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  • Biochemistry and Biotechnology  (1,524)
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  • 101
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 45 (1995), S. 374-378 
    ISSN: 0006-3592
    Keywords: cell culture ; patterened surfaces ; cell adhesion ; hydrogel ; polyHEMA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chinese hamster ovary (CHO) cells were attached to tiny adhesive sites in poly-2-hydroxyethyl methacrylate(polyHEMA-) coated glass, and their divison properties were examined. The adhesive sites were produced by placing a metal mask, containing 8-μm-diameter holes arranged in a regular pattern, on top of the coated glass and exposing the sandwich to glow discharge treatment. This treatment produced an ordered array of circular cavities in the polyHEMA down to the glass. These adhesive sites were smaller in diameter than a newborn CHO cell, so that, upon division, there would theoretically be room for only one of the two new daughter cells to remain attached. It was found that individual CHO cells attached to, and grew upon, the sites, and that division normally resulted in the releas of one of the two new daughters. It is concluded that this culture technique has applications in research on the mammalian cell cycle, cell partitioning, and cellular senescence. © 1995 John Wiley & Sons, Inc.
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  • 102
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    Biotechnology and Bioengineering 45 (1995), S. 387-397 
    ISSN: 0006-3592
    Keywords: transesterification ; water activity ; lipolytic enzymes ; gas ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani cutinase and Candida cylindracea lipase were used to catalyze a transesterification reaction in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrate and removed reaction products simultaneously. Different conditions of immobilization were used and compared to the results obtained with a nonsupported enzyme. The enzymatic activity was found to be highly dependent of a key parameter: water activity (aw). Biocatalyst stability was greatly influenced by water activity and the choice of immobilization technique for the enzymatic material. For free and adsorbed enzymes, water requirements exhibited optima which corresponded to the complete hydration coverage of the protein. These optima presented a good correlation with the isotherm sorption curves obtained for the different preparations. In this work are reported the results concerning the possibility of using a continuous system able to operate at controlled water activity in a heterogeneous medium. Lipolytic enzyme in such a system appears to be a new process for the biotransformation of volatile esters. © 1995 John Wiley & Sons, Inc.
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  • 103
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    Biotechnology and Bioengineering 45 (1995), S. 398-405 
    ISSN: 0006-3592
    Keywords: anaerobic digestion ; full-scale ; granule activity ; multiplate reactor ; solid retention ; whey permeate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A 450-m3 multiplate anaerobic reactor (MPAR) has been started-up in April 1992 for treating wastewater (whey permeate and domestic wastewater) at the Nutrinor (Lactel) cheese factory in Chambord (Québec, Canada). The MPAR consists of four superimposed sections. The liquid flows upwards from one section to the next, while the gas is collected below each plate and evacuated through side-outlets. The wastewater is concurrently distributed at the bottom of the first, second, and third sections, as 50%, 33%, and 17% of the total influent stream, respectively. Granular anaerobic sludge at an initial concentration of 30 kg of volatile suspended solids (VSS) per cubic meter of reactor liquid volume was used to inoculate the reactor. Under normal operation of the factory, the chemical oxygen demand (COD) concentration of the influent ranged from 20 to 37 kg COD m-3. The reactor organic loading rate (OLR) fluctuated between 9 and 14.7 kg COD m-3 d-1 for hydraulic retention times (HRT) maintained between 55 and 68 h. At the highest OLR, the MPAR showed an efficiency of 98% and 92% for soluble and total COD removal, respectively, and a methane production rate averaging around 4 m3 m-3 d-1.Biomass-specific activities ranged between 7 and 51, 1.3 and 8.5, 5.3 and 12.2, 60 and 119, and 119 and 211 mmol g-1 VSS d-1 for glucose, propionate, acetate, formate, and hydrogen, respectively. Average equivalent-diameter of the granules was around 0.65 mm. The MPAR reactor generally showed a large capacity for solid retention with a biomass content between 32 and 37 kg VSS m-3. © 1995 John Wiley & Sons, Inc.
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  • 104
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    Biotechnology and Bioengineering 45 (1995), S. 379-386 
    ISSN: 0006-3592
    Keywords: propionic acid fermentation ; Propionibacterium acidipropionici ; immobilized cell bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took ∼55 h to ferment whey permeate containing ∼45 g/L lactose to ∼20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L · h to 0.47 g/L · h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L · g cell. The productivity increased to 0.68 g/L · h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. © 1995 John Wiley & Sons, Inc.
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  • 105
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    Biotechnology and Bioengineering 45 (1995), S. 415-425 
    ISSN: 0006-3592
    Keywords: plant cell suspension culture ; capillary shear loop ; Morinda citrifolia ; shear susceptibility ; morphology ; stirred tank reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The shear susceptibility of cell suspension cultures of the plant cell Morinda citrifolia was investigated by subjecting the cells to the well-defined shear field generated in turbulent flow through a capillary. Suspensions were circulated using a peristaltic pump and average shear stresses between 25 and 350 N m-2 were generated in the capillary test section. Control experiments were performed to assess the possible contribution of the peristaltic pump to the observed cell damage. There was clear evidence of pump-induced damage at the more severe test conditions and all viability measurements were corrected accordingly. Both shake flask suspension cultures (aged between 9 and 15 days) and repeated batch fermentation cultures, grown in a stirred tank reactor (STR) under a variety of controlled agitation conditions, were tested in the capillary shear loop. The cell damage incurred was evaluated in terms of suspension viability, as determined by a dye exclusion technique. Viability loss was found to conform closely to a first-order model in which the rate constant was observed to increase with the imposed shear stress. Furthermore, a linear relationship was identified between the specific death constant and the cumulative energy dissipated. Post-shear morphological measurements showed that the chain length distribution is shifted toward markedly lower values. In comparison with shake flask cultures, repeated batch fermentation cultures exhibited a marked increase in sensitivity to capillary shear. Based upon the determined morphological characteristics, this result is primarily attributable to the increased chain lengths characteristic of the repeated batch cultures. © 1995 John Wiley & Sons, Inc.
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  • 106
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    Biotechnology and Bioengineering 45 (1995), S. 406-414 
    ISSN: 0006-3592
    Keywords: chymotrypsin ; differential scanning calorimetry ; ligands ; lipase ; organic media ; sorbitol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of the addition of sorbitol on the activity and stability of enzymes was examined by monitoring transesterification reactions performed in organic media at various water activities (aw = 0.08 to 0.97). Lipases from Chromobacterium viscosum and Candida rugosa immobilized on celite, and chymotrypsin, free or immobilized on celite, were used. When the sorbitol-containing enzymes were employed, higher reaction rates and less hydrolysis were observed. Immobilization of chymotrypsin resulted in high activity and operational stability, while the nonimmobilized enzyme was stable only in the presence of sorbitol. The activity of all preparations diminished after washing them with pyridine to remove sorbitol. Furthermore, severe stability problems occurred in the preparations lacking sorbitol. Sorbitol treatment, even after removal of the sorbitol itself, improved the activity of nonimmobilized chymotrypsin relative to the washed control. On the other hand, washing to remove sorbitol had a negative effect on the activity of both coimmobilized lipase and coimmobilized chymotrypsin. Addition of a substrate analogue, N-acetyl-L-phenylalanine, to chymotrypsin yielded a preparation that exhibited higher activity than both the control and its sorbitol-containing counterpart. Differential scanning calorimetry measurements revealed that the chymotrypsin-sorbitol complex was stable against thermal denaturation, undergoing transition at a high temperature (89°C). The transition temperatures of the substrate-containing chymotrypsin and of the control were identical (72°C). © 1995 John Wiley & Sons, Inc.
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  • 107
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    Biotechnology and Bioengineering 45 (1995), S. 435-439 
    ISSN: 0006-3592
    Keywords: Catharanthus roseus ; ajmalicine production rate ; dissolved oxygen concentration ; kinetic model ; high-density culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The relation between dissolved oxygen (DO) and the ajmalicine production rate of Catharanthus roseus was investigated in 15-L tank reactors at constant stirrer speed and gas flow rate. Below a DO concentration of 29% of air saturation the ajmalicine production rate was less than 0.06 μmol/g/d. Above a DO of 43% the ajmalicine production rate was constant at 0.21 μmol/g/d. Between a DO of 29% and 43% there was a strong relation between the ajmalicine production rate and the DO concentration. After a period of at least 12 days at DO ≤29% the culture lacked the ability to adapt to a DO ≥57%. A kinetic equation is proposed for the relation between DO and the specific ajmalicine production rate. © 1995 John Wiley & Sons, Inc.
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  • 108
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    Biotechnology and Bioengineering 45 (1995), S. 426-434 
    ISSN: 0006-3592
    Keywords: regioselectivity of enyzmatic catalysis ; sucrose acylation in organic solvents ; molecular modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The rational control over enzyme-catalyzed regioselectivity has been studied using sucrose acylation by vinyl esters in organic media as a model. Subtilisins BPN' and Carlsberg preferentially acylate at the 1′-hydroxyl of sucrose with some acylation observed at the 6-hydroxyl. The preference for the 1′-hydroxyl is strongly affected by the hydrophobicity of the organic solvent and the chain length of the vinyl ester. Increasingly hydrophobic solvents and longer chain lengths lower the favorable formation of the 1′-acylation and improve 6-acylation. Molecular modeling of sucrose in the binding pocket of subtilisin BPN' shows that the 1′-acylation is favored in solvents that can solvate sugars (such as pyridine) as the glucose moiety is exposed to the medium, whereas 6-acylation leaves the entire sucrose molecule buried within the enzyme's binding pocket. Thus, 1′-acylation is sterically more favorable than 6-acylation. Increasingly hydrophobic solvents affect regioselectivity by changing the degree of solvation of the glucose moiety in the medium and forcing the sucrose 1′-ester completely into the binding pocket. In a related modeling, the vinyl ester chain length was shown to modulate regioselectivity by controlling the bond angles between the resulting acylenzymes and the sucrose thereby affecting the positioning of the sucrose in the binding pocket of subtilisin BPN'. This study shows that control over enzymic regioselectivity can be achieved by rational choices of substrate and solvent. © 1995 John Wiley & Sons, Inc.
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  • 109
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    Biotechnology and Bioengineering 45 (1995), S. 454-457 
    ISSN: 0006-3592
    Keywords: copper ; diffusion ; alginate gel beads ; diffusion models ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The diffusivity of Cu2+, as determined by previous authors from analysis of experimental data in terms of the shrinking core (SCM) and linear absorption (LAM) models, is examined in light of the ability of the models to curve fit all the data. It is concluded from this further analysis that previous conclusions depicting the LAM to have an advantage over the SCM for predictive value are not justified. It is also shown that equally good curve fits can be obtained with a recent absorption/desorption model of diffusion which considers directly, through distribution theory, the effect of heterogeneity of material properties on the rate of diffusion. © 1995 John Wiley & Sons, Inc.
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  • 110
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    Biotechnology and Bioengineering 45 (1995), S. 458-462 
    ISSN: 0006-3592
    Keywords: molecular recognition ; in vitro selection ; metal-binding protein ; molecular process engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We propose an in vitro selection strategy to identify bacteriophage variants that recognize metal ions in solution. In 6 M urea, phage T7 loses 99.9% of its activity in less than 5 min. Inactivation is accelerated by gold, but slowed by zinc and magnesium. Selection of phage over five generations in the presence of gold, zinc, and magnesium increases phage half-lives 4-, 10-, and 70-fold, respectively. As selections are repeated, phage become increasingly dependent on the specific metal employed in the selection, indicating the suitability of the strategy for optimization of metal-ion recognition. © 1995 John Wiley & Sons, Inc.
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  • 111
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    Biotechnology and Bioengineering 45 (1995), S. 440-449 
    ISSN: 0006-3592
    Keywords: transformation capacity ; product toxicity ; oxygenase enzymes ; chlorinated organics ; trichloroethylene ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The degradation of trichloroethylene (TCE), chloroform (CF), and 1,2-dichloroethane (1,2-DCA) by four aerobic mixed cultures (methane, propane, toluene, and phenol oxidizers) grown under similar chemostat conditions was measured. Methane and propane oxidizers were capable of degrading both saturated and unsaturated chlorinated organics (TCE, CF, and 1,2-DCA). Toluene and phenol oxidizers degraded TCE but were not able to degrade CF, 1,2-DCA, or other saturated organics. None of the cultures tested were able to degrade perchloroethylene (PCE) or carbon tetrachloride (CC4). For the four cultures tested, degradation of each of the chlorinated organics resulted in cell inactivation due to product toxicity. In all cases, the toxic products were rapidly depleted, leaving no toxic residues in solution. Among the four tested cultures, the resting cells of methane oxidizers exhibited the highest transformation capacities (Tc) for TCE, CF, and 1,2-DCA. The Tc for each chlorinated organic was observed to be inversely proportional to the chlorine carbon ratio (Cl/C). The addition of low concentrations of growth substrate or some catabolic intermediates enhanced TCE transformation capacities and degradation rates, presumably due to the regeneration of reducing energy (NADH); however, addition of higher concentrations of most amendments reduced TCE transformation capacities and degradation rates. Reducing energy limitations and amendment toxicity may significantly affect Tc measurements, causing a masking of the toxicity associated with chlorinated organic degradation. © 1995 John Wiley & Sons, Inc.
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  • 112
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    Biotechnology and Bioengineering 45 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 113
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    Biotechnology and Bioengineering 45 (1995), S. 450-453 
    ISSN: 0006-3592
    Keywords: ethanol production ; albumin hydrolysate ; plasma membrane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The enhancing effect of albumin hydrolysate on ethanol production was investigated in ethanol fermentations using Saccharomyces sake. In batchwise ethanol production, addition of supplemental albumin hydrolysate and phosphatidylcholine, or albumin hydrolysate alone, brought about a more than 60% increase in final ethanol concentration (148 or 144 g/L compared with 88 g/L with no supplementation [control] after 72 h). The effect of the supplements is believed to be due to an enhanced alcohol tolerance of cells grown in media containing the supplements. Cells grown in media containing albumin hydrolysate were enriched in phenyalanine, tyrosine, and methionine in their plasma membranes. All three amino acids were also present in considerable amounts in the albumin hydrolysate. This fact suggests that the three amino acids, which are present in albumin hydrolysate, are incorporated into the plasma membranes of cells. Under ethanol production conditions in which only one amino acid among the components of albumin hydrolysate was excluded, namely phenlalanine, tyrosine, or methionine, significant reductions in ethanol production resulted. © 1995 John Wiley & Sons, Inc.
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  • 114
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    Biotechnology and Bioengineering 45 (1995), S. 481-487 
    ISSN: 0006-3592
    Keywords: biofilms ; detachment ; substrate loading ; airlift reactor ; abrasion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The dynamic change in the overall detachment rate of spherical biofilms in a biofilm airlift suspension reactor was measured after a downshift of the substrate loading rate to zero while all other conditions remained constant. In contrast to the expectations, the overall detachment rate decreased rapidly to a nearly stable level. Correlations available from literature were not able to describe this phenomenon. Concepts were formulated which can describe the observations from this study. Research under dynamic conditions and careful monitoring of the biofilm surface area and biofilm morphology are necessary to elucidate and discriminate biofilm detachment mechanisms. © 1995 John Wiley & Sons, Inc.
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  • 115
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    Biotechnology and Bioengineering 45 (1995), S. 473-480 
    ISSN: 0006-3592
    Keywords: cell adhesion ; protective additives ; interfacial tensions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A significant degree of cell damage is observed during suspension cell culture with air sparging. Protective agents can be added to the culture medium to protect the cells from damage. It has been observed that cells tend to adhere to air-medium interfaces and cell damage is mainly due to this cell-bubble interaction; protective additives have been found to prevent this cell adhesion to the bubble surfaces. In this article, it is demonstrated that the interfacial tension between the air and medium is related to the effectiveness of the protective additives to prevent adhesion of cells to this interface. Five different types of additives (Pluronic F-68, Methocels, dextran, Polyvinyl alcohol, and polyethylene glycols) were studied in an effort to determine their protective characteristics. Liquid-vapor interfacial tensions of the culture medium, with and without the additives, were measured by two different techniques (maximum bubble pressure method and Wilhelmy plate method). In addition, visualization techniques showed that in the presence of certain protective additives cells do not adhere to the bubble surface. Results obtained from these experiments indicate that the additives which rapidly lower the liquid-vapor interfacial tension of the culture medium also prevent adhesion of cells to the bubble surface. Experiments have also been conducted to determine the number of cells killed due to bubble rupture, and it was observed that this number is related to the amount of cells adhering to the bubble surface. © 1995 John Wiley & Sons, Inc.This article is a US Government Work and, as such, is in the public domain in the United States of America.
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  • 116
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    Biotechnology and Bioengineering 45 (1995), S. 463-472 
    ISSN: 0006-3592
    Keywords: apoptosis ; animal cell death ; hybridoma cells ; agitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The question is addressed as to whether cells which are subject to high-energy dissipation rates in agitated bioreactors show an apoptotic response. Murine hybridoma cells in batch culture were agitated in bench-scale (1-L) bioreactors without gas sparging. At an energy dissipation rate of 1.5 W m-3 there was no apparent damage. At 320 W m-3 cell viability declined, and increasing proportions of the dead cells displayed the morphological features of apoptosis, but necrosis also remained as a significant mechanism of death. When cells were subjected to the intensive energy dissipation rate of 1870 W m-3 in a bioreactor without gas headspace, the cell number dropped by 50% within 2 h and a subpopulation of smaller-sized cells emerged. This excluded trypan blue but showed some apoptotic characteristics such as reduced and condensed DNA content and low F-actin content. The incidence of apoptotic activity was further demonstrated by the appearance of numerous apoptotic bodies. Analysis of the cell cycles of both small and normal size populations indicated that greater proportions of S and G2 cells had become apoptotic and there was evidence of preferential survival of G1 cells. It is suggested that two mechanisms of cell death are apparent in hydrodynamically stressful situations, but their relative expression depends on the energy dissipation rate. © 1995 John Wiley & Sons, Inc.
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  • 117
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    Biotechnology and Bioengineering 45 (1995), S. 495-502 
    ISSN: 0006-3592
    Keywords: optical cell density probes ; turbidity probes ; on-line monitoring ; in situ probes ; mammalian cell bioreactors/fermentors ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: On-line optical cell density probes were implemented to continuously monitor the cell densities in mammalian cell bioreactor and to achieve advanced bioreactor controls. We tested cell density probes from six manufacturers in high cell density bioreactors. When externally calibrated, Aquasant and Ingold backscattering probes produced the most linear probe responses (PR) versus cell density (CD), followed by the ASR and Cerex laser probes. Monitek and Wedgewood transmission probes had lower resolutions. All probes were tested in two murine hybridoma fermentations. Cell densities varied between 1 × 106 cells/mL to 20 × 106 cells/mL and the bioreactors were operated for 5 to 7 weeks. For our bioreactors, Aquasant, Ingold, ASR, Wedgewood, and Monitek probes gave satisfactory responses. Little fouling was observed with any probe at the end of 2 weeks. Fouling was a possibility after 3 weeks in one bioreactor but its effect can be easily corrected. Cell density control and specific perfusion control of bioreactors based on the Aquasant probe were achieved. Implementation of cell density probe based perfusion control, instead of “step perfusion adjustments” based on manual hemacytometer control, will result in smoother operation, healthier cultures, increased medium delivery efficiency, and reduced operational excursions. © 1995 John Wiley & Sons, Inc.
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  • 118
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    Biotechnology and Bioengineering 47 (1995), S. 461-469 
    ISSN: 0006-3592
    Keywords: trichloroethylene ; bioscrubber ; bubble column ; cometabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A bubble column bioreactor was used as bioscrubber to carry out a feasibility study for the cometabolic degradation of trichloroethylene (TCE). Phenol was used as cosubstrate and inducer. The bioreactor was operated like a conventional chemostat with regard to the cosubstrate and low dilution rates were used to minimize the liquid outflow. TCE degradation measurements were carried out using superficial gas velocities between 0.47and 4.07 cm s-1 and TCE gas phase loads between 0.07 and 0.40 mg L-1 Depending on the superficial gas velocity used, degrees of conversion between 30% and 80% were obtained. A simplified reactor model using plug flow for the gas phase, mixed flow for the liquid phase, and pseudo first order reaction kinetics for the conversionof TCE was established. The model is able to give a reasonable approximation of the experimental data. TCE degradation at the used experimental conditions is mainly limited by reaction rate rather than by mass transfer rate. The model can be used to calculate the reactor volume and the biomass concentration for a required conversion. © 1995 John Wiley & Sons Inc.
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  • 119
    ISSN: 0006-3592
    Keywords: cyclin E expression ; CHO cells ; insulin ; fibroblast growth factor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Exogenous growth factors normally required in cell culture activate cell proliferation via the molecular controls of cell-cycle progression. Highly differing influences of mitogenic stimulation of Chinese hamster ovary (CHO) cells by insulin and basic fibroblast growth factor(bFGF) have been clearly observed in a defined protein-free medium. CHO K1 cells stimulated only with insulin grow with flattened cell morphology and extensive cell-cell contact, whereas stimulation with only bFGF or bFGF plus insulin results in loss of cell-cell contact and a transformed and rounded-up morphology. Compared with insulin-stimulated cells, bFGF-stimulated cells exhibit a relatively long G1, and short S phase, and contain higher levels of cyclin E. Observation of elevated levels of cyclin E in wild-type CHO K1 cells mitogenically stimulated by basic fibroblast growth factor motivated transfection of these cells by a cyclin E expression vector. These transfectants grew rapidly in protein-free basal medium and had similar cyclin b levels, distributions of nuclear cell-cycle times, and cell morphologies as bFGF-stimutated CHO K1 culture. Metabolic engineering of cell-cycle regulation thus bypasses exogenous growth factor requirements, addressing a priority objective in economical, reproducible, and safe biopharmaceutical manufacturing. © 1995 John Wiley & Sons Inc.
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  • 120
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    Biotechnology and Bioengineering 47 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
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    Biotechnology and Bioengineering 47 (1995), S. 520-524 
    ISSN: 0006-3592
    Keywords: bacillus subtilis ; plasmid ; continuous culture ; CAT ; recombinant cultures ; acid formation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The feasibility of continuous production of proteins in chemostat cultures of Bacillus subtilis was investigated. An expression system consisting of the bacterium B. subtilis BR151 carrying plasmid p602/19 was used. The plasmid contains the cat (chioramphenicol acetyltrans-ferase) gene downstream of a strong vegetative T5 promoter. It was found that, at a dilution rate of 0.2 h-1 production of relatively high levels of CAT protein (about 4% ofcellular protein) can be sustained. But, experiments at a higher dilution rate of 0.4 h-1 were unproductive because of high acidformation and washout. Combination of low cell yield, which results from excessive acid formation, and low dilution rate led to a low volumetric CAT productivity. Our recent work with the nonrecombinant cells, has demonstrated that uptake of small amounts of citrate significantly reduces or entirelyeliminates the acid formation. This superior performance in the presence ofcitrate was hypothesized, based on strong experimental evidence, to be the result of a reduction in glycolysis flux through a sequence of events leading to a reduction in pyruvate kinase and phosphof- ructokinase activities, the regulatory enzymes of glycol-ysis. In this study, it is demonstrated that cofeeding of glucose and citrate substantially reduces theorganic acid formation and significantly increases the recombinant culture productivity. The combination of high specific CAT activity and cell density resulted in a total of six- to tenfold higher culture productivitywhen citrate and glucose were cometabolized than when glucose was the only carbon source. © 1995 John Wiley & Sons Inc.
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    Biotechnology and Bioengineering 47 (1995), S. 550-556 
    ISSN: 0006-3592
    Keywords: spheroids ; porous and solid microcarriers ; CHO ; controlled release ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of the microcarrier type on the performance of a controlled release process used to produce a recombinant glycosyl-phosphatidylinositol anchored protein was investigated. Chinese hamster ovary (CHO) cells expressing the human melanoma tumor antigen (p97) were cultured in 10% serum on Cultispher-GH porous microcarriers and then, for protein production, maintained in 2% serum. Cells were harvested every 48 h and p97 was recovered at 90 μg/mL and 40% purity. Harvested p97 concentrations were increased by harvestingfrom spheroid (241 μg/mL) and smaller porous microcarrier, Cultispher-G (167 μg/mL) cultures. The low total cell specific p97 production of cells cultured on Cultispher-GH was due to necrosis of cells within the beads, decreased p97 expression of the immobilized cells, dilution by the liquid (up to 40% volume) associated with settled beads, and incomplete recovery of p97 from within the beads. Cells cultured on solid microcarriers, Cytodex-1, had the highest cell viability and cell specific p97 production, It is recommended that a two-stage cyclic harvesting process of cells cultured on small Cultispher-G or on Cytodex-1 beads would minimize protein loss and maximize cell specific protein recovery. © 1995 John Wiley & Sons Inc.
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  • 123
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    Biotechnology and Bioengineering 47 (1995), S. 567-574 
    ISSN: 0006-3592
    Keywords: immobilized Lactococcus diacetylactis ; alginate beads ; diffusionl/reactionl/growth model ; lactosecitrate cometabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A dynamic diffusion-reaction-growth model is proposed for the study of lactic fermentation, the bioconversion of citric acid, and cell release in an immobilized cell reactor [pH-stat continuous stirred tank-reactor (CSTR)]. The model correctly simulates the onset of fermentation and colonization of the gel, followed by the steady state. External diffusion is nonlimiting and internal diffusion is limited by high cell densities at the periphery of the gel beads. Lactose-citrate cometabolism in the gel is related to the distribution of active included biomass within the gel and to gradients of substrates (lactose, citrate) and products (lactate, pH) in the beads. The utilization of lactose is limited by reaction, whereas that of citrate is limited by diffusion. Cell release from gel to the liquid medium occurs in the external spherical cap of the beads. In this peripheral zone viability is maintained at around 90%. © 1995 John Wiley & Sons Inc.
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  • 124
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    Biotechnology and Bioengineering 47 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 125
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    Biotechnology and Bioengineering 47 (1995), S. 617-625 
    ISSN: 0006-3592
    Keywords: cross-flow ultrafiltration ; polysulfonic membranes ; proteins ; concentration-polarization ; pore size distributions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Flux and retention of 0.1%w/w aqueous solutions of several proteins [lysozyme, pepsin, bovine serum albumin (BSA), lipase, and γ-globulin] with molecular weights of 14.6, 36, 67, 801 and 150 kDa are studied when they are tangentially filtered, with transmembrane pressure differences until 1 MPa and circulation velocities in the re-tentate loop from 0.04 to 1.98 m/s (laminar regime), through two asymmetric polysulfone commercial membranes (E-100 with a nominal pore size of 0.01 μm and E-500 with a nominal pore size of 0.04 μm). Results are analyzed with the film theory for the concentration-polarization phenomenon, obtaining the mass transfer coefficient along with the apparent and true retention coefficients for the cell used, as a function of the feed circulation velocity and the molecular weight of the solute. The standard retention curves lead to pore size distributions differing from the nominal ones. These differences can be attributed to the modifications of the membranes when they are in operational conditions, probably due to protein adsorption. © 1995 John Wiley & Sons, Inc.
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  • 126
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    Biotechnology and Bioengineering 47 (1995), S. 651-665 
    ISSN: 0006-3592
    Keywords: abiotic proton balance ; acid, base ; bicarbonate buffer system ; fermentation ; pH ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The linear relation between the pH control reagent addition rate, the net conversion rates of metabolites, the carbon dioxide mass transfer rate and the feed rates is developed and shown to have the same form for batch, fed-batch, and continuous reactors, regardless of the number of feeds. The magnitudes of various biological and solution chemistry effects on the pH control reagent addition rate are estimated to be negligible, facilitating the use of the pH control reagent addition rate as a straightforward indicator of primary metabolism. Finally, application of the linear relation, termed the abiotic proton balance, is discussed for a number of fermentation processes. © 1995 John Wiley & Sons, Inc.
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  • 127
    ISSN: 0006-3592
    Keywords: taxol ; plant cell culture ; bioreactors, kinetics ; Taxus baccata ; secondary metabolite ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of biomass accumulation, nutrient uptake and taxol production of Taxus baccata cell suspensions were examined in three bioreactor configurations, viz. 250-mL Erienmeyerflasks, 1-L working volume pneumatically mixed (PMB), and stirred tank (STB) bioreactors. Qualitatively similar kinetics were observed in all three bioreactor types. Biomass accumulation and specific nutrient uptake rates exhibited biphasic characteristics. Carbohydrate uptake and biomass accumulation substantially ceased when phosphate was depleted from the medium. Phosphate was identified as a possible growth-limiting nutrient. Taxol accumulated exclusively in the second phase of growth. A maximum taxol concentration of 1.5 mg/L was obtained in the PMB which was fivefold greater than that obtained in the Erienmeyer flasks and the STB, but the relative kinetics of taxol production was the same in all three reactor types. Biomass yields were calculated from the kinetic data and a stoichiometry for biomass formation was evaluated. The similarity of kinetics in the three bioreactor configurations suggests that taxol production by T. baccata cell suspensions is amenable to scateup. © 1995 John Wiley & Sons, Inc.
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  • 128
    ISSN: 0006-3592
    Keywords: acidic fibroblast growth factor ; Escherichia coli ; sterilization ; comparative reasoning tools ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of various medium sterilization conditions on fermentations of a recombinant, acidic fibroblast growth factor (aFGF) producing Escherichia coli have been studied. Changes in the medium resulting from sterilization were monitored by pH and absorption spectra. This simple experiment provided excellent data for the demonstration of the usefulness of comparative reasoning tools in order to evaluate the effect of sterilization on fermentation performance. The time profiles of the main parameters (e.g., carbon dioxide evolution rate, dissolved oxygen, pH, and aFGF productivity) were simplified into piecewise contiguous linear segments, each of which was sequentially numbered. The length, position, and slope of each tine were then characterized. Application of the comparative reasoning tools confirmed that separate sterilization of the glucose was necessary for the success of the process, despite adding to the cost and complexity. The comparative data analysis also showed that scaleup with longer sterilization holding and cooling times would not be detrimental to aFGF production. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995), S. ii 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 130
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    Biotechnology and Bioengineering 47 (1995), S. 703-712 
    ISSN: 0006-3592
    Keywords: biofilm modeling ; detachment ; porous media ; biobarriers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A packed bed biofilm reactor inoculated with pure culture Pseudomonas aeruginosa was run under high substrate loading and constant flow rate conditions. The 3.1-cm-diameter cylindrical reactor was 5 cm in length and packed with 1-mm glass beads. Daily observations of biofilm thickness, influent and effluent glucose substrate concentration, and effluent dissolved and total organic carbon were made during the 13-day experiment. Biofilm thickness appeared to rech quasi-steady-state condition after 10 days. A published biofilm process simulation program (AQUASIM) was used to analyze experimental data. Comparison of observed and simulated variables revealed three distinct phases of biofilm accumulation during the experiment: an initial phase, a growth phase, and a mature biofilm phase. Different combinations of biofilm and mass transport process variables were found to be important during each phase. Biofilm detachment was highly correlated with shear at the biofilm surface during all three phases of biofilm development. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995), S. 25-35 
    ISSN: 0006-3592
    Keywords: DNA purification ; DNA-binding fluorophore ; Fluorescent ligand ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The fluorescent probe PO-PRO-3 was investigated as a potential ligand for the affinity immobilization and purification of genomic or plasmid DNA fragments. Affinities and mechanisms for PO-PRO-3 binding to superhelical and linearized pUC 18 plasmid DNA were examined through measurement of binding isotherms, continuous-variation analysis, and DNA titrations. In addition, the effects of DNA conformation, protein and RNA contaminants, solvent polarity, and ionic strength are examined with the aim of optimizing binding and elution conditions and of assisgning limits to the range of applicability of the affinity purification. © 1995 John Wiley & Sons, Inc.
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  • 132
    ISSN: 0006-3592
    Keywords: hollow fiber ; bioreactor ; immobilized enzymes ; porosity ; phospholipase A2 ; low densitylipoprotein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Previous studies have shown that the modification of low density lipoprotein (LDL) by the enzyme phospholipase A2(PLA2)results in a reduction of cholesterol levels in the plasma of hypercholesterolemic rabbits, due to accelerated clearance of the modified LDL. In the current study, we established techniques and optimized the ratio of enzyme to support for the immobilization of PLA2 on a polymeric support. Hollow fiber bioreactors made from polytetrafluoroethylene (PTFE) polymers were used to encapsulate immobilized PLA2. This design was adopted to eliminate hemolysis of red blood cells by the enzyme. Characterization of the resulting immobilized enzyme in terms of its activity, Michaelis-Menten kinetic constants, and the variation of its activity with incubation time is presented. The enzyme activity was not significantly altered upon incubation at 37°C in lipoprotein-deficient serum (LPDS), over the course of 2 months. The Michaelis-Menten kinetics constants are KM = 8.9 mM, Vmax = 6434.2 for the free enzyme and KappM = 16.7 mM, Vappmax = 619.7 for the immobilized enzyme. These data suggest that a system based on immobilized PLA2 in conjunction with hollow fiber bioreactors (HFBs) may be a good candidate for lowering LDL levels in plasma. © 1995 John Wiley & Sons, Inc.
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  • 133
    ISSN: 0006-3592
    Keywords: crystalline surface layers ; synthetic medium ; Bacillus stearotherrnophilus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bacterial cell surface layers (S-layers) which show a crystalline structure, defined pores, and a regular arrangement of functioal groups can be used for production of isoporous ultrafiltration membranes and as a matrix for immobilization of macromolecules. S-layer-carrying cell wall fragments from thermophilic Bacillaceae possess an extremely thin peptidoglycan-containing layer with pores larger than those in the S-layer lattice. Thus, they can directly be used for biotechnological applications, when an S-layer protein pool is stored in the rigid cell wall layer which is released during cell wall preparation, forming an inner S-layer. In the present study, a synthetic medium for Bacillus stearothermophilus PV 72 was developed by applying the pulse and shift technique with the aim to produce cell wall fragments with before-mentioned properties by varying the growth conditions in condtinuous culture. The organism was grown at 57°C in a bioreactor with 1 L working volume equipped with exhaust gas analysis and connected to a PC-based process control system. Biomass concentration was 2.2 g/L out of 8 g/L glucose at a dilution rate of 0.3 h-1, giving a biomass productivity of 0.66 g/L h. Although the organism was grown under different conditions, no change in peptidoglycan composition, extent of peptidoglycan crosslinking, and content of secondary cell wall polymers was observed. The amount of S-layer protein pool stored in the rigid cell wall layer and the autolytic activity depended mainly on the specific growth rate. Cell wall fragments with properties required for ultrafiltration membrane production could be produced by parameter settings in continuous culture. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995), S. 89-107 
    ISSN: 0006-3592
    Keywords: fibrin ; collagen ; proteolysis ; plasminogen ; diffusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A numerical model based on the convective-diffusive transport of reacting and adsorbing proteolytic enzymes within erodible fibrous biopolymers was used to predict lysis fronts moving across biogels such as fibrin or collagen. The fiber structure and the transport properties of solutes in fibrin (or collagen) were related to the local extent of dissolution within the dissolving structure. An accounting for solubilization of adsorbed species into solution from the eroding fiber phase provided for complete conservation of mass in reacting systems containing over 10 species. At conditions of fibrinolysis typical of clinical situations, the model accurately predicted the dynamic rate of lysis front movement for plasmin, urokinase, and tissue plasminogen activator (tPA)-mediated lysis of fibrin gels measured in vitro. However, under conditions of extremely fast fibrinolysis using high enzyme concentrations, fibrinolytic fronts moved very rapidly (〉0.1 mm/mm) - faster than predicted for diffusionlimited reactions - at nearly constant velocity for over 2 h, indicating non-Fickian behavior. This was due to proteolysis-mediated retraction of dissolving fibrin fibers that resulted in fiber convection and front-sharpening within 3 μm of the reaction front, as observed by digitally enhanced microscopy. In comparing the model to fibrinolysis measurements using human lys77-plasmin, the average first order rate constant for non-crosslinked fibrin bond cleavage by fibrin-bound plasmin was calculated to be 5s-1 assuming that 10 cleavages per fibrin monomer were required to solubilize each monomer. The model accurately predicted lysis front movement using pressure-driven permeation of plasmin or urokinase into fibrin as well as literature data obtained under well- mixed conditions for tPA-mediated fibrinolysis. This numerical formulation provides predictive capability for optimization of proteolytic systems which include thrombolytic therapy, wound healing, controlled drug release, and tissue engineering applications. © 1995 John Wiley & Sons, Inc.
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  • 135
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    Biotechnology and Bioengineering 48 (1995), S. 123-132 
    ISSN: 0006-3592
    Keywords: secondary metabolite ; taxol ; ethylene ; carbon dioxide ; oxygen ; taxus cuspidata ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. © 1995 John Wiley & Sons, Inc.
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  • 136
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    Biotechnology and Bioengineering 48 (1995), S. 158-168 
    ISSN: 0006-3592
    Keywords: Vero ; vaccinia virus expression ; MOI ; βhCG ; mRNA ; immunoligical and biological activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Synthesis of the β-subunit of human chorionic gonado-tropin (βhCG) in Vero cells by the recombinant vaccinia virus has been studied. The yield of βhCG was a function of the multiplicity of infection (MOI), and was highest at 25 MOI. The kinetics of synthesis and initial secretion of βhCG, deduced from the pulse-chase experiments were “zero order.” At 30 h postinfection, the relative values of net synthesis and secretion rates were 4.0 AU. mm2 βhCG/106 cells. h and 1.55 AU. mm2 βhCG/106 cells. h, respectively. The time required to secrete 50% of intracellular βhCG was 210 min. Pulse-chase data also showed that 24% of βhCG was degraded intracelluiarly within 10 h, of which 17% was detected in the autoradiogram. Along with 30 kD βhCG, a satellite band of 28 kD was evident among the peptide synthesized in Vero cells. The molecular weight of vaccinia-derived βhCG was 13 kD more that its nonglycosylated form, indicating extensive glycosylation in Vero cells. The mRNA levels in infected Vero cells at different postinfection times were quantified by excess DNA dot-blot hybridization. It appears that the Vero cell possesses some host cell-associated factor(s), which prevented the transcription of early βhCG-mRNA promoted by the early signal of the vaccinia P 7.5 promoter. The half-life of βhCG-mRNA, as determined by follow-up of decay after blocking transcription initiation, was found to be 6.4 h. The synthesized βhCG was immunoreactive as it reacted with monoclonal and polyclonal monospecific antibodies. The subunit was also biologically active, as it combined with native βhCG to form heterodimer βhCG, which competed with 125I-hCG for radioreceptors and stimulated testosterone synthesis in Leydig cells. © 1995 John Wiley & Sons, Inc.
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  • 137
    ISSN: 0006-3592
    Keywords: biphasic system ; activity coefficient ; organic solvents ; solvation ; hydrolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Kinetics of lipase-catalyzed hydrolysis of esters were modeled using reactant activities for aqueous-organic, biphasic systems. By using thermodynamic activities of the substrates in ordinary rate equations, the kinetic parameters were corrected for the contribution of substrate-solvent interactions and a uniform quantification of the substrates for lipase attached to the interface can be achieved. The kinetic parameters, on the basis of their thermodynamic activities, should be constant in different systems, provided that the solvents do not interfere with the binding of the substrates to the enzyme nor affect the catalytic mechanism. Experimental and computational methods on how to obtain the thermodynamic activities of the substrates are presented. Initial rates were determined for Pseudomonas cepacia lipase (PcL)-catalyzed hydrolysis of decyl chloroacetate in dynamic emulsions with various solvents. The thermodynamic equilibrium and corrected kinetic constants for this reaction appeared to be similar in various systems. The kinetics of PcL in an isooctane-aqueous biphasic system could be adequately described with the rate equation for a ping-pong mechanism. The observed inhibitory effect of decanol appeared to be a consequence of this mechanism, allowing the backreaction of the decanol with the chloroacetyl-enzyme complex. The kinetic performance of PcL in systems with toluene, dibutyl ether, and methyl isobutyl ketone could be less well described. The possible causes for this and for the remaining differences in corrected kinetic parameters are discussed. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995), S. 207-213 
    ISSN: 0006-3592
    Keywords: Bacillus thuringiensis ; glucose ; thuringiensin production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fed-batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed-batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995), S. 234-245 
    ISSN: 0006-3592
    Keywords: phosphorus removal, biological ; metabolic model ; polyphosphate ; PHB, ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A metabolic model of the biological phosphorus removal process has been developed and validated previously for complex conversions during the process under anaerobic and aerobic conditions at different growth rates in sequencing batch reactors in steady state. For additional validation of the metabolic model, the model was applied to the dynamic conditions which occur during the start-up phase of the biological P removal in the presence and absence of non-polyP heterotrophic microorganisms. In a laboratory scale sequencing batch reactor, experiments were performed to examine the enrichment of the population with polyphosphate organisms during the start-up and the subsequent shift from non-polyP, heterotrophic organisms to polyP organisms in the sludge. The effect of different influent loading patterns for acetate and phosphate was studied. In these experiments, the maximal growth rate of the polyP organisms and the behavior of the internal storage compounds could be derived. The metabolic model was capable of describing the experimental results, without the need to adjust the kinetic or stoichiometric parameters obtained under steady state conditions. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995) 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 141
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    Biotechnology and Bioengineering 48 (1995), S. 278-288 
    ISSN: 0006-3592
    Keywords: affinity chromatography ; biomimetic dye ; Candida boidinii ; enzyme purification ; oformate dehydrogenase ; triazine dye ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Formate dehydrogenase (FDH, EC 1.2.1.2) was purified from Candida boidinii cells in a single step by biomimetic-dye affinity chromatography. For this purpose, seven' biomimetic analogues of the monochlorotriazine dye, Cibacron® Blue 3GA (CB3GA), and parent dichloro-triazine dye, Vilmafix® Blue A-R (VBAR), bearing a car-boxylated structure as their terminal biomimetic moiety, were immobilized on crosslinked agarose gel, Ultrogel® A6R. The corresponding new biomimetic-dye adsorbents, along with nonbiomimetic adsorbents bearing CB3GA and VBAR, were evaluated for their ability to purify FDH from extracts obtained after press-disintegration of C. boidinii cells. Optimal conditions for maximizing specific activity of FDH in starting extracts (1.8 U/mg) were realized when cell growth was performed on 4% methanol, and press disintegration proceeded in four consecutive passages before the homogenate was left to stand for 1 h (4°C). When compared to nonbiomimetic adsorbents, biomimetic adsorbents exhibited higher purifying ability. Furthermore, one immobilized biomimetic dye, bearing as its terminal biomimetic moiety mercap-topyruvic acid linked on the chlorotriazine ring (BM6), displayed the highest purifying ability. Adsorption equilibrium data which were obtained for the BM6 adsorbent in a batch system corresponded well to the Langmuir isotherm and, in addition, breakthrough curves were taken for protein and FDH adsorption in a fixed bed of BM6 adsorbent. The dissociation constant ( KD) of the complex between immobilized BM6 and FDH was found to equal 0.05 μM. Adsorbent BM6 was employed in the purification of FDH from a 18-L culture of C. boidinii in a single step (60% overall yield of FDH). The purified FDH afforded a single-band on sodium dodecyl sulphate poly-acrylamide gel electrophoresis, and a specific activity of 7,0 U/mg (30°C). © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995), S. 324-332 
    ISSN: 0006-3592
    Keywords: plasma fractionation ; mixing ; protein precipitation ; metal affinity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Previous work by us and others has shown that mixing impacts apparent protein solubility in single protein precipitations. In this work, we probe the effects of contacting conditions on fractional precipitation behavior at the bench scale. We have chosen metal affinity precipitation as our model system; the kinetics of this mode of precipitation are very rapid and largely irreversible and, consequently, mixing conditions govern the extent of fractionation and purity of the product in such a process. Our experimental strategy involved a three-pronged approach to control the effects contacting conditions on precipitate yield, purity, and particle size distribution. First, we studied the impact of process variables that control precipitant concentrations in the reactor including impeller speed and precipitant addition rate. Second, we controlled the rate of precipitation by changing the initial protein concentration to alter the protein-protein collision rate. Third, we examined the role of the molecular-level kinetics of affinity precipitation by using modifiers that compete with surface moieties to bind the metal ion, thereby reducing its availability. Our model process and protein system consisted of zinc precipitations of mixtures of bovine serum albumin and bovine γ-globulins, carried out at a nominal 1-L scale; glycine was examined as a modifier. Faster impeller speeds and lower precipitant addition rates increased the desired protein yields, decreased purities, and reduced average precipitate particle size. Higher initial protein concentrations were found to produce precipitates with higher yields, lower purities and diminished particle size. Experiments with glycine indicated that modifiers in the precipitant solution serve to increase product purity, decrease yield, and increase the average particle size in bench-scale precipitations. © 1995 John Wiley & Sons, Inc.
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  • 143
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    Biotechnology and Bioengineering 46 (1995), S. 99-108 
    ISSN: 0006-3592
    Keywords: microemulsions, nonionic ; protein, separations ; sorbitan esters ; alkyl ethoxylates ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, the extraction of cytochrome c utilizing various nonionic surfactant microemulsions has been tested to determine the effect of surfactant structure on protein partitioning. Surfactants tested include a linear alcohol ethoxylate (Neodol 91-2.5), two alkyl phenol ethoxylates (lgepal CO-520, Trycol 6985), and a series of alkyl sorbitan esters that are either ethoxylated (Tweens) or un-ethoxylated (Spans). Initial attempts to extract hemoglobin into Neodol 91-2.5 Winsor II microemulsions (oil-continuous) appeared successful based on heme estimation. Careful analysis showed that the hemoglobin had dissociated prior to extraction and that only the heme was extracted with false positive results. In fact, Neodol 91-2.5 microemulsions were unable to extract a variety of proteins with differing biophysical properties. Among all the other nonionic surfactant microemulsions tested only those made using sorbitan esters extracted significant amounts of cytochrome c. The partition coefficients achieved in this study are more than an order of magnitude higher than that seen previously in the literature for comparable sorbitan systems. However, this partition coefficient is extremely sensitive to ionic strength. At an ionic strength as low as 0.001 M, the partition coefficient is reduced to that seen in previous studies. We have found that protein partitioning in sorbitan ester microemulsions is not a function of water content. In addition, extraction is not a function of either alkyl chain length, or polyethylene oxide molecular weight. Hence, the sorbitan group appears to have an important role in extraction, possibly through a weak electrostatic protein-surfactant interaction. © 1995 John Wiley & Sons, Inc.
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  • 144
    ISSN: 0006-3592
    Keywords: glucosylation ; alcohol ; hydrolysis, reverse ; galactoside ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Alkyl β-D-glucosides were synthesized from D-glucose and alcohols by reverse hydrolysis using the commercially available almond β-D-glucosidase in 9:1 (v/v) acetonitrile-water medium. The main characteristics of this enzyme-catalyzed glucosylation were established by using 2-hydroxybenzyl alcohol. The reaction is entirely regio- and stereoselective. The solvent plays a fundamental role because, by decreasing the water concentration in the medium, the shift of the reaction equilibrium toward synthesis is realized without using an excessive amount of alcohol. Nevertheless, a minimum amount of water is necessary to maintain the enzyme activity. In contrast to the use of the enzyme in aqueous medium, the pH of the added water in acetonitrile did not influence the synthesis. Using this procedure, we have conducted systematic glucosylation of numerous alcohols and we have investigated enzyme specificity and alcohol reactivity. The enzyme has a pronounced affinity for the alcohols containing a phenyl group, and enantioselectivity for the aglycon is obtained with 1-phenylethyl alcohol. Moreover, by using almond β-D-glucosidase it was also possible to synthesize alkyl β-D-galactosides. © 1995 John Wiley & Sons, Inc.
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  • 145
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    Biotechnology and Bioengineering 46 (1995), S. 93-98 
    ISSN: 0006-3592
    Keywords: photosynthetic reaction center ; liquid crystals ; cubic phases ; immobilization ; Chloroflexus aurantiacus ; photochemistry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Photosynthetic reaction centers, isolated and purified from the facultative phototrophic bacterium Chloroflexus aurantiacus, were immobilized in optically transparent lipidic cubic phases composed of 42% (w/w) 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine and 58% (w/w) water. The immobilized photosynthetic protein retains its native properties, as indicated by visible and circular dichroic spectra. The ground state visible spectrum of the immobilized reaction centers is very similar to the corresponding spectrum in aqueous solution, indicating that the protein pigments are not extracted into the lipidic regions of the cubic phase. The secondary structure of the protein is maintained in the immobilized state, as determined by far-UV circular dichroism spectroscopy in the 200- to 250-nm range. Moreover, immobilized reaction centers retain their photochemical activity: a reversible photo-oxidation of the primary electron donor (P) is seen upon continuous illumination. Furthermore, the entrappment of reaction centers does not affect the kinetics of charge recombination between the photo-oxidized primary donor (P+) and the photoreduced primary quinone acceptor, generated by a short flash of light. Reaction centers devoided of the secondary quinone acceptor can be easily reconstituted in cubic phases by means of their coimmobilization with 1,4-naphtoquinone. Indeed, the kinetics for charge recombination in reconstituted reaction centers is dramatically slower than the corresponding kinetics in the unreconstituted protein. Interestingly, immobilized reaction centers are significantly stabilized as compared with reaction centers in aqueous solution: the integrity of the protein in the cubic phase is maintained for at least 5 months, whereas in water solution 50% of the activity is lost within 2 months. © 1995 John Wiley & Sons, Inc.
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  • 146
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    Biotechnology and Bioengineering 46 (1995), S. 132-138 
    ISSN: 0006-3592
    Keywords: centrifuge ; disk stack ; mammalian cell separation ; hybridoma cells ; cell harvest ; debris ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A prototype disc stack centrifuge was tested for the separation of mammalian cell cultures from 80- and 2000-L fermentations. The clarification capacity for mammalian cells was excellent, but some smaller particles remained in the supernatant and reduced its usefulness for downstream processing. In order to identify the source of such particle formation, several parameters were assessed and minimum particle size for separation was calculated. An analysis of particle distribution was performed. Temperature and pressure effects inside the centrifuge bowl were measured. Some modifications of mechanical engineering can be suggested for the improvement of the use of standard disc stack centrifuges for mammalian cells. © 1995 John Wiley & Sons, Inc.
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  • 147
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    Biotechnology and Bioengineering 46 (1995), S. 139-146 
    ISSN: 0006-3592
    Keywords: fluidized-bed bioreactor ; concentration profile ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A fully predictive mathematical description of a three-phase, tapered, fluidized-bed bioreactor is developed. This mathematical model includes the effects of the tapered bed, variable dispersion coefficient, and variable solid holdup upon the concentration profiles developed in the bed. In addition, the effect of the concentration profile which is developed inside the biocatalyst bead is included by means of an effectiveness factor calculation. Using accepted correlations for the dispersion coefficient and for the liquid, gas, and solid holdup in the bed, the model is fully predictive. The model was found to adequately predict experimental obtained concentration profiles. Then, the model was used to examine the various phase holdups through the bed and the degree to which the dispersion coefficient varied through the bed. The effect of changes in these calculated variables upon the reaction rate is discussed. © 1995 John Wiley & Sons, Inc.
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  • 148
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    Biotechnology and Bioengineering 46 (1995), S. 147-158 
    ISSN: 0006-3592
    Keywords: CHO cell ; cell aggregation ; recombinant human interferon-γ ; mammalian cell culture ; cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-γ (IFN-γ), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-γ. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-γ within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-γ are heterogeneous in their environment, with variable access to O2 and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. © 1995 John Wiley & Sons, Inc.
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  • 149
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    Biotechnology and Bioengineering 46 (1995), S. 117-131 
    ISSN: 0006-3592
    Keywords: biochemical model ; Penicillium chrysogenum ; flux analysis ; penicillin ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Based on a review of the Penicillium chrysogenum biochemistry a stoichiometric model has been set up. The model considers 61 internal fluxes and there are 49 intracellular metabolites which are assumed to be in pseudo-steady state. In addition to the intracellular fluxes the model considers the uptake of 21 amino acids. From the stoichiometric model the maximum theoretical yield of penicillin V is calculated to 0.43 mol/mol glucose. If biosynthesis of cysteine is by direct sulfhydrylation rather than by transsulfuration, the maximum theoretical yield is about 20% higher, i.e., 0.50 mol/mol glucose. The theoretical yield decreases substantially if α-aminoadipate is converted to 6-oxo-piperidine-2-carboxylic acid (OPC). If only 40% of the α-aminoadipate is recycled, the maximum theoretical yield is 0.31 mol/mol glucose. The uptake rates of glucose, lactate, γ-aminobutyrate, and 21 amino acids were measured during fed-batch cultivations. The rates of formation of penicillin V, δ-(L-α)-aminoadipyl-L-cysteinyl-D-valine (ACV), OPC, and the pool of isopenicillin N, 6-APA, and 8-HPA were also measured. Finally the synthesis rates of the biomass constituents RNA/DNA, protein, lipid, carbohydrate, and amino carbohydrate were measured. From these measured rates and the stoichiometric model the metabolic fluxes through the different intracellular pathways are calculated. The calculations show that penicillin formation is accompanied by a large flux through the pentose phosphate (PP) pathway due to a large requirement for nicotinamide-adenine dinucleotide phosphate (NADPH) used in the biosynthesis of cysteine. If cysteine is added to the medium, the flux through the PP pathway decreases. From the stoichiometric model YxATP is calculated to 87 mmol adenosine triphosphate (ATP)/g dry weight (DW), and from the flux calculations mATP is found to 3 mmol ATP/g DW/h. © 1995 John Wiley & Sons, Inc.
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  • 150
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    Biotechnology and Bioengineering 46 (1995), S. 172-174 
    ISSN: 0006-3592
    Keywords: reversed micelles ; extraction ; trypsin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: By using trypsin as the model protein and AOT as the model surfactant, the effect of a variety of solvents on protein transfer and activity recovery during the liquid-liquid reversed micellar extraction was investigated. It was found that several solvents, including isooctane, octane, heptane, and kerosene, had a similar effect on the recovery of trypsin activity after a full cycle of forward and backward extraction, and could all be used as the solvents for AOT-reversed micelles in trypsin extraction. Two other solvents (hexane and cyclohexane), however, were not so efficient. © 1995 John Wiley & Sons, Inc.
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  • 151
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    Biotechnology and Bioengineering 46 (1995), S. 159-171 
    ISSN: 0006-3592
    Keywords: denitrification ; nitrate ; nitrite ; cyclic bioreactors ; wastewater treatment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biological reduction of nitrate and nitrite was studied with a continuously operated cyclic reactor. The medium was fed to the reactor during the first phase of the cycle, and the effluent was drawn from the reactor during the third phase of the cycle; reaction occurred throughout the cycle. The process was described mathematically based on kinetic expressions revealed in an independent study. The model equations were subjected to detailed analysis with numerical codes based on the bifurcation theory for forced systems. The analysis has shown that in the operating parameter space there are extensive regions where the system can reach up to three different periodic states. The results of this analysis are shown in the form of two-dimensional operating diagrams. Numerical results have also shown that under certain operating conditions nitrate can be completely eliminated, while nitrite remains practically untreated. An experimental unit was designed, constructed, and used in experiments with a strain of Pseudomonas denitrificans [American Type Culture Collection (ATCC) 13867] under different operating conditions. The experimental results confirmed the theoretical predictions both qualitatively and quantitatively. Conditions under which complete reduction of both nitrate and nitrite is achieved, were found and experimentally verified. The results of this study suggest a methodology for analysis and design of cyclically operated bioreactors employed in denitrification of wastewaters. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 46 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 153
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    Biotechnology and Bioengineering 46 (1995), S. 175-179 
    ISSN: 0006-3592
    Keywords: catalase ; oxidation ; hydroperoxide ; enantioselectivity ; alcohol ; organic solvents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The catalytic properties of bovine liver catalase have been investigated in organic solvents. In tetrahydrofuran, dioxane, and acetone (all containing 1% to 3% of water), the enzyme breaks down tert-butyl hydroperoxide several fold faster than in pure water. Furthermore, the rate of catalase-catalyzed production of tert-butanol from tert-butyl hydroperoxide increases more than 400-fold upon transition from aqueous buffer to ethanol as the reaction medium. The mechanistic rationale for this striking effect is that in aqueous buffer the rate-limiting step of the enzymatic process involves the reduction of catalase's compound I by tert-butyl hydroperoxide. In ethanol, and additional step in the reaction scheme becomes available in which ethanol, greatly outcompeting the hydroperoxide, is oxidized by compound I regenerating the free enzyme. In solvents, such as acetonitrile or tetrahydrofuran, which themselves are not oxidizable by compound I, catalase catalyzes the oxidation of numerous primary and secondary alcohols with tert-butyl hydroperoxide to the corresponding aldehydes or ketones. The enzymatic oxidation of some chiral alcohols (2,3-butanediol, citronellol, and menthol) under these conditions occurs enantioselectively. Examination of the enantioselectivity for the oxidation of 2,3-butanediol in a series of organic solvents reveals a considerable solvent dependence. © 1995 John Wiley & Sons, Inc.
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  • 154
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    Biotechnology and Bioengineering 48 (1995), S. 452-460 
    ISSN: 0006-3592
    Keywords: chromatography ; displacement ; protein purification ; cation exchange systems ; amino acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Although the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low-molecular-weight dendritic polymers can be successfully employed as displacers for protein purification in ion-exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation-exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low-molecular-weight compounds under dis-placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low-molecular-weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low-molecular-weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. © 1995 John Wiley & Sons, Inc.
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  • 155
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    Biotechnology and Bioengineering 48 (1995), S. 461-475 
    ISSN: 0006-3592
    Keywords: countercurrent gradient chromatography ; protein separation ; human serum albumin ; chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The continuous separation of proteins was performed in a countercurrent gradient chromatography (CGC) system. A magnetically stabilized fluidized bed (MSFB) was used to establish true countercurrent contact of a solid resin with a liquid buffer. STable pH gradients were formed in the system in less than 10 min and remained stable throughout the course of the separation experiment (〉2 h). The shape of the pH gradient, which ultimately controls the resolution and purity of the separation, can be controlled by making simple adjustments in the interstitial velocities of the liquid and solid phases. We have performed the separation of myoglobin and human serum albumin (HSA) using this device and achieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been developed that predicts the focusing behaviour and capabilities of the CGC system. Using the model, we have estimated the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations. © 1995 John Wiley & Sons, Inc.
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  • 156
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    Biotechnology and Bioengineering 48 (1995), S. 481-489 
    ISSN: 0006-3592
    Keywords: bioseparation ; protein refolding ; reverse micelles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The recombinant production of proteins leads to inclusion bodies which contain aggregated proteins in active, partially active, and inactive conformational states. These aggregated proteins must be extracted from the inclusion bodies, unfolded, and carefully refolded to the active and the stable conformational state. Mechanistic models for protein refolding are briefly presented. Different strategies and protocols are presented that lead to the active and stable protein conformational state. The techniques presented include chaperonin-assisted refolding, amino acid substitution, polyethylene glycolassisted refolding, protein refolding in reverse micelles, and antibody-assisted refolding of proteins. The techniques presented together provide a reasonable framework of the state-of-the-art and may be carefully applied to the bioseparation of other proteins and biological macromolecules of interest. © 1995 John Wiley & Sons, Inc.
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  • 157
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    Biotechnology and Bioengineering 48 (1995), S. 520-528 
    ISSN: 0006-3592
    Keywords: human growth hormone ; animal cell culture ; purification ; serum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Human growth hormone (hGH) is a polypeptide with 191 amino acids and a molecular mass of 22 kilodaltons. With the aid of computer molecular simulation, an hGH analog was created by altering an hGH gene to reflect the change of one amino acid (glycine [G] 120 to arginine [R]) within the third α-helix of the hGH molecule. This hGH analog, named hGHG120R, was found to be an hGH antagonist. It may have important implications in treating human conditions in which hGH levels are abnormally high, as found in type I diabetics. Several hundred milligrams of purified hGHG120R were needed to determine the biological activity of the antagonist in animal models. A multistep downstream process was developed to purify hGHG120R from cultured mouse L cells transfected with the hGHG120R gene. The process consisted of cell clarification, salt precipitation, membrane ultrafiltration, reversed phase high performance liquid chromatography, phase separation, and lyophiliation. This work discusses the rationale for the design of the process and experimental results on the purification of hGHG120R using the process. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 48 (1995), S. 557-557 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 159
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    Biotechnology and Bioengineering 48 (1995), S. 573-584 
    ISSN: 0006-3592
    Keywords: state estimation ; structured modeling ; lipase ; Candida rugosa ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple structured mathematical model coupled with a methodology of state and parameter estimation is developed for lipase production by Candida rugosa in batch fermentation. The model describes the system according to the following qualitative observations and hypothesis: Lipase production is induced by extracellular oleic acid present in the medium. The acid is transported into the cell where it is consumed, transformed, and stored. Lipase is excreted to the medium where it is distributed between the available oil-water interphase and aqueous phase. Cell growth is modulated by the intracellular substrate concentration. Model parameters have been determined and the whole model validated against experiments not used in their determination. The estimation problem consists in the estimation of three state variables (biomass, intra- and extracellular substrate) and two kinetic parameters by using only the on-line measurement provided by exhaust gas analysis. The presented estimation strategy divides the complex problem into three subproblems that can be solved by stable algorithms. The estimation of biomass (X) and the specific growth rate (μ), is achieved by a recursive prediction error algorithm using the on-line measurement of the carbon dioxide evolution rate. μ is then used to perform an estimation of intracellular substrate and the other kinetic parameter related to substrate transport (A) by an adaptive observer. Extracellular substrate is then evaluated by means of the estimated values of intracellular substrate and biomass through the material balance of the reactor. Simulation and experimental tests showed good performance of the developed estimator, which appears suitable to be used for process control and monitoring. © 1995 John Wiley & Sons, Inc.
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  • 160
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    Biotechnology and Bioengineering 48 (1995), S. 585-591 
    ISSN: 0006-3592
    Keywords: phenoloxidases ; enzyme immobilization ; reverse micelles ; organic gels ; biotic detoxification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4°C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 45 (1995) 
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    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 162
    ISSN: 0006-3592
    Keywords: nitrification ; immobilized cells ; activation energy ; diffusion limitation ; temperature ; Chemistry ; Biochemistry and Biotechnology
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Activation energies of suspended and immobilized nitrifying bacteria were determined and compared to determine if diffusion limitation results in decreased sensitivity for temperature. The activation energy for the respiration activity of suspended Nitrosomonas europaea and Nitrobacter agilis was found to be 86.4 and 58.4 kJ mol-1, respectively. The activation energy for oxygen diffusion in the support material, κ-carrageenan, determined from the effect of temperature on the effective diffusion coefficient (D), was 17.2 kJ mol-1. Consequently, the apparent actvation energy of diffusion limited cells should be lower. It was indeed shown that due to the effect of diffusion limitation and to temperature effects on the Monod constant Ks, the immobilized-cell activity was less sensitive to temperature. The apparent activation energy for immobilized Ns. europaea was between 28.6 and 94.2 kJ mol-1 and for immobilized Nb. agilis between 1.4 and 72.9 kJ mol-1, depending on the oxygen concentration and temperature. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 45 (1995), S. 33-41 
    ISSN: 0006-3592
    Keywords: lipase ; reverse micelles ; surfactants ; esterification ; glycerides ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The activity of purified Pseudomonas cepacia lipase has been investigated in esterification reactions of various aliphatic alcohols with natural fatty acids. The reactions were carried out in microemulsions formed in isooctane by bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT). Kinetic studies showed that the reaction follows a ping-pong bi-bi mechanism with inhibition by both substrates. The apparent kinetic parameters of the reaction were found to be Km octanol = 310 mM, Km lauric acid = 78 mM, and Vmax = 250 μmol min-1 mg-1. The same system was used for the synthesis of mono- and diglycerides from glycerol and lauric acid, which was successful at very low wo values. The catalytic behavior of P. cepacia lipase was also studied in esterification reactions performed in a nonionic microemulsion system formulated by tetraethyleneglycoldodecylether (C12E4). The optimum activity was found at about wo = 8. The apparent values of Vmax app and Km app for octanol were calculated and found to be 100 μmol min-1 mg-1 and 76 mM, respectively. © 1995 John Wiley & Sons, Inc.
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  • 164
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    Biotechnology and Bioengineering 45 (1995), S. 63-68 
    ISSN: 0006-3592
    Keywords: dissolved-hydrogen probe ; anaerobic digestion ; hydrogen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The concentration of diatomic hydrogen in the liquid phase of an anaerobic digester was used to determine the onset of digester failure induced by substrate overloading. The construction of an inexpensive probe to measure dissolved hydrogen, having a partial pressure detection limit of 30 Pa, is described. An increase in the partial pressure of dissolved hydrogen, from less than 30 Pa to 400 Pa, was observed when the D-glucose concentration in a laboratory-scaled digester was increased rapidly to 10 mM. However, when the digester was gradually overloaded, an increase in the dissolved-hydrogen partial pressure was not observed until after the digester failed. The accumulation of volatile fatty acids and digester failure were observed at dissolved-hydrogen partial pressures below 30 Pa. © 1995 John Wiley & Sons, Inc.
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  • 165
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    Biotechnology and Bioengineering 45 (1995), S. 95-95 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 166
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    Biotechnology and Bioengineering 45 (1995), S. 91-94 
    ISSN: 0006-3592
    Keywords: mass transfer ; Monod equation ; growth rate ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An alternative interpretation of the growth rate-substrate concentration dependence is presented. This is based on the assumption that the main factors affecting growth rate are transfer of substrate from the medium and the maximum growth velocity, which is that observed when no substrate limitations occur. This approach allows the approximate prediction of one of the two kinetic constants required, and may be of great use, especially for continuous cultures. It is the first attempt to provide a phenomenological explanation for the large variations observed in the values of the Monod constant, Ks, reported in the literature. © 1995 John Wiley & Sons, Inc.
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  • 167
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    Keywords: biosensor ; insecticides ; phycoerythrin ; alkaline phosphatase ; immobilization and chemiluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A molecular assembly in which a conjugated polymer is interfaced with a photodynamic protein is described. The conjugated polymer, functionalized with biotion, is designed such that it can be physisorbed on or chemically grown off a glass surface. The streptavidin-derivatized protein is immobilized on the biotinylated polymer matrix through the strong biotin-streptavidin interactions. The assembly, built on the surface of an optical fiber or on the inside walls of a glass capillary, forms an integral part of a biosensor for the detection of environmental pollutants such as organophosphorus-based insecticides. The Protein in the system can be replaced by any biological macromolecule of interest. We study one specific case, the enzyme alkaline phosphatase. The enzyme catalyzes a reaction producing an intermediate compound that chemiluminesces, and the chemiluminescence singnal is monitored to detect and quantify insecticides such as paraoxon and methyl parathion. Preliminary results indicate ppb level detection with response time less than 1 minute. © 1995 John Wiley & Sons, Inc.
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  • 168
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    Biotechnology and Bioengineering 45 (1995), S. 129-134 
    ISSN: 0006-3592
    Keywords: water activity ; Lipozyme ; diglyceride isomerization ; multiresponse lipase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of the thermodynamic activity of water (aw)on the synthesis of triolein catalyzed by Mucor miehei lipase was investigated. Its effect on the equilibrium and on the rates of the different reactions present, esteification and mono- and diglyceride isomerization, was evaluated through measurements made in controlled water activity atmosphere. The apparent equilibrium constants were measured from the concentration of the different species as a function of the intial glycerol-to oleic-acid ratio using all the values at once with a multi-response nonlinear regression technique. Rate constants were determined from kinetic measurements and non-linear regression uning the variation of the concentration of all significant species in the system. Except for the synthesis of diolein from monoolein, which shows a maximum for aw ≈ 0.5, the apparent rate constants of the various reactions are not significantly affected by the value of the water activity. The equilibrium is shifted to-ward the synthesis of triolein for low values of aw, indicating that in the design of a process for triglyceride synthesis, using M. miehei lipase as a catalyst, the water activity can be lowered to extreme values to favor the synthesis, without any sacrifice on the productivity of the process. © 1995 John Wiley & Sons, Inc.
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  • 169
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    Biotechnology and Bioengineering 47 (1995), S. 165-173 
    ISSN: 0006-3592
    Keywords: error vector ; physiological state recognition ; fuzzy inference ; metabolic reaction model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The physiological states with respect to cell growth and ethanol production in a yeast fed-batch culture expressed in linguistic form could be recognized on-line by fuzzy inferencing based on error vectors. The error vector was newly defined here in a macroscopic elemental balance equation. The physiological states for cell growth and ethanol production were characterized by error vectors using many experimental data from fed-batch cultures. Fuzzy membership functions were constructed from the frequency distributions of the error vectors and state recognition was performed by fuzzy inferencing. In particular, an unusual physiological state for a yeast cultivation, in which aerobic ethanol production was accompanied by very low cell growth, could be recognized accurately. According to the results of the state recognition, an energy parameter, the P/O ratio in the metabolic reaction model was adaptively estimated, and the cell growth was successfully evaluated with the estimated P/O. © 1995 John Wiley & Sons, Inc.
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  • 170
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    Keywords: promoter system ; recombinant proteins ; Escherichia coli ; plasmid construct ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A pH-inducible promoter system was characterized and its potential applicability in recombinant protein production was evaluated using a plasmid construct, pSM552-545C-, in which the promoter and activator coding sequences of the cad operon were inserted into the upstream region of a lacZ′ reporter gene. Graded gene expression levels with respect to culture pH between 8.0 and 5.5 were observed and the induction range can be as high as 200-fold. The effects of several cultivation parameters, including pH, temperature, induction cell density, and inoculum size, were systematically examined. The practical application of this expression system to high level production of recombinant proteins was successfully demonstrated using a rich medium, superbroth. An extremely high recombinant protein productivity at a value of approximately 1.4 g/L with a specific expression level as high as 35% of total cellular protein can be obtained in a simple batch cultivation. The behavior of this expression system was further investigated using chemostat cultures. An uncommon relationship between the volumetric or specific recombinant protein activity and the dilution rate, with a maximal activity at a dilution rate of approximately 0.4 h-1was observed. © 1995 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 47 (1995), S. 234-242 
    ISSN: 0006-3592
    Keywords: gene expression ; Bacillus subtilis ; transcriptional state heterogeneity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Using a flow cytometry-based technique for detecting p-galactosidase. lacZ fusions have been used to study the pattern of gene expression exhibited by stationary phase cultures of Bacillus subtilis that have initiated the developmental pathway of spore formation. We have found that within such cultures there exist two distinct cell types: one that has induced the developmental program of gene expression and one that has not. This heterogeneity among transcriptional states is shown to be established early during the stationary phase and plays a significant role in influencing later stationary phase events. Additionally, when this technique is used to study gene expression in mutants that display altered patterns of gene expression, we are able to conclude that the cellular apparatus responsible for sensing and transducing stationary phase developmental signals represents a bottleneck that controls the expression of certain early stationary phase genes. These findings provide a demonstration of the utility of single cell measurements of gene expression and indicate that unless culture heterogeneity is properly taken into account, standard measurements of gene expression may not provide information suitable for the analysis of events occurring at the cellular level. © 1995 John Wiley & Sons, Inc.
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  • 172
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    Biotechnology and Bioengineering 47 (1995), S. 243-251 
    ISSN: 0006-3592
    Keywords: transfectoma ; chimeric antibody ; stability ; flow cytometry ; RT-PCR ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To design the scheme of large-scale production of chimeric antibody for the postexposure prophylaxis of hepatitis B virus (HBV) infection, the stability of transfectomas (H69K-1 and 6-31) in regard to antibody production was examined during a long-term, repeated fed-batch culture without selection pressure using antibiotics. Although the H69K-1 transfectoma was more stable than the 6-31 transfectoma, both displayed gradual decreases in specific antibody productivity (qAb) for the first several weeks of cultivation. During this period, qAb was de-creased by 40% to 50%. This loss of qAb was due mainly to the appearance of a nonproducing population (NP) of transfectoma, which was monitored throughout the culture by flow cytometry and the limiting dilution method. However, an NP did not overtake the culture and was balanced with a producing population (P) of transfectoma, resulting in stable antibody production. The subclones of NP obtained at the end of long-term culture were further characterized by reverse transcription-polymerase chain reaction assay of the heavy and light chain mRNA. All the subclones of NP derived from H69K-1 transfectoma had only light chain mRNA. On the other hand, an NP in the 6-31 transfectoma culture was heterogeneous. Some subclones of NP derived from 6-31 transfectoma had only heavy chain mRNA and other subclones had only light chain mRNA. Taken together, the results obtained here suggest that selection pressure is necessary for a long-term, continuous culture, because stable antibody production in a long-term culture was achieved only after a significant loss of antibody productivity. Accordingly, a batch culture appears to be more appropriate for large-scale chimeric antibody production without selection pressure. © 1995 John Wiley & Sons, Inc.
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  • 173
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    Biotechnology and Bioengineering 47 (1995), S. 308-318 
    ISSN: 0006-3592
    Keywords: hybridoma ; cell growth ; antibody production ; toxic waste removal ; electrical technique ; electrokinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ammonium and lactate are two known toxic products detrimental to mammalian cell growth and productivity. An electrokinetic technique, utilizing an electrophoretic mechanism, was developed to remove these cellular wastes in-situ from suspension hybridoma (ATCC CRL-1606) cultures to enhance cell growth and productivity. This technique applies continuously a dc electric field to selectively remove the electrically charged wastes. The experiments were shown to be successful in the removal of externally added 10 rnM ammonium and 45 mM lactate while maintaining the chemostatic condition of culture medium in a cell-free condition under an electric current density of 50 A/m2. Toxic levels of ammonium were added, ranging from 7.5 to 12.5 mM, at the start of the hybridoma culture, and the applied dc electric fields were able to completely remove these added materials. This in turn released the inhibition and restored the cell growth. Finally, this electrokinetic technique was applied to the batch and glutamine fed-batch hybridoma cultures. At an applied electric current density of 50 A/m2, this was able to completely remove cell-produced ammonium and increased the cell growth and antibody titer by 30% to 50%, respectively, compared to the control experiment in the absence of the electric field. Lastly, the applied electric current density of 50 A/m2 did not affect cellular functionalities such as glucose and glutamine consumption and antibody productivity.© 1995 John Wiley & Sons, Inc
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  • 174
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    Biotechnology and Bioengineering 47 (1995), S. 327-333 
    ISSN: 0006-3592
    Keywords: sulphur ; thiosulphate ; Thiobacilli ; fed-batch reactor ; oxygen limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This study shows that, in a sulphide-oxidizing bioreactor with a mixed culture of Thiobacilli, the formation of sulphur and sulphate as end-products from the oxidation of sulphide can be controiledinstantaneously and reversibiy by the amount of oxygen supplied. It was found that at sulphide loading rates of up to 2.33 mmol7/L · h, both products can be formed already at oxygen concentrations below 0.1 mg/L. Because the microorganisms tend to form sulphate rather than forming sulphur, the oxygen concentration is not appropriate to optimize the sulphur production. Within less than 2 h, the system can be switched reversibly from sulphur to sulphate formation by adjusting the oxygen flow. This is below the minimum doubling time (2.85 h) of, e.g., Thiobacillus neapolitanus and Thiobacillus 0,18 which indicates that one metabolic type of organism can probably perform both reactions. Under highly oxygen-limited circumstances, that is, at an oxygen/sulphide consumption ratio below 0.7 mol · h-1 mol · h-1 thiosulphate is abundantly formed. Because the chemical sulphide oxidation results mainly in the formation of thiosulphate, it is concluded that, under these circumstances, the biological oxidation capacity of the system is lower than the chemical oxidation capacity. The oxidation rate of the chemical sulphide oxidation can be described by a first-order process (k =-0.87 h-1).© 1995 John Wiley & Sons, Inc
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  • 175
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    Biotechnology and Bioengineering 47 (1995), S. 355-372 
    ISSN: 0006-3592
    Keywords: steric mass action ; step gradient ; ion exchange ; protein purification ; nonlinear adsorption ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nonlinear adsorption plays an important role in determining the chromatographic behavior of proteins in preparative ion-exchange chromatography. In this article, the steric mass action (SMA) isotherm is used in conjunction with a mass transport model to describe nonlinear cation-exchange chromatography. Excellent agreement is observed between simulated and experimental step gradient separations of the proteins α-chymotryp-sinogen A, cytochrome C, and lysozyme. A systematic method of selecting the optimum step gradient program for a given separation problem is presented and employed to study optimization of step gradient chromatography under conditions of high mass loading. This article includes consideration of the effects of the adsorption properties of the feed stream, the feed stream concentration, protein solubility, and otherconstraints on the optimum separation conditions.© John Wiley & Sons, Inc.
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  • 176
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    Biotechnology and Bioengineering 47 (1995), S. 401-404 
    ISSN: 0006-3592
    Keywords: crossflow filtration ; periodical stopping ; air bubbling ; baker's yeast ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The periodical stopping of permeation flow was applied to increase the permeation flux in crossflow filtration of commercially available baker's yeast cell suspension. The permeation flux after 3 h filtration in the crossflow filtration increased to 8 × 10-5 m3 /m2 s (290 L/m2 h) from 2 × 10-5 m3/m2 s (72 L/m2 h) by applying the periodical stopping of permeation. Introduction of air bubbles during the stopping period of permeation further increased the flux.© John Wiley & Sons, Inc.
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  • 177
    ISSN: 0006-3592
    Keywords: nitrogenase ; mass spectrometry ; fermentation monitoring ; Azotobacter vinelandii ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This study evaluated the feasibility of monitoring nitro-genase activity in situ through measurement of N2 uptake rate (NUR) using off-gas mass spectrometry. Four 50-L cultures of Azotobacter vinelandii were grown aer-obically in nitrogen-free medium to cell densities of 1.0-1.3gL-1 magnetic-sector mass spectrometer was used to monitor NUR along with other gas exchange rates. The small specific uptake rate (1.2 mmol g-1 h-1) and low cell density were found to lead to a NUR below the measurement accuracy limits under normal conditions. An operating strategy and feed gas mixture (40% O2, 45% N2 15% Ar) were designed to improve the signal-to-noise ratio while maintaining dissolved O2 and N2 levels in desired ranges. The fraction of N2 removed from the air stream was increased approximately 5-fold from 0.2% to 1.0% and the measurement noise was reduced 25-fold from a baseline of ±5to ±0.2 mmol L-1 h-1. The NUR measurements were compared against in vivo and in vitro acetylene reduction assays as well as on-line cell growth rate measurements. While electron transfer requirements predict an NUR-to-acetylene reduction rate ratio of 0.33, measured ratios for the in vivo and in vitro assays were 0.8 and 0.44, respectively. This suggests that other rate-limiting steps were present in the case of the in vivo assay. In accordance with reports in the literature, no concomitant hydrogen evolution was detected. This is the first reported continuous and direct measurement of NUR in fermentation and demonstrates a novel approach for improving measurement accuracy through rational adjustment of operating conditions. The technique has potential to provide useful insight for development and control of microbial nitrogen fixation processes.© John Wiley & Sons, Inc.
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  • 178
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    Biotechnology and Bioengineering 47 (1995), S. 431-443 
    ISSN: 0006-3592
    Keywords: AtT-20 spheroids ; alginate entrapment ; 31P and 1H NMR ; pancreas, bioartificial ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Transformed, insulin-secreting endocrine cells have been proposed as an alternative to islets for the development of a bioartificiat pancreas. With appropriate immunoprotection, such cells may be implanted without the need for patient immunosuppression. Use of continuous cell lines alleviates the cell availability limitation, but poses questions regarding thestability and biochemical and secretory function of the preparation, especially in the longterm. We have developed a bioreactor/perfusion system, compatible with a horizontal-bore NMR instrument, that can maintain immunoprotected endocrine cells for prolonged periods of time. 31P NMR spectroscopy was used to study the bioenergetics of recombinant, insulin-secreting mouse pituitary AtT-20 cells entrapped as spheroids in calcium alginate/poly-L-lysine/alginate beads. NMR provided data verifying the macroscopic homogeneity within the bioreactor and allowing the evaluation of changes in cellular bioenergetics for a period of 70 days under different culture conditions. Levels of high-energy phosphates changed slightly during the first 40 days of the experiment, then decreased considerably as cell death occurred. Rates of glucose consumption and insulin-related peptide secretion also remained constant for 40 days and decreased rapidly thereafter. This study constitutes the beginning of an extensive quantitative analysis of the biochemistry of transformed endocrine cell lines in a sequestered, artificial tissue environment. © 1995 John Wiley & Sons Inc.
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  • 179
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    Biotechnology and Bioengineering 47 (1995), S. 451-460 
    ISSN: 0006-3592
    Keywords: biosorption ; cadmium ; copper ; zinc ; two-systems ; ascophyllum nodosum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A biosorbent prepared from Ascophyllum nodosum seaweed biomass, FCAN2, was examined for its sorption capacity. Equilibrium batch sorption studies were performed using two-matal systems containing either (Cu + Zn), (Cu + Cd), or (Zn + Cd). In the evaluation of the two-metal sorption system performance, simple isotherm curves had to be replaced by three-dimensional sorption isotherm surfaces. In order to describe the isotherm surfaces mathematically, three Langmuir-type models were evaluated. The apparent one-parameter Langmuir constant (b) was used to quantify FCAN2 “affinity” for one metal in the presence of another one. The uptake of Zn decreased drastically when Cu or Cd were present. The uptake of Cd wasmuch more sensitive to the presence of Cu than to that of Zn. The presence of Cd and Zn alter the “affinity” of FCAN2 for Cu the least at high Cu equilibrium concentrations. The mathematical model of the two-metal sorption system enabled quantitative estimation of one-metal (bio)sorption inhibition due to the influence of a second metal. © 1995 John Wiley & Sons Inc.
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    Biotechnology and Bioengineering 47 (1995), S. 470-475 
    ISSN: 0006-3592
    Keywords: chlorophenol ; cresol ; cell growth ; flavobacterium species ; cell death ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of three aromatic compounds, p-cresol, 2,4-dichlorophenol (DCP), and 2,4,6-trichlorophenol (TCP), on cell growth and pentachlorophenol (PCP) degradation bya Flavobacterium species were investigated. While p-cresol was not degraded by this bacterium, DCP and TCP were simultaneously degraded with PCP. Both DCP and TCP lowered cell growth and PCP degradation rate. Cell growth was modeled by cell death, because p-cresol, DCP, and TCP were toxic to the organism. A new model was used to predict cell death rate in a mixture of two toxic compounds from the cell death kinetics for each individual compound. PCP degradation rates were modeled by conventional inhibition models, but only over a small concentration range for the secondary toxic compound. However, a new empirical model described PCP degradation over a wider concentration range of the secondary toxic compound. © 1995 John Wiley & Sons Inc.
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  • 181
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    Keywords: diffusion ; gel ; alginate ; lactose ; lactic acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A non-steady-state method has been used for determining the effective diffusion coefficient, De, and a distribution constant, Ki, of small molecules in alginate gel beads. A mathematical model based on Pick's law and includingexternal film diffusion resistance describe the diffusion process. Criticalexperimental parameters for the estimation of De and Ki, for both one- and two-parameter methods were the initial solute concentration in the bulk liquid, the void fraction inthe reactor, and the experimental starting point. In our analysis, the two-parameter method is preferable. Incorporation of an estimate of the film resistance into the overall model increased the estimated values of De significantly and improved the stability of the term over a range of reactor agitation rates. © 1995 John Wiley & Sons Inc.
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    Biotechnology and Bioengineering 47 (1995), S. 509-519 
    ISSN: 0006-3592
    Keywords: reverse micelles ; lysozyme ; ribonuclease-a ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experiments are reported here on the equilibrium partitioning of lysozyme and ribonuclease-a between aqueous and reversed micellar phases comprised of an anionic surfactant, sodium di-2-ethylhexyl sulfosuccinate (AOT), in isooctane. A distinct maximum, [P]rm,max was found for the quantity of a given protein that can be solubilized in the reverse micelle phase by the phase-transfer method. This upper limit depended upon the size of the protein, the surfactant concentration, and the aqueous phase ionic strength, and was determined by complex formation between protein and surfactant molecules to form an insoluble interfacial precipitate at high values of [P]rm. In this work, it was found to be possible to dissociate the protein-surfactant complex and recover the precipitated protein. The kinetics of protein-surfactant complex formation depended upon the nature and concentration of the solubilized protein and on the surfactant concentration. Calculations of micellar occupancy and the relative surface areas of protein molecules and surfactant head-groups suggested that it was the exposure of the solubilized protein to the bulk organic solvent which promoted protein-surfactant complex formation as [P]rm → [P]rm,max. In the light of the experimental results and calculations described above, a mechanistic model is proposed to account for the observed phenomena. This is based upon the competing effects of increasing the solubilized protein concentration and the corresponding increase in the rate of protein-surfactant complex formation. The dynamic nature of the reverse micelles is inherent in the model, explaining the formation of the interfacial precipitate with time and its dependence on the internal phase volume of the micellar phase. Experiments on the co-partitioning of water and measurement ofthe AOT concentration in both phases verified the loss of protein, water, and surfactant from the organic phase at high values of [P]rm. © 1995 John Wiley & Sons Inc.
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    Biotechnology and Bioengineering 47 (1995), S. 541-549 
    ISSN: 0006-3592
    Keywords: solanum aviculare ; immobolized plant cells ; oxygen mass transfer ; effective diffusivity ; solasodine ; Thiele modules ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Oxygen transfer characteristics of self-immobilized Solanum aviculare cells were measured using aggregates 3.0 to 12.5 mm in diameter. Apparent specific oxygen uptake rates in the absence of external boundary layers varied from 5.9 × 10-11 to 8.5 × 10-7 kg kg-1 s-1 dry weight, but did not decline continuously with increasing particle size. The effective diffusivity of oxygen in deactivated aggregates increased with particle diameter, varying from 5.0 × 10-11 to 1.0 × 10-9 m2 s-1 or between 2% and 40% of the molecular diffusivity in water at the same temperature. Gas spaces detected in the larger aggregates were confined to the central core and were not distributed throughout the tissue to facilitate oxygen transfer. Oxygen consumption rates in the absence of diffusional limitations were estimated using the relationship between the observable Thiele modulus and effectiveness factor for zero-order reaction. The calculated results indicated severe oxygen limitations in the aggregates, but were inconsistent with the observation that relatively large S. aviculare aggregates contained a high fraction of viable cells and were capable ofactive growth and steroidal alkaloid synthesis. This work suggests that oxygen delivery is facilitated in living plant cell aggregates by mechanisms which depend on metabolic activity and which do not function in deactivated cells. © 1995 John Wiley & Sons Inc.
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    Biotechnology and Bioengineering 47 (1995), S. 535-540 
    ISSN: 0006-3592
    Keywords: hybridoma ; flow cytometry ; cell cycle ; surface lgG ; antibody secretion rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Previous experiments have shown that population average surface lgG content is correlated with the specific antibody production rates of batch hybridoma cultures. Therefore, surface associated lgG content of single hybridoma cells might indicate antibody secretion rates of individual cells. Moreover, the surface lgG content should reflect the pattern of secretion rates during the cell cycle. To probe for lgG secretion rates during the cellcycle, a double staining procedure has been developed allowing simultaneousflow cytometric analysis of surface lgG content and DNA content of murine hybridoma cells. Crosslinking of the surface associated immunofluorescence with the cell by paraformaldehyde fixation permits subsequent DNA staining without loss of immunofluorescence. The optimized protocol has been used to determine the pattern of the surface lgG fluorescence as a function of the cell cycle position. It is highest during the G2+M cell cycle phase and the experimental data are in excellent agreement with the previously predicted secretion pattern during the cell cycle. © 1995 John Wiley & Sons Inc.
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  • 185
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    Biotechnology and Bioengineering 47 (1995), S. 585-595 
    ISSN: 0006-3592
    Keywords: biofilm ; wastewater treatment ; airlift reactor ; nitrification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: For a stable and reliable operation of a BAS-reactor a high, active biomass concentration is required with mainly biofilm-covered carriers. The effect of reactor conditions on the formation of nitrifying biofilms in BAS-reactors was investigated in this article. A start-up strategy to obtain predominantly biofilm-covered carriers, based on the balancing of detachment and a biomass production per carrier surface area, proved tp be very successful. The amount of biomass and the fraction of covered carrier were high and development of nitrification activity was fast, leading to a volumetric conversion of 5 kgN · m-3 · d-1 at a hydraulic retention time of 1h. A 1-week, continuous inoculation with suspended purely nitrifying microorganisms resulted in a swift start-up compared with batch addition of a small number of biofilms with some nitrification activity. The development of nitrifying biofilms was very similar to the formation of heterotrophic biofilms. In contrast to heterotrophic bio-films, the diameter of nitrifying biofilms increased during start-up. The detachment rate from nitrifying biofilms decreased with lower concentrations of bare carrier, in a fashion comparable with heterotrophic biofilms, but the nitrifying biofilms were much more robust and resistant. Standard diffusion theory combined with reaction kinetics are capable of predicting the activity and conversion of biofilms on small suspended particles. © 1995 John Wiley & Sons Inc.
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  • 186
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    Biotechnology and Bioengineering 47 (1995), S. 596-608 
    ISSN: 0006-3592
    Keywords: amino acid addition ; protein stability ; protease ; fed-batch ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this work, feeding policies aimed to avoid cellular stress responses as indicated by an increase in ATP-dependent proteolysis are tested. A set of experiments was carried out where glucose, IPTG (inducer), and phenylalanine (rate-limiting precursor) were added gradually in a fed-batch fashion. A significant increase in CAT activity was found compared with pulse-induction. In addition, there was a substantial increase in the rate of CAT synthesis as well as in the final specific CAT activity when phenylatanine and the inducer were added simultaneously. CAT degradation was confirmed through Western blotting analysis. Protease analysis (SDS-GPAGE) indicated lower proteolytic activity for the IPTG and phenylalanine fed-batch cases. GroEL immunoas-says indicated that amplification of stress proteins occurred upon CAT induction. This research impacts the yield of soluble cytoplasmic proteins in Escherichia coli and suggests that metabolically based induction/feeding policies are beneficial. © 1995 John Wiley & Sons Inc.
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  • 187
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    Biotechnology and Bioengineering 47 (1995), S. 626-632 
    ISSN: 0006-3592
    Keywords: adaptive estimation ; specific growth rate ; aerobic fermentation processes ; nonlinear systems ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The specific growth rate of the biomass, a very important parameter of almost every fermentation process, cannot be measured directly or estimated from related variables, as the concentrations of biomass, substrates, or products, due to the lack of reliable and cheap sensors. In this article a stable adaptive estimator of the specific growth rate is designed for those aerobic processes where the measurement of the oxygen uptake rate is available on-line. This particular approach can be applied also for other reaction rates if the model of the process satisfies some very general assumptions, which make the dynamics of the measured reaction rate a nonlinear function only of two unknown parameters, the specific growth rate and its time derivative. With respect to a previous similar approach, the new estimator has one additional parameter and a different nonlinear structure. From the analysis of the dynamics of the estimation error, a tuning criterion is derived, by which the two different algorithms can be compared under similar conditions. Simulation results show a good performance of both estimators for various kind of processes and disturbances. © 1995 John Wiley & Sons, Inc.
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  • 188
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    Biotechnology and Bioengineering 47 (1995), S. 575-584 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The starch-binding domains of glucoamylase I (SBD of GA-I) from Aspergillus awamori and of cyclodextrin glucanotransferase (domain E of CGTase) from Bacillus macerans were fused to the C-terminus of β-galactosidase (β-gal) The majority of the fusion proteins produced in Escherichia coli were found as inclusion bodies. Active fusion proteins were purified by partial solubilization of the inclusion bodies with 2 M urea followed by affinity chromatography. Adsorption isotherms of purified fusion proteins on corn starch and cross-linked amylose were generated. The β-gal fusion proteins had similar affinities for cross-linked amylose and corn starch but significantly different saturation capacities on corn starch. The adsorption and elution data from the potato starch column as well as the adsorption isotherms of p-gal-domain E fusion protein (BDE109) on corn starch and cross-linked amylose demonstrated that domain E of CGTase is an independent domain, which retained its starch-binding activity when separated from the other four (A-D) domains in CGTase. © 1995 John Wiley & Sons Inc.
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  • 189
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    Biotechnology and Bioengineering 47 (1995), S. 609-615 
    ISSN: 0006-3592
    Keywords: Trichoderma reesei ; image analysis ; morphology ; modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The microscopic morphology of Trichoderma reesei QM 9414, growing in submerged culture, was studied by image analysis. The morphology was characterized by the total hyphal length, the total number of tips, the number of actively growing tips, and the length of the main hypha. To describe the growth of a single mycelium a simple model is set-up. The main features of the model are: (1) saturation type kinetics for the tip extension of the individual branches within the mycelium; and (2) random branching with a frequency function, which is proportional to the total hyphal length. The model is used to simulate a population of mycelia, where spore germination is described with a log-normal distribution. From the simulation of the population, the average properties of the mycelia, e.g., the average total hyphal length, are calculated, and by fitting the model to experimental data the model parameters are estimated. Finally, the distribution function with respect to the mycelia properties, that is, number of tips and total hyphal length, is calculated, and it corresponds well with the experimental determination of the distribution function. © 1995 John Wiley & Sons Inc.
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  • 190
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    Biotechnology and Bioengineering 47 (1995), S. 633-639 
    ISSN: 0006-3592
    Keywords: protein adsorption ; sequential ; polymer colloids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The aim of the present work is to study the sequential adsorption of F(ab′)2 and bovine serum albumin (BSA) molecules adsorbed onto positively and negatively charged polystyrene latexes. Cationic and anionic latexes were prepared by emulsifier-free emulsion polymerization. Adsorptions of F(ab′)2 on both latexes at a low ionic strength and different pHs were performed. The cationic latex showed a higher adsorption of F (ab′)2 molecules over a range of pH, which could be due to the formation of multilayers. Sequential adsorption of anti-CRP F(ab′)2 and monomeric BSA were performed at two different pre-adsorbed F(ab′)2 amounts on both types of latex. Displacement of F(ab′)2 occurred only when the preadsorbed amounts were larger than a certain critical value, which depends on the adsorption pH. A greater displacement of larger preadsorbed amounts might be the result of a weaker contact between the protein molecules and the polystyrene surface. The displacement of F(ab′)2 previously adsorbed onto both latexes occurred due to pH changes, an increase of ionic strength and the presence of BSA molecules. The effect caused by these three factors was studied independently. The main factors in the desorption of F(ab′)2 on the anionic latex are the changes in pH and ionic strength, whereas on the cationic latex the desorption is mainly caused by the increase of the ionic strength and the presence of BSA. The colloidal stability of the immunotatex was improved by BSA adsorption, especially on cationic latex. © 1995 John Wiley & Sons, Inc.
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  • 191
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    Biotechnology and Bioengineering 47 (1995), S. 677-687 
    ISSN: 0006-3592
    Keywords: surface treatment ; polycation ; aggregation ; sedimentation ; flow ; Phanerochaete chrysosporium ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Immobilization of conidiospores of Phanerochaete chrysosporium by adhesion was investigated in static and flow conditions on flat and on porous supports. Reducing the electrostatic repulsion between the spores and the support by adsorption of polycations on the support allows a better adhesion efficiency and a higher density of adhering spores and does not affect germination and growth. Formation of spore aggregates either in the suspension (high ionic strength) or on the support tends to decrease the surface coverage and to give an inhomogeneous distribution of adhering spores due to detachment of aggregates. The density of spores adhering from a flowing suspension is lower as compared with static conditions and does not exceed about 2% of surface coverage; this is due to the influence of tangential forces, to the short contact time with the surface, and to perturbation of the hydrodynamics along the surface by the previously immobilized spores. Obtaining a high coverage of the support by immobilized spores requires the absence of a tangential motion. © 1995 John Wiley & Sons, Inc.
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  • 192
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    Biotechnology and Bioengineering 46 (1995), S. 43-53 
    ISSN: 0006-3592
    Keywords: acetate ; anaerobic biofilms ; mass transfer ; pH ; biofilm modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A detailed model acetate-utilizing methanogenic biofilms accounting for the diffusion of neutral and ionic species, chemical equilibrium, electroneutrality, gas production within the biofilm, pH-dependent Monod kinetics, and the presence of a concentration boundary layer is presented. The model qualitatively fits the pH profiles that are reported for acetate-utilizing methanogenic aggregates. A sensitivity analysis on the biological parameters showed that the flux of acetate is sensitive to the maximum utilization rate, half-saturation constant, and biofilm density for the bulk conditions investigated. Criteria when traditional biofilm models can be used to predict the flux of acetate into the biofilm are established. If the maximum pH change predicted using a hypothetical system is within ±0.05, the traditional model predicts the flux to within ±5% of the value calculated with the model developed in this study. © 1995 John Wiley & Sons, Inc.
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  • 193
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    Biotechnology and Bioengineering 48 (1995), S. 17-24 
    ISSN: 0006-3592
    Keywords: spheroid ; polymer ; temperature-responsive ; collagen ; crosslinkage ; gradient surface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple method for preparing multicellular spheroids from varied cell types has been successfully developed by using a stepwise gradient surface in cell attachability or detachability. The surface was composed of poly-N-isopropylacrylamide (PNIPAAm), a temperature responsive polymer, as a cell detaching component, and collagen as a cell attaching component. The surface functions as a culture substratum at 37°C; then, when lowering the temperature of culture medium, the cells attached to it detach as a self-supporting sheet. This is because PNIPAAm dissolves into the culture medium below the lower critical solution temperature (LCST; about 30°C), but it is insoluble above the LCST. The detached cell sheet forms a multicellular spheroid. The stepwise gradient surface which consisted of six different sectors was prepared by exposing a surface of the PNIPAAm-collagen mixture to ultraviolet (UV) irradiation six times using a photomask, sliding the hole position in the photomask, and changing the energy of UV irradiation. This was because crosslinking of collagen depended on the energy of UV irradiation, then, cell attachability to and detachability from the surface were tightly controlled by changing the energy.The stepwise gradient surface allowed us to easily determine optimal surface conditions to obtain good cell attachment and detachment as a self-supporting sheet from the surface to prepare multicellular spheroids. According to the evaluation of the attachability and detachability of 23 cell types, the optimal surface condition remarkably depended on each cell type. The detached cells under optimal surface conditions, including fibroblasts, osteoblastic cells, smooth muscle cells, and measangial cells, which were very difficult to form spherioids using conventional methods, were able to form multicellular spheroids. The results clearly demonstrate that the above-described method for preparing multicellular spheroids can be applied to varied cell types. © 1995 John Wiley & Sons, Inc.
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  • 194
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    Keywords: daunorubicin biosynthesis ; enzyme kinetics ; enzyme immobilization ; Streptomyces peucetius ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4°C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent Km (K′m) for carminomycin while the K′m for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the Vmax value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. © 1995 John Wiley & Sons, Inc.
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  • 195
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    Biotechnology and Bioengineering 48 (1995) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 196
    ISSN: 0006-3592
    Keywords: hydrolysis ; esterification ; Humicola lanuginosa ; Rhizomucor miehei ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lipolase and Lipozyme are produced in large quantities (as a result of genetic engineering and overexpression) for the detergents market and provide a cheap source of highly active biocatalysts. Humicola lanuginosa lipase (HIL) and Rhizomucor miehei lipase (RmL) have been isolated in partially purified form from commercial preparations of Lipolase and Lipozyme, respectively. These lipases were solubilized in Aerosol-OT (AOT)-stabilized water-in-oil (w/o) microemulsions in n-heptane. HIL and RmL activity in these microemulsions was assayed by spectrophotometric measurement of the initial rate of p-nitophenyl butyrate hydrolysis, and by chromatographic determination of the initial rate of octyl decanoate synthesis from 1-octanol and decanoic acid. The hydrolytic activity of HIL in microemulsions measured as a function of buffer pH prior to dispersal, followed a sigmoidal profile with the highest activities observed at alkaline pHs. This broadly matches the pH-activity profile for tributyrin hydrolysis by Lipolase in an aqueous emulsion assay. The hydrolytic activity of RmL in the same microemulsions, measured as a function of pH, gave a bell-shaped profile with a maximum activity at pH 7.5. Again, the observed pH-activity profile was similar to that reported for a purified RmL in a tributyrin-based aqueous emulsion assay. In contrast, the esterification activity exhibited by both HIL and RmL in AOT microemulsions over the available range pH 6.1 to 10.4, decreases as the pH increases, most likely reflecting the effect of substrate ionization. The dependence of the hydrolytic and condensation activity of HIL on R, the mole ratio of water to surfactant, were similar with both profiles exhibiting a maximum at R = 5. The hydrolytic and esterification activities of RmL followed similar R-dependent profiles, but the profiles in this case exhibited a maximum at R = 10. The water activities at these R values were directly measured as 0.78 and 0.9, respectively. Measured water activities were unperturbed by the presence of lipase at the concentrations used in these studies. © 1995 John Wiley & Sons, Inc.
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  • 197
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    Biotechnology and Bioengineering 48 (1995), S. 108-117 
    ISSN: 0006-3592
    Keywords: microfiltration ; hydrulic resistance ; fouling ; yeast ; depressurization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The hydraulic resistance and membrane fouling effects of Candida utilis in fermentation broth were investigated using Millipore PVDF 0.22-μm membranes (GVWP and GVHP) in a stirred-cell system at 50 kPa and 700 rpm. With the various components of broth, spent medium, which contains colloidal particles and macromolecules having sizes (0.32 to 2.67 μm) comparable with the membrane pores (actual range 0.26 to 0.63 μm), was found to be the major contributing factor to the membrane fouling by broth through pore plugging. This led the spent medium to exhibit the highest hydraulic resistance (Rsm of 5.8E + 12 m-1) and percentage flux loss (81.0%) when compared with either intact cells alone in buffer or to whole broth. Intact cells appeared to physically block and protect the pores without significant adhesion, because of the relatively hydrophilic nature of their cell walls (hydrophobicity of 5.9% at hour 36), resulting in the lowest hydraulic resistance (Rsbc of 7.5E + 11m-1) and percentage flux loss (19.3%).However, the hydraulic resistance and percentage flux loss of broth increased as cells aged. This was attributed to the increase in particle loading (intact cells by 15.37%, released cell contents and cell fragments) and in the hydrophobicity of cell walls. Autoclaved broth, lysed broth and aged broth, which contained a larger portion of colloidal particles and released cell contents caused a more pronounced fouling effect. This was revealed by the absence of flux recovery after depressurization with continuous stirring, even when a hydrophilic membrane was used. Furthermore, the hydrophobicity of C. utilis was found to increase with yeast extract present in medium, and use of hydrophobic membranes helped enhance the fouling effect. Overall, the degree of irreversible membrane fouling could be revealed by the value of Rsm/Rt′ and the hydraulic resistance, which resulted from concentration polarrzation, could be revealed by the value of Rc/Rt′ where Rt = Rm + Rsm + Rc′ and Rm is the clean membrane resistance. © 1995 John Wiley & Sons, Inc.
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  • 198
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    Biotechnology and Bioengineering 48 (1995), S. 141-148 
    ISSN: 0006-3592
    Keywords: protien stability ; protein surface charge ; 6- amino-penicillanic acid ; biocatalysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. © 1995 John Wiley & Sons, Inc.
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  • 199
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    Biotechnology and Bioengineering 48 (1995), S. 341-354 
    ISSN: 0006-3592
    Keywords: affinity chromatography ; perfluoropolymers ; expanded bed ; dye-ligand ; dehydrogenases ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Malate dehydrogenase (MDH) and glucose 6-phosphate dehydrogenase (G6PDH) have been partially purified from preparations of homogenized yeast cells using Procion Yellow H-E3G and Procion Red H-E7B, respectively, immobilized on solid perfluoropolymer supports in an expanded bed. A series of pilot experiments were carried out in small packed beds using clarified homogenate to determine the optimal elution conditions for both MDH and G6PDH. Selective elution of MDH using NADH was effective but the yields obtained were dependent on the concentration of NADH used. Selective elution was found to be most effective when a low concentration of NaCl (0.1 M) was present. MDH could be recovered in 84% yield with a purification factor of 94 when this strategy was adopted. In the case of G6PDH, specific elution using NADP+ was successful in purifying G6PDH 178-fold in 96% yield. The dynamic capacity of both affinity supports was estimated by frontal analysis, in an expanded bed with unclarified homogenate, and corresponded to 17 U MDH/mL of settled Procion Yellow H-E3G perfluoropolymer support and 7.7 U H6PDH/mL of settled Procion Red H-E7B perfluoropolymer support. Expanded bed affinity chromatography of MDH resulted in an eluted fraction containing 89% of the applied activity with a purification factor of 113. Expanded bed affinity chromatography of G6PDH resulted in an eluted fraction containing 84% of the applied activity with a purification factor of 172. With both enzymes, the overall recovery of enzyme activity was greater than 94%, showing that the expanded bed approach to purification was nondenaturing. © 1995 John Wiley & Sons, Inc.
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  • 200
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    Biotechnology and Bioengineering 48 (1995), S. 406-414 
    ISSN: 0006-3592
    Keywords: membrane filtration ; protein separations ; albumin ; hemoglobin ; electrostatic interactions ; diafiltration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: One of the critical factors limiting the development of membrane systems for protein fractionation has been the poor selectivity that has generally been obtained with these membrane devices. We have demonstrated that it is possible to dramatically improve the selectivity of available membrane systems by exploiting the different electrostatic interactions between the two proteins and the membrane. The separation factor for the albumin-hemoglobin system could be increased to more than 70 simply by reducing the salt concentration and adjusting the pH to around 7 (near the isoelectric point of hemoglobin). This very high selectivity was a direct result of the strong electrostatic exclusion of the charged albumin from the membrane pores under these conditions. This high selectivity makes it possible to very effectively separate these albumin-hemoglobin mixtures using membrane filtration, and this was demonstrated experimentally using both a simple batch filtration process and a continuous diafiltration system. The hemoglobin recovery in the diafiltration experiment was greater than 70% after a 3-diavolume filtration, with the Hb purification factor being around 100 under these conditions. These results clearly demonstrate the potential of membrane systems for the fractionation of proteins even with very similar molecular weights. © 1995 John Wiley & Sons, Inc.
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