed to result from the shell-side convective flow and its influence on the inoculum and high-molecular-weight growth factor distributions. The heterogeneous distribution of cells leads to reduced cell numbers and reactor productivities. The mechanisms responsible for these phenomena have been investigated and their implications in process design and operation are considered. The heterogeneous protein and cell distributions on the shell side of hollow fiber bioreactors have been reduced significantly by periodic alternation of the direction of recycle flow and the reactor antibody productivities have been doubled.
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  • 59
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    Biotechnology and Bioengineering 35 (1990), S. 123-131 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A carrier-mediated counter transport process is proposed to separate and to purify an amino acid produced by microbial fermentation. The case of L-valine permeation through a liquid membrane, constituted by a solution of Aliquat 336 in decanol and supported by a hydrophobic microporous membrane, is reported. A mathematical model was developed to estimate distribution coefficients and permeabilities and to predict the influence of hydrodynamic and pH conditions on supported liquid membrane (SLM) performances. Optimum conditions for the transport and the concentration of valine were achieved with synthetic aqueous valine solutions. Series of experiments on fermentation broths, where molasses and biomass contents were varied, permitted pointing out the role of the broth composition on the kinetics and yields of separation. The selectivity of transport of valine by an Aliquat 336/decanol liquid membrane was about 10 toward molasses dyes, 100 toward glucose, and beyond 1000 toward sucrose. This allowed us to achieve the recovery and one step of purification of the product in a single operation. The stability of the Aliquat 336/decanol liquid membrane was sufficient to ensure a selective transport of valine during a continuous run lasting 18 days.
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  • 60
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    Biotechnology and Bioengineering 35 (1990), S. 138-145 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pirt's maintenance model has been widely accepted for the effects of growth rate and maintenance on growth yield. However, the interpretation of parameters in Pirt's model as biological constants is difficult for energy-sufficient culture growth. In this study, a mechanistic model for the growth energetics of energy-sufficient chemostat cultures is proposed and verified with literature data. In the model, the overutilization of the energy substrate in energy-sufficient culture growth is attributed to the defective regulation of the energy substrate metabolism and energy uncoupling. The model also uses an “energy surplus” concept to collectively represent the effects of energy excessiveness. The proposed model provides a better quantitative understanding of the maximum growth yield and maintenance of energy-sufficient cultures. It also explains the glucose concentration effect reported in the literature.
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  • 61
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    Biotechnology and Bioengineering 35 (1990), S. 160-184 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model for the growth process of the bacterium Bacillus subtilis is described. The model is a highly structured one. The driving motivation for development of the model and explicit accounting of major interactions of metabolic networks in the model is related to our eventual goal that the model will be used in the analysis of complex biological patterns. Bacillus subtilis was chosen in our study due to the interesting sporulation process that these cells undergo in response to adverse environmental conditions including nutrient limitation. Sporulation process in B. subtilis represents a primordial prototype of cellular differentiation in higher cellular systems. Thus a model for the B. subtilis growth process should prove extremely useful for understanding questions of developmental biology. The model is capable of simulating the transition between the exponential and stationary phase of growth in a batch culture. Since during the transition period the growth process and the metabolism become decoupled and many transient processes are taking place, such predictions are a severe test for the validity of any model. A strategy to examine the leading hypothesis on B. subtills sporulation implementing GTP as a component which signals sporulation initiation is described.
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  • 62
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    Biotechnology and Bioengineering 35 (1990), S. 201-206 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The fermentation of glucose by a strain of Saccharomyces cerevisiae was studied in a continuous single-stage process with recycle of the cells via cross-flow micro-filtration membranes. Operating conditions were selected such that the culture was not carbon limited and inhibition by ethanol and cell death were minimized.Steady states were obtained for various biomass bleeding rates, i.e., various specific growth rates. From the experimental data, the stoichiometry of the simultaneous reactions, cell growth, ethanol production and maintenance were established using mass and degree of reduction balance relative to substrates (carbon source and oxygen) and products (biomass, ethanol, carbon dioxide etc.), and the growth parameters, yields, and maintenance cofficients were determined. It was shown that the oxygen consumption was not linked to the kinetics of the fermentation. The calculated growth constants were discussed and compared to the currently reported values.
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  • 63
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    Biotechnology and Bioengineering 35 (1990), S. 793-801 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The activities of lipase from Candida cylindracea and Rhizopus delemar have been investigated in water/AOT/iso-octane reverse micellar media through the use of two esterification reactions: fatty acid-alcohol esterification and glyceride synthesis. Such media promotes the occurrence of these two lipase-catalyzed reactions due to its low water content. The effect of various parameters on the activity of lipase from C. cylindracea in reverse micelles was determined and compared to results where alternate media were employed. It was observed that the structure of the media, as dictated by the type and concentration of the substrates and products and by the water/AOT ratio, w0, had a strong impact on enzyme activity. Strong deactivation of both typase types occurred in reverse micelles, especially in the absence of substrates and for w0 values greater than 3.0. Glyceride synthesis was realized with lipase from R. delemar, but not with that from C. cylindracea; the temperature and concentration of substrates and water strongly dictated the reaction rate and the percent conversion.
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  • 64
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    Biotechnology and Bioengineering 35 (1990), S. 966-975 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel process has been developed to improve the refolding yield of denatured proteins. It uses reversed micelles to isolate denatured protein molecules from each other and thus, upon refolding, reduces the intermolecular interactions which lead to aggregation. The feasibility of this process was first demonstrated with Ribonuclease A as a model protein. In the present work, we expanded the scope of this study to better understand both the general mechanisms of protein refolding in reversed micelles and the biotechnological applicability of the process. First, we investigated the interactions between the individual components of the reversed micellar system (the protein molecule, the denaturant guanidine hydrochloride (GuHCl), and the surfactant (AOT)) during the refolding process. We then extended our studies to a more hydrophobic protein, γ-interferon, which aggregates upon refolding in aqueous solution. However, it was also found to aggregate in our reversed micelle process during the extraction step. Since γ-interferon is a much more hydrophobic protein than RNase, we hypothesize that interactions between hydrophobic amino acids and the surfactant layer may interfere with refolding. This hypothesis was tested by studying the refolding of chemically modified RNase. The substitution of 55% of the surface lysine residues with hydrophobic caproyl groups caused a significant decrease in the refolding yield of RNase in the reversed micellar system without affecting aqueous solution renaturation. In addition, the extraction efficiency of the enzyme from reversed micelles back into aqueous solution was severely reduced and resulted in aggregation. These experiments indicate that unfolded hydrophobic Proteinsinteract with the Surfactant molecules, which limits their ability to refold in reversed micelles.
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  • 65
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    Biotechnology and Bioengineering 36 (1990), S. 759-762 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 66
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    Biotechnology and Bioengineering 36 (1990), S. 1041-1048 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents an introduction to the use of neural network computational algorithms for the dynamic modeling of bioprocesses. The dynamic neural model is used for the prediction of key fermentation variables. This relatively hew method is compared with a more traditional prediction technique to judge its performance for prediction. Illustrative simulation results of a continuous stirred tank fermentor are used for this comparison. It is shown that neural network models are accurate with a certain degree of noise immunity. They offer the distinctive ability over more traditional methods to learn very naturally complex relationships without requiring the knowledge of the model structure.
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  • 67
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    Biotechnology and Bioengineering 36 (1990), S. 1056-1062 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production of IGF-1 (insulin-like growth factor 1), expressed in Escherichia coli as a secreted fusion protein with affinity for the Fc region of IgG, was monitored automatically during fermentations. A sampling device was used to automatically inject filtered culture medium from the fermentor onto a small affinity column (IgG Sepharose® Fast Flow) connected to a chromatographic system. The area of the eluted peak was proportional to the concentration of the fusion protein. The relationship was linear over the range 25-630 μg/mL with relative standard deviation of around 1% at the higher concentrations. Samples could be monitored automatically every half hour during fermentation (48 h). The method of analysis is nondestructive, allowing further analysis of product quality. A complete evaluation of the monitoring system is described. With this system, fermentations based on the described expression system can be optimized on the basis of product concentration; this will lead to more effective fermentations and higher product yields. It should also be possible to monitor other secreted products with this system by using other affinity gels.
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  • 68
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    Biotechnology and Bioengineering 36 (1990), S. 1083-1089 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The protein releases, the particle size distribution and the viscosity of disrupted E. coli suspensions from Dyno Mill KDL, Manton Gaulin 15 M-8TA and Microfluidizer M-110 were determined. The effects of these parameters on separation of the cell debris from the protein solution by centrifugation and by filtration were also examined. All three disintegration methods investigated give approximately the same protein and enzyme releases but considerably different physical properties of the cell disintegrates which influences centrifugation and filtration. The separation degree of biomass during centrifugation is only slightly affected by increasing degree of disruption (increasing protein releases) in the bead mill, while an increase in the degree of disruption in the two high pressure homogenizers drastically reduces the centrifugal degree of separation. However, increasing degrees of disruption result in shorter filtration times during filtration for all three disintegration methods. The results show further that the cell concentration only has a minor influence on protein releases in the Microfluidizer high-pressure homogenizer, while an increase in the biomass content reduces the separability of the cell disintegrate both in filtration and in centrifugation.
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  • 69
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    Electrophoresis 11 (1990), S. 140-147 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The program “IPGMAKER” is a computational aid for creating and testing recipes for near-linear immobilized pH gradients. It was written for fast IBM personal computers (with a Type 80386 processor and 80387 coprocessor) and compatibles equipped with a VGA, EGA or Hercules (mono) graphics card. The program is limited to the use of up to 10 acids and/or bases, and to ranges spanning between pH 2 and 12. The resulting recipes are presented either as final concentrations in the 2 chambers of a mixing device for linear gradients or as volumes from 0.2 moles/L stock solutions adjusted to a user-defined average buffering power. One of the subroutines determines the pH, gradient slope and buffering capacity at any location of the gradient and includes a facility to estimate the pI of proteins from the composition of their primary structure.
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  • 70
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    Electrophoresis 11 (1990), S. 147-151 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Isoelectric focusing on carrier ampholyte-containing immobilized pH gradient gels was applied (i) to gels submerged in silicone oil on a Peltier cooled apparatus, (ii) to the separation of the higher molecular weight (HMW, Mr 140 000) and the lower molecular weight (LMW, Mr 38 000) acid phosphatases (AcPases) from frog livers. (i) Electrofocusing was conducted on gels submerged under silicone oil cooled and stirred on a Peltier-thermoregulated horizontal gel support plate. This procedure aimed at (a) improving the temperature control of the gel by direct contact of coolant with the gel surface, and thus at being able to focus at the maximal field strength and consequently highest resolution; (b) preventing evaporation from the gel and (c) excluding atmospheric carbon dioxide. Silicone oil submersion did not abolish water loss from the gel into the electrolyte strips during isoelectric focusing, or a rippled gel surface. Absence of water exudation on the ripples noted previously by Altland [1] was observed. (ii) The electrofocusing of AcPases on immobilized pH gradients yielded patterns which remained stationary as a function of time, by contrast to previous analyses on carrier ampholyte generated pH gradients. The total number of enzymatically active components found in the enzyme preparations from different stages of purification and in the isolated HMW and LMW AcPases was 18. The HMW and LMW AcPases focused in characteristic pH ranges and exhibited qualitative and quantitative pattern differences. Their band patterns add up to that of a crude preparation containing both enzymes. Neither polyacrylamide gel electrophoresis (PAGE) at any nondenaturing pH, nor isoelectric focusing in carrier ampholytes with pattern changes due to the pH gradient drift were able to yield that result.
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  • 71
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    Electrophoresis 11 (1990) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 72
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    Electrophoresis 11 (1990), S. 15-18 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The electrophoretic properties of purified calmodulin were investigated. High performance capillary electrophoresis of this Ca2+-binding protein in free solution at pH 2.5 resulted in an elution of a single peak with a retention time of approximately 4.7 min. Addition of [ethylene-bis(oxyethylenenitrilo)]N, N, N', N'-tetraacetic acid (EGTA) to the protein prior to capillary electrophoresis completely abolished this electrophoretic profile. Polyacrylamide gel electrophoresis of calmodulin under denaturing and nondenaturing conditions also revealed a single polypeptide band. However, the relative electrophoretic mobilities of this protein could vary, depending on the presence or absence of Ca2+. The pI of calmodulin was estimated to be 3.7 by using isoelectric focusing techniques. Analysis of this acidic protein by high performance capillary electrophoresis at pH 8.0 revealed that it could be resolved into two major and one minor polypeptide peaks, regardless of the presence or absence of Ca2+. These findings suggest that capillary electrophoresis at near physiological pH may differentiate the microheterogeneity of calmodulin.
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  • 73
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Nondenaturing polyacrylamide gel electrophoresis was used to separate protein kinases in crude extracts and subcellular fractions of murine erythroleukemic cells. The kinases were detected using an in situ phosphorylation assay. The electrophoretic patterns obtained using gel bound to GelBond and prepared with AcrylAide differed from those seen without GelBond and with N, N'-methylenebisacrylamide as cross-linker. In an attempt to improve the resolution of the bands in the membrane fractions, detergent-treated preparations were electrophoresed through gels which contained either 0.1 % Triton X-100 or 0.1 % Nonidet P-40. The resolution of the bands in this fraction was not, however, improved with the inclusion of the nonionic detergent in the gels. When cytosol was electrophoresed through gels containing detergent, a major band of cAMP-dependent protein kinase activity showed a marked shift in mobility. This may have been the result of a structural change, altering the shape and possibly affecting the charge on the molecule, or the enzyme may have formed aggregates with the detergent.
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  • 74
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Enzyme-linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 μm). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetetive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1-5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.
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  • 75
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The separation of enantiomeric forms of dansylated amino acids by isoelectric focusing in immobilized pH gradients (IPG) is demonstrated for the first time. Separations occur in a pH 3.0-4.0 IPG interval, in presence of 7murea, 10% methanol and 60 mM β-cyclodextrin (CD) as chiral discriminator. It is found that the inclusion complex formed between the D-form and CD has a lower pI than the uncomplexed form (ΔpI = 0.05 for DL-Phe and ΔpI = 0.025 for DL-Trp); from this, it is calculated that the pK of the tertiary amino group in the dansyl moiety is lowered by 0.1 pH unit in the former case (D-Phe) and by 0.05 in the case of D-Trp (both values referring to 60 mM CD gels). For some racemates (e.g., DL-Phe) the separation mechanism is still operative with CD concentrations as low as 20 mM. In our system 60 mM CD appears to be the solubility limit of CD. As the complex is stable in the electric field for at least 15 h, this separation mechanism could be exploited for purifying large quantities of pure D and L forms racemates in multicompartment electrolyzers with isoelectric Immobiline membranes.
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  • 76
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    Electrophoresis 11 (1990), S. 5-15 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments ≤ 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained ≤ 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incroporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments ≤ 12 kbp in size in agarose gels ≤ 1.4 % in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments ≤ 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments ≥ 1 kbp in size in agarose gels ≥ 2 % in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an “intrinsic” DNA mobility of 2.7 x 10-4 cm2/Vs at 20°C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments. If the volume occupied by each DNA molecule is approximated by its root-mean-square (rms) radius of gyration, the median pore radius, rp, of an LE agarose gel cast and run in TAE buffer is rp = 98 A-0.90, where A is the agarose concentration in % and rp is given in nm. For gels cast and run in TBE buffer, the apparent median pore radius is rp = 66 A-0.95. If the volume occupied by each DNA molecule is represented by its mean geometric radius instead of the rms radius of gyration, the median gel pore radius in both buffers depends on A-0.5, as predicted by the extended Ogston theory. From the corresponding retardation coefficients, the gel fiber parameters were estimated.
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  • 77
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Myoglobin CNBr peptides, constituting the commercially available molecular weight calibration kits for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were analyzed by microsequencing after electroblotting on polyvinylidene difluoride (Immobilon) membranes. An obious disagreement was found between peptide identification and the data provided by the manufacturers. We observed 6 peptides from Mr 2500 to 17 000 corresponding, in increasing size order, to the 3 peptides resulting from the total CNBr digestion, to 2 incompletely cleaved peptides and to the intact myoglobin. Using a corrected calibration curve, a linear relationship was established from Mr 6000 to 43 000 and a second one for shorter peptides. This method of electrophoresis and electroblotting, easily adapted for peptides, is a powerful tool for peptide identification correlated with size determination. It is especially useful for CNBr-cleaved peptides.
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.
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    Electrophoresis 11 (1990), S. 187-187 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 80
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    Electrophoresis 11 (1990), S. 219-227 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Infection of tissue culture cells with certain viruses results in the shutoff of host cell protein synthesis. We have examined virally infected cell lysates using two-dimensional gel electrophoresis and immunoblotting to ascertain whether initiation factor protein modifications are correlated with translational repression. Moderate increases in eukaryotic initiation factor (eIF)-2α phosphorylation are detected in reovirus- and adenovirus-infected cells, as reported previously (Samuel et al., 1984; O'Malley et al., 1989). Neither vesicular stomatitis virus, vaccinia virus, frog virus III, rhinovirus, nor encephalomyocarditis virus caused significantly increased 2α phosphorylation. There were no reproducible, significant changes in eIF-4A, eIF-4B, or eIF-2β in cells infected by any of these viruses. The cleavage of eIF-4F subunit p220, such as has been previously demonstrated to occur in poliovirus (Etchison et al., 1982) and rhinovirus (Etchison and Fout, 1985), was not detected in any of the other virus infections analyzed.
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  • 81
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    Electrophoresis 11 (1990), S. 213-219 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2-D PAGE and [125I]concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2-D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combinat on of 2-D PAGE with electroblotting and 45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2-D PAGE and 45Ca overlay has been used to demonstrate the presence of a calcium-binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2-D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2-D PAGE when used in combination with detection methods which select specific subsets of proteins such as glycoproteins.
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  • 82
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    Electrophoresis 11 (1990) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 83
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    Electrophoresis 11 (1990) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 84
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    Electrophoresis 11 (1990), S. 292-298 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The isotachophoretic behavior of serum albumin is examined for three anionic and one cationic electrolyte systems by (i) computer simulation, (ii) capillary isotachophoresis (ITP) and (iii) continuous flow ITP. The theoretical relationship between pH of the leading electrolyte and the steady state protein plateau concentration is presented for one of the anionic systems. With leading ion concentrations of the order of 10 mm, experimental protein plateau concentrations of 1.3-2.3 % w/v are obtained. The computer predictions are approximately half these values.
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  • 85
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    Electrophoresis 11 (1990), S. 304-309 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The principles and some applications of free flow zone electrophoresis and isotachophoresis are described. The influence of (i) carrier electrolyte conductivity on the migration velocity and (ii) band shape on zone electrophoresis was investigated. The technique was found convenient for studying the effect of pH on the mobility of proteins to create a mobility curve. The purification of alcohol dehydrogenase from a crude yeast extract revealed the separation power of zone electrophoresis for complex protein mixtures. Without additional steps, a purification factor of 5.4, with a recovery of 97 % alcohol dehydrogenase, was achieved. Free flow isotachophoresis was applied to the purification of immunoglobulins from human serum. Disadvantages of this technique are the time-consuming development of an optimized separation system and the empirical search for suitable spacers. Also, reaching of the steady state becomes increasingly difficult as the number of sample components increases.
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  • 86
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    Electrophoresis 11 (1990), S. 310-314 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Gliadins, defined as wheat kernel storage proteins soluble in 70% ethanol, possess an electrophoretic diversity permitting us to identify different varieties. Because of genetic proximity, however, it sometimes proves impossible to distinguish between different varieties using standard methods. An electrophoresis method, utilizing a discontinuous buffer system with aluminum lactate and potassium lactate in the gel and sodium lactate in the electrode vessels, is described for two types of gels 18 cm and 32 cm in length. The number of bands in higher than with standard methods, and is significantly increased with long gels, facilitating the distinction between varieties. A nomenclature for the new bands is presented for several widely different cultivars.
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  • 87
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    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A new and simplified procedure is described for apolipoprotein E (APO E) phenotyping from native plasma or serum samples. Diluted or dialyzed samples are separated on agarose isoelectric focusing gels followed by protein blotting on nitrocellulose membranes. APO E banding patterns are localized immunologically using polyclonal goat anti-APO E antiserum as the primary antibody and rabbit anti-goat IgG conjugated with alkaline phosphatase as the secondary antibody. The method was used in parallel with our previously described polyacrylamide gel system to screen 110 unrelated and healthy US whites. Both gel systems gave identical APO E phenotypes, and allele frequencies were comparable with reported US white values. This simplified method can be used on a large number of population and clinical samples with minimum cost and effort.
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  • 88
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Human heart muscle proteins have been analyzed by two-dimensional electrophoresis. Twenty five autopsy heart muscle samples obtained from individuals who had died in accidents and who had no signs of cardiovascular pathology have been compared with biopsy and autopsy myocardium samples of patients with: dilated cardiomyopathy (5 cases), hypertrophical cardiomyopathy (2 cases) and myocarditis (2 cases). In dilated cardiomyopathy in 3 out of 5 cases an additional protein spot was found in the myocardial myosin light chain 1 area.
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  • 89
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    Electrophoresis 11 (1990), S. 343-344 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A reddish-brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11-12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2-2h with 7% acetic acid at 60°C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R-250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity.
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  • 90
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    Electrophoresis 11 (1990), S. 350-350 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 91
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    Electrophoresis 11 (1990) 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 92
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    Electrophoresis 11 (1990), S. i 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
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  • 93
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    Electrophoresis 11 (1990), S. 367-375 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Gas counters based on the multiwire proportional counter (MWPC) and multistep avalanche (MSA) technologies can combine the functions of imaging and quantitation (or assay) of radioactively labelled electrophoretograms and blots. Submillimeter spatial resolutions combined with good quantitation have been achieved for the most common beta-emitting radio labels. The current state of development of this technology is reviewed and assessments of the performance of these devices relative to the more traditional systems (X-ray film and the scintillation counter) presented. Particular attention is paid to the potential of these devices to enhance the productivity of the scanning of blots and the performance of assays.
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  • 94
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    Electrophoresis 11 (1990), S. 344-346 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Six new protein spots were detected by two-dimensional electrophoresis in cultured human fibroblasts infected with mycoplasma. No changes were observed in other protein spots present in normal and pathological cells. After treatment with an antimycoplasma drug for a week, the new spots disappeared and the cells became negative for mycoplasma stain.
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  • 95
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    Electrophoresis 11 (1990), S. 361-366 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A modernized schlieren optics system was applied to follow protein bands visually during polyacrylamide gel electrophoresis. This approach is suitable for quantitative measurement of electrophoresis as well as for detection of protein bands because of the absence of distortion of the electrophoretic pattern during the staining process. The optics are also suitable for the detection of various background phenomena which usually escape observation.
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  • 96
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A numerical analysis of one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoretic protein patterns of three separate bacterial groups is described. Similarity between patterns was estimated using a correlation coefficient and is represented diagramatically by the unweighted pair-group with arithmetic averages method of clustering. Protein patterns were scanned using a laser densitometer interfaced directly to a microcomputer, where calculations were performed semiautomatically with specially written software. A method for the normalization of patterns from different gels is utilized. Whole cell protein patterns clearly distinguished strains representing various groups of Achromobacter. Similarly the two biovars of the Group EF-4 bacteria could be separated after analysis of partial „background patterns“ as could the different species of Providencia. In addition, whole patterns could be used to type the various strains of P. rettgeri. These examples serve to illustrate the power, versatility and potential of the technique when applied to problems of classification, identification and typing of bacteria of medical interest. Its advantages and disadvantages, when compared to other available electrophoretic methodologies, are discussed, as is the future application of such technology to the hospital laboratory.
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  • 97
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    Electrophoresis 11 (1990), S. 400-406 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The process of quantitative densitometry is analyzed with methods developed in information theory. It is shown that the steps involved in densitometry, e. g. gel staining, mechanical, optical and electronic processing, as well as all the steps of data processing, can be viewed as communication channels. The factors affecting both the relevant and irrelevant part of the total information passed through these channels are discussed in the consistent frame provided by information theory. This view leads to a unifying context for analyzing the performance of quantitative densitometers.
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  • 98
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    Electrophoresis 11 (1990), S. 425-431 
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The separation of DNA by gel electrophoresis provides a rapid method for determining size distributions of DNA in solution. Densitometric scanning of photographs of gels has been the standard method of analysis of agarose gels. However, analysis of photographs is complicated by the non-linear response of photographic film. Charged-coupled device video cameras have become popular for quantitative densitometry and we have used a charge-coupled device camera to image agarose gels to quantitate DNA damage. We compare video and photographic densitometry for quantitation of ultraviolet radiation (UV)-induced DNA damage and find that the two methods give equivalent results.
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  • 99
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Results of the purification of alcohol dehydrogenase (ADH) by field step electrophoresis and combined field step-zone electrophoresis are presented. In field step electrophoresis, optimization of voltage, residence time and pH of the sample solution led to a maximal purification factor of 2.8 and a yield of 89% ADH. The limit of loading capacity was reached at a protein concentration of the sample solution of approximately 4 g/L, allowing a maximal throughput of 1.14 g/h with a yield of 86% and a 2.8-fold purification in the Elphor VaP 22 apparatus. With a production scale apparatus a throughput of 2.07 g/h without any loss of separation quality could be achieved. By introducing the sample solution into the separation chamber through 3 inlets, simultaneously, the throughput was increased to 3.2 g/h with a purification factor of 2.7 and a yield of 82% ADH. For the combined field step-zone electrophoresis method a maximum purification factor of 3.6 and a yield of 80% ADH were achieved. The loading capacity was limited to a 4.13 g/L protein concentration of the sample solution, resulting in a throughput of 440 mg/h. Injecting the sample solution simultaneously into 3 inlets resulted in a maximum throughput of 1.92 g/h with 3.1-fold purification and a yield of 80% ADH. Zone electrophoresis, field step electrophoresis and a combination of both are compared with respect to resolution, throughput and the application potential in a protein purification scheme. A scale-up to 3 g/h is possible in zone electrophoresis and field step electrophoresis. The highest yields and the best purification were achieved in zone electrophoresis with a purification factor of 4.7 while on field step electrophoresis this factor was only 2.8. The combination of both technique was less efficacious with respect to throughput, yield and purification factor. Due to the different sample concentrations used, zone electrophoresis and field step electrophoresis are complementary in a purification scheme.
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  • 100
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin-layer polyacrylamide gel isoelectric focusing in a pH 4.5-5.4 carrier ampholyte generated pH gradient, additionally containing N-(2-acetamido)-2-aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25-hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.
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