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  • Biochemistry and Biotechnology  (879)
  • Inorganic Chemistry  (608)
  • 1995-1999  (1,487)
  • 1990-1994
  • 1950-1954
  • 1997  (1,487)
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  • 1990-1994
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 1-10 
    ISSN: 0006-3592
    Keywords: metal biosorption ; acetone-washed yeast biomass ; Saccharomyces uvarum ; lead binding mechanisms ; X-ray photoelectron spectroscopy ; Fourier transform infrared photoacoustic spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The mechanism of lead cation biosorption by acetone-washed biomass of Saccharomyces uvarum was investigated by chemical modifications and spectroscopic monitoring of the cell components. Reacting the carboxyl groups with propylamine, which neutralizes these anions, considerably decreased the metallic ion uptake, indicating that negatively charged carboxyl groups play an important role in lead bisorption due to electrostatic attraction. After lead biosorption the photoacoustic Fourier transform infrared spectroscopy showed a change in the symmetrical stretch of the carboxylate groups of the acetone-washed yeast biomass, and the X-ray photoelectron spectroscopy oxygen peak was also found to be shifted. These findings support the hypothesis that lead uptake occurs mainly through binding to the carboxyl group. In X-ray photoelectron spectroscopy the nitrogen peak decreased after the biosorption of lead, suggesting that nitrogen-containing groups are also involved in the biosorption process. Acylation of amino groups was shown to increase the lead biosorption capacity. The acylation reaction converts the positively charged amino group to an amide capable of coordination to lead cations. Deproteination by boiling the biosorbent with NaOH increased the lead uptake. The acetone-washed biomass uptake of lead from an aqueous solution at ph 5.5 was 48.9 mg/g dry weight. Pure chitin adsorbed 48.8 mg lead/g dry weight. Mannan isolated from S. uvarum did not adsorb lead at all. Electrostatic attraction of the carboxyl groups and other anions present in the acetone-washed biomass, and complexation with nitrogen atoms, especially in chitin, appear to be the main mechanisms involved in lead cation biosorption. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 1-10, 1997.
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  • 2
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 16-27 
    ISSN: 0006-3592
    Keywords: uranium ; aluminum ; biosorption ; Rhizopus arrhizus ; mechanism ; competing ions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Uranium competitive uptake experiments by Rhizopus arrhizus were carried out at three different solution pH levels and in the presence of different concentrations of competing aluminum ions in order to examine the competing ion effect. The coion effect became more pronounced as the coion concentration in solution and pH level increased. A preliminary examination of the effect of aluminum on the rate of uranium uptake was also completed. Results showed that the presence of aluminum does not interfere with the kinetics of uranium uptake by R. arrhizus. Electron microscopic and energy dispersive X-ray analyses were also performed on samples of the biomass. The combination of spectral data and the information from the equilibrium studies and the kinetic studies suggested that aluminum interferes with the uranium biosorptive uptake capacity of R. arrhizus by the precipitation of a metastable amorphous hydroxy polymeric precipitate through a mechanism we refer to as steric competition. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 16-27, 1997.
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  • 3
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    Biotechnology and Bioengineering 55 (1997), S. 28-32 
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; poly(3-hydroxybutyrate) ; fed-batch fermentation ; phosphate limitation ; dissolved oxygen concentration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH4OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L · h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.
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  • 4
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    Biotechnology and Bioengineering 53 (1997), S. 10-16 
    ISSN: 0006-3592
    Keywords: microfiltration ; fouling ; yeast ; antifoam agents ; depressurization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The fouling effects of yeast fermentation broths of Candida utilis in the presence of various commercial antifoam agents (PPG2000, B5600, and G832) up to 4.0 mL/L were studied, using Millipore polyvinylidene fluoride 0.22-μm hydrophilic membranes (GVWP), in a stirred-cell system at 50 kPa and 700 rpm. PPG2000, which has a low value of work of adhesion (Wa of 0.81 mN/m), gave a steady flux of broth of 29 L/(h m2) and was found to have no significant fouling effect on the microfiltration of broth. G832, which has a high Wa, (26.0 mN/m) reduced the flux of the broth to 17 L/(h m2); i.e., by 42% when only 1.0 mL/L was used. However, B5600, which has a Wa of 14.3 mN/m, was found to enhance the flux of broth to 54 L/(h m2); i.e., by 86%, due to the preferential adsorption of the B5600 components onto the hydrophobic cell contents released. These results were reinforced by the depressurization experiments performed with both hydrophilic (GVWP) and hydrophobic (GVHP) membranes, using both young and aged broths. B5600 was found to be the optimum antifoam agent in this study in terms of membrane performance and defoaming efficiency. © 1997 John Wiley & Sons, Inc.
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  • 5
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    Biotechnology and Bioengineering 53 (1997), S. 21-25 
    ISSN: 0006-3592
    Keywords: starch fermentation ; recombinant yeast ; ethanol production ; glucoamylase activity ; fed-batch culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. © 1997 John Wiley & Sons, Inc.
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  • 6
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    Biotechnology and Bioengineering 55 (1997), S. 909-920 
    ISSN: 0006-3592
    Keywords: baculovirus ; insect cells ; metabolism ; Sf-9; high five™ ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five™) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five™ cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five™ cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five™ cell cultures, but not in Sf-9 cell cultures. In addition, High Five™ cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five™ cell cultures. It was also found that the medium had a significant effect on High Five™ cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.
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  • 7
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    Biotechnology and Bioengineering 55 (1997), S. 921-926 
    ISSN: 0006-3592
    Keywords: green fluorescent protein ; sensor ; on-line monitoring ; quantitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:921-926, 1997.
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  • 8
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    Biotechnology and Bioengineering 56 (1997), S. 1-8 
    ISSN: 0006-3592
    Keywords: transesterification ; hydrolysis ; water activity ; cutinase ; gas ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters 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 substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (aw = 0.2, aw = 0.4 and aw = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from aw = 0.2 to aw = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.
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  • 9
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    Biotechnology and Bioengineering 53 (1997), S. 372-378 
    ISSN: 0006-3592
    Keywords: glucose ; lactate ; on-line monitoring ; mammalian cell culture ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: On-line monitoring and control of cell culture fermentation is important for optimal and consistent production of biologicals. In this work, glucose and lactate concentrations are monitored on-line using a commercially available analyzer (Model 2700, Yellow Springs Instruments, Yellow Springs, OH) during batch and perfusion hybridoma cell fermentation. Cell free samples from the reactor are obtained using a 0.45 μm hollow fiber filtering system placed in a circulation loop. The samples were analyzed at specified times and the data are collected on a computer. A process control strategy was developed to control the concentrations of glucose and lactate in a perfusion reactor where the feed rate is adjusted to maintain their concentrations at desired set points. Hybridoma cells (A10G10) were cultivated in a high density perfusion culture where cell density increased from 2 to 14 million cells/mL. During this period the control algorithm successfully adjusted the perfusion rate while maintaining constant glucose and lactate concentrations. Glucose consumption and lactate accumulation rates as well as net lactate yield on glucose were monitored continuously during perfusion culture. These metabolic rates were observed to be independent of cell concentration and were used for the estimation of viable cell density in the reactor. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 372-378, 1997.
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  • 10
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    Biotechnology and Bioengineering 53 (1997), S. 406-408 
    ISSN: 0006-3592
    Keywords: bioaffinity separation ; reverse micelles ; trypsin-trypsin inhibitor ; nonionic surfactant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Trypsin inhibitor was converted to hydrophobic states by covalently combining cholesteryl groups using an acylation reaction, and was immobilized in reverse micelles composed of a nonionic surfactant. Using this reverse micellar phase containing trypsin inhibitor as an affinity ligand, trypsin was selectively separated with high recoveries from a mixture of several kinds of contaminating proteins by forward and backward extraction. No loss of activity of the recovered trypsin was observed through these operations. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 406-408, 1997.
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  • 11
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    Biotechnology and Bioengineering 53 (1997), S. 409-414 
    ISSN: 0006-3592
    Keywords: acylation ; pea isolate ; plant protein ; torus bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Acetylation, which acts on the amino groups of proteins, allows to increase the solubility and the emulsifying properties of pea isolate. Acetylation by acetic anhydride was carried out in a torus microreactor in semibatch and continuous conditions. The mixing characteristics, obtained by a residence time distribution (RTD) method, are the same in batch and continuous processes. The maximum acetylation degree reached by the torus reactor is higher than with the stirred reactor. Torus reactors are more efficient than stirred ones as shown by a conversion efficiency, defined by the quantity of modified lysine groups by consumed acetic anhydride. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 409-414, 1997.
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  • 12
    ISSN: 0006-3592
    Keywords: malate dehydrogenase ; protein chromatography ; Saccharomyces cerevisiae ; direct extraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel technique for protein chromatography has been developed, which can be used to extract proteins from particulate-containing solutions (such as fermentation broths or preparations of disrupted cells) on a continuous basis, and delivers clarified streams of purified product. Adsorbents deployed in this type of contactor are based on PVA-coated perfluorocarbons derivitized with affinity ligands such as triazine dyes. In this article, we describe the application of this equipment for the continuous purification of malate dehydrogenase from an unclarified homogenate of Saccharomyces cerevisiae, using a Procion Red HE-7B-derivitized adsorbent. Although operating conditions were not optimized to produce a product of maximized purification factor, concentration, and yield, we have shown that MDH can be purified continuously in 78% yield at a rate of 70 U/min, with a purification factor of approximately 10. This corresponds to specific productivity of approximately 0.35 U/min per milliliter of settled adsorbent, a higher specific productivity than was feasible with the same adsorbent using expanded bed adsorption (EBA). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 427-441, 1997.
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  • 13
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    Biotechnology and Bioengineering 53 (1997), S. 453-458 
    ISSN: 0006-3592
    Keywords: chemical permeabilization ; cell disruption ; urea ; EDTA ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetet-raacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 453-458, 1997.
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  • 14
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    Biotechnology and Bioengineering 56 (1997), S. 130-137 
    ISSN: 0006-3592
    Keywords: manganese peroxidase ; Phanerochaete chrysosporium ; pulsed packed-bed bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The bottleneck of the application of manganese peroxidase (MnP) on an industrial scale in pulp biobleaching or in degradation of hazardous compounds is the lack of an efficient production system. Three main problems arise for the continuous production of MnP during secondary metabolism of Phanerochaete chrysosporium: enzyme production occurs only under specific physiological conditions corresponding to C or N limitation, high O2 tension, and adequate Mn+2 concentration; the enzyme that is produced is destabilized by extracellular proteases; and excessive growth of the mycelium blocks effective oxygen transfer. To overcome these drawbacks, continuous production of MnP was optimized by selecting a suitable bioreactor configuration and the environmental and operating conditions affecting both enzyme production and stability. The combination between a proper feed rate and the application of a pulsation in a packed-bed bioreactor permitted the maintenance of continuous secretion of MnP while limiting mycelial growth and avoiding bed clogging. Environmental factors as an Mn+2 concentration of 5000 μM and high oxygen tension enhanced MnP production. The hydraulics of the bioreactor corresponding to a plug flow model with partial mixing and an operating hydraulic rentention time of 24 h were optimal to achieve stable operating conditions. This policy allowed long operation periods, obtaining higher productivities than the best reported in the literature. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 130-137, 1997.
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  • 15
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    Biotechnology and Bioengineering 53 (1997), S. 487-496 
    ISSN: 0006-3592
    Keywords: bacterial chemotaxis ; Escherichia coli ; random motility ; mathematical model ; sand core ; porous media ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The migration of chemotactic bacteria in liquid media has previously been characterized in terms of two fundamental transport coefficients - the random motility coefficient and the chemotactic sensitivity coefficient. For modeling migration in porous media, we have shown that these coefficients which appear in macroscopic balance equations can be replaced by effective values that reflect the impact of the porous media on the swimming behavior of individual bacteria. Explicit relationships between values of the coefficients in porous and liquid media were derived. This type of quantitative analysis of bacterial migration is necessary for predicting bacterial population distributions in subsurface environments for applications such as in situ bioremediation in which bacteria respond chemotactically to the pollutants that they degrade.We analyzed bacterial penetration times through sand columns from two different experimental studies reported in the literature within the context of our mathematical model to evaluate the effective transport coefficients. Our results indicated that the presence of the porous medium reduced the random motility of the bacterial population by a factor comparable to the theoretical prediction. We were unable to determine the effect of the porous medium on the chemotactic sensitivity coefficient because no chemotactic response was observed in the experimental studies. However, the mathematical model was instrumental in developing a plausible explanation for why no chemotactic response was observed. The chemical gradients may have been too shallow over most of the sand core to elicit a measurable response. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 487-496, 1997.
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  • 16
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    Biotechnology and Bioengineering 53 (1997), S. 515-522 
    ISSN: 0006-3592
    Keywords: RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine ; Serratia marcescens ; biotransformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biotransformation of hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) has been observed in liquid culture by a consortium of bacteria found in horse manure. Five types of bacteria were found to predominate in the consortium and were isolated. The most effective of these isolates at transforming RDX was Serratia marcescens. The biotransformation of RDX by all of these bacteria was found to occur only in the anoxic stationary phase. The process of bacterial growth and RDX biotransformation was quantified for the purpose of developing a predictive type model. Cell growth was assumed to follow Monod kinetics. All of the aerobic and anoxic growth parameters were determined: μmax, Ks, and Yx/s. RDX was found to competitively inhibit cell growth in both atmospheres. Degradation of RDX by Serratia marcescens was found to proceed through the stepwise reduction of the three nitro groups to nitroso groups. Each of these reductions was found to be first order in both component and cell concentrations. The degradation rate constant for the first step in this reduction process by the consortium was 0.022 L/g cells · h compared to 0.033 L/g cells · h for the most efficient isolate. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 515-522, 1997.
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  • 17
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    Biotechnology and Bioengineering 53 (1997), S. 547-559 
    ISSN: 0006-3592
    Keywords: recombinant protein production ; protein folding ; protein secretion ; human antithrombin III ; Chinese hamster ovary cells ; serum-free culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 547-559, 1997.
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  • 18
    ISSN: 0006-3592
    Keywords: recombinant protein production ; Escherichia coli ; heparinase ; bioprocess simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Heparinase I from flavobacterium heparinum has several potential clinical applications; the resulting high demands on protein purity and quantity can be met by recombinant expression in Escherichia coli. Based on laboratory scale experiments with insoluble heparinase I expression followed by renaturation, a process for production of 3 kg/year of heparinase I was designed. We present a comparative analysis of the production costs of soluble and insoluble heparinase I expression, as well as a generalized approach to sensitivity analysis, based on perturbation around a base case design scenario. This may assist focusing further development on process steps for which improvements both are feasible and result in significant cost saving. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 575-582, 1997.
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  • 19
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    Biotechnology and Bioengineering 53 (1997), S. 594-600 
    ISSN: 0006-3592
    Keywords: CHO ; mammalian cells ; metalloproteinase ; recombinant protein production ; expression stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Stability of heterologous protein expression during production is critical for regulatory approval of vaccine and therapeutic products. Leishmania GP63, a zinc metalloproteinase that is a potential vaccine candidate, has been expressed on the surface of Chinese hamster ovary (CHO) cells. Flow cytometry was used to follow the stability of GP63 expression. Expression of proteolytically active GP63 (GP63WT) was unstable whether or not methotrexate (MTX) selection was maintained. In contrast, expression of an active site mutant (GP63E265D) was stable under MTX selection. In the absence of selection, the decline in GP63E265D expression was more gradual than the loss of GP63WT expression. Different molecular mechanisms accounted for these losses and resulted in higher growth rate nonproducer populations. A dynamic population model was used to calculate the conversion rates of GP63WT producers to nonproducers. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 594-600, 1997.
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  • 20
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    Biotechnology and Bioengineering 53 (1997), S. 583-593 
    ISSN: 0006-3592
    Keywords: bovine serum albumin ; α-chymotrypsin ; extraction ; lysozyme ; microemulsions ; reverse micelles ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The extraction of solid-phase α-chymotrypsin, bovine serum albumin (BSA), and lysozyme by water-in-oil microemulsion (w/o-ME) solution containing Aerosol-OT (AOT) was thoroughly examined as a means to maximize protein solubilization in organic solvent media. Protein extraction occurred simultaneously with the adsorption of water and AOT by the solid protein. Water and AOT were desorbed at nearly equal rates, suggesting that both materials were desorbed together as micreomulsions. The solubilization of protein increased linearly with the ratio of solid protein to extractant solution except at a high value of the ratio, where most protein-containing microemulsions were desorbed. Based on our results, a mechanistic model was developed to describe the solid-phase extraction procedure. First, microemulsions are desorbed from solution by the solid protein, resulting in the formation of a solid protein-AOT-water aggregate. Second, when a protein in the solid phase binds to a sufficient number of microemulsions, the resulting aggregate's increased hydrophobicity drives its solubilization into lipophilic solvent. Third, through the exchange of materials between the solubilized precipitate and the remaining microemulsions, protein-containing w/o-MEs are formed. The presence of adsorption is further indicated by an isotherm existing between the water, AOT, and protein content of the resulting solid phase for each protein. The driving force behind adsorption is either AOT-protein interactions or the protein's affinity for microemulsion-encapsulated water, depending on the properties of the protein and the size of the microemulsions, in agreement with the model of P. L. Luisi [Chimia, 44: 270-282 (1990)]. The second step of our model is mass transfer limited for the extraction of solid α-chymotrypsin and BSA. The extraction of solid lysozyme was limited by the occurrence of an irreversible precipitation process. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 583-593, 1997.
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  • 21
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    Biotechnology and Bioengineering 56 (1997), S. 268-278 
    ISSN: 0006-3592
    Keywords: Thermotoga maritima ; Methanococcus jannaschii ; hydrogen transfer ; hyperthermophiles ; coculture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Interactions involving hydrogen transfer were studied in a coculture of two hyperthermophilic microorganisms: Thermotoga maritima, an anaerobic heterotroph, and Methanococcus jannaschii, a hydrogenotrophic methanogen. Cell densities of T. maritima increased 10-fold when cocultured with M. jannaschii at 85°C, and the methanogen was able to grow in the absence of externally supplied H2 and CO2. The coculture could not be established if the two organisms were physically separated by a dialysis membrane, suggesting the importance of spatial proximity. The significance of spatial proximity was also supported by cell cytometry, where the methanogen was only found in cell sorts at or above 4.5 μm in samples of the coculture in exponential phase. An unstructured mathematical model was used to compare the influence of hydrogen transport and metabolic properties on mesophilic and hyperthermophilic cocultures. Calculations suggest the increases in methanogenesis rates with temperature result from greater interactions between the methanogenic and fermentative organisms, as evidenced by the sharp decline in H2 concentration in the proximity of a hyperthermophilic methanogen. The experimental and modeling results presented here illustrate the need to consider the interactions within hyperthermophilic consortia when choosing isolation strategies and evaluating biotransformations at elevated temperatures. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 268-278, 1997.
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  • 22
    ISSN: 0006-3592
    Keywords: methanol sensor ; methanol monitoring and control ; methylotrophic yeast fermentation ; Pichia pastoris ; transferrin ; shake-flask cultures ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 279-286, 1997.
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  • 23
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    Biotechnology and Bioengineering 56 (1997), S. 287-294 
    ISSN: 0006-3592
    Keywords: specific growth rate ; hyphal morphometry ; Aspergillus niger ; surface growth kinetics ; glucose effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Development of surface grown cultures of Aspergillus niger no. 10 was studied at two experimental levels: (a) following the time course of the biomass density (X [=] mg cm-2) and fitting the data by the logistic expression, which yielded a macroscopic specific growth rate expressed as μobs = (dX/Xdt)[1-(X/Xmax)]-1; and (b) measuring morphometric parameters like the specific elongation rate (k) of the germ tubes and their diameters (Dh), the colony rate of radial extension (ur), and the mean length of distal hyphae (Lav) to estimate the specific growth rate with the following proposed expression: μcalc = urln2[Lavln(Lav/Dh)]-1. Increases in the initial glucose concentration (10, 40, 70, 120, 200, and 300 g L-1) caused reductions in the specific growth rates, the elongation kinetics of the germ tubes, and the hyphal diameter, nevertheless, ur and Xmax presented parabolic behavior, showing their maxima in the interval of 90 to 120 g L-1 of glucose. The overall macroscopic effect of the tested concentrations of glucose on surface grown cultures of A. niger was to produce densely packed and slowly extending colonies, where changes in hyphal lengths and diameters were significant. There was good agreement between μobs and μcalc values. Hence, this work validates a kinetic model based on morphometric data to estimate the specific growth rate of molds, obtained from dry weight data, using mold cultures grown in the same solid medium i.e., agar plates. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 287-294, 1997.
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  • 24
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    Biotechnology and Bioengineering 56 (1997), S. 295-303 
    ISSN: 0006-3592
    Keywords: three phase fluidized bed reactor ; immobilized biomass ; quinoline degradation ; Comamonas acidovorans ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Quinoline degradation by Comamonas acidovorans was investigated in a three phase fluidized bed reactor at dilution rates below and above the critical value (μmax = 0.42 h-1). Quinoline was used as the sole source of carbon, nitrogen, and energy. Two attachment carriers, polyurethane foam (Bayvitec®) and modified cellulose (Aquacel®), and a gel entrapment carrier (polyvinyl alcohol) were studied and compared with regard to their effectiveness to immobilize cells. Attachment and biofilm formation was best at higher dilution rates, regardless of carrier type used. Except for the maximum biomass concentration on the carrier, YV (biomass per volume of solid particles), there was no significant difference in reactor performance between the investigated carriers under stationary conditions. The highest value for YV was found for the gel entrapment carrier (YV = 35 g L-1). In a long-term run (66 days), the gel entrapment carrier established a permanent biofilm on the surface of the gel beads after 900 h of cultivation time. Complete quinoline mineralization was achieved at a dilution rate of 2.0 h-1, which is 4.7 times higher than the critical dilution rate. Identical substrate overloads were applied to the gel entrapment and the cellulose carrier by a step increase of the quinoline feed concentration at a dilution rate of 0.8 h-1 (D ∼2μmax). The cells survived the overload, but the accumulation of quinoline and quinoline degradation products and the degradation efficiency were different for the two systems during the overload, showing the influence of the carrier type on the dynamic performance and stability of the process. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 295-303, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 304-310 
    ISSN: 0006-3592
    Keywords: crossflow membrane filtration ; Escherichia coli ; high pressure homogenization ; inclusion bodies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Crossflow membrane filtration was used to process recombinant Escherichia coli cell lysates containing protein inclusion bodies after high pressure homogenization. The number of passes through the high pressure homogenizer changed the viscosities and average particle sizes of the cell lysates. The different cell lysates were processed with a hollow fiber unit containing microfiltration membranes and a plate and frame unit with either ultrafiltration or microfiltration membranes. There were differences in permeate flux and protein transmission for the various membranes with the best performing membranes giving permeate fluxes greater than 60 L m-2 h-1 and protein transmissions greater than 90%. For a given membrane, no differences were observed between the cell lysates following homogenization with one, two, and three passes at 83 MPa. The lack of a difference between the three lysates is due to their similarities with respect to the released macromolecules and the presence of small (〈0.1 μm) cell debris. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 304-310, 1997.
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  • 26
    ISSN: 0006-3592
    Keywords: micelle ; water-soluble polymer ; protein extraction ; phase separation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A water-soluble polymer such as polyethylene glycol (PEG), Dextran T-500 (Dx), or diethylaminoethyl-Dextran (DEAE-Dx) induced aqueous micellar solutions of octyl-β-D-thioglucoside (OTG) to phase separation at 0°C. One of the two phases thus formed is a surfactant-depleted aqueous solution (aqueous phase) of a water-soluble polymer and the other a concentrated OTG solution (surfactant-rich phase). In a combination of OTG with PEG or Dx, cytochrome P450 (P450) and cytochrome b5 (b5) were well extracted into the surfactant-rich phase. The extraction yield of P450 was slightly greater than that of b5. In contrast to PEG and Dx, DEAE-Dx markedly reduced the extraction of b5, while that of P450 remained almost unchanged. DEAE-Dx served the dual functions of inducing the phase separation and preventing the extraction of b5 into the surfactant-rich phase. This depressed extraction of b5 was reversed by the addition of potassium phosphate. DEAE-Dx and potassium phosphate proved effective in controlling the extractability of b5. The polymer-induced phase separation provides a new basis for highly efficient extraction of membrane proteins under mild conditions that should be acceptable for thermolabile membrane proteins under physiological conditions. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 311-318, 1997.
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  • 27
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    Biotechnology and Bioengineering 56 (1997), S. 340-344 
    ISSN: 0006-3592
    Keywords: subtilisin ; chymotrypsin ; substrate specificity ; organic solvents ; lyophilized enzymes ; stereoselectivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A simple methodology has been successfully employed to explain the solvent dependence of the substrate specificity of enzymes in organic media. This methodology, which does not require the knowledge of the enzyme structure and is thus applicable to lyophilized and other noncrystalline enzyme preparations, predicts that the kcat/KM ratio for two substrates should be proportional to their Raoult's law activity coefficients. This approach has been validated for two enzymes, subtilisin Carlsberg and α-chymotrypsin, catalyzing the propanolysis of unnatural (in addition to natural) ester substrates in a variety of anhydrous solvents. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 340-344, 1997.
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  • 28
    ISSN: 0006-3592
    Keywords: Primatone RL ; sialylation ; interferon-γ ; serum substitutes ; cell ; CHO cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Although serum-free media have been widely used in mammalian cell culture for therapeutic protein production, the effects of serum-substitutes on product quality have not been extensively examined. This study observed an adverse effect of Primatone RL, an animal tissue hydrolysate commonly used as a serum-substitute to promote cell growth, on sialylation of interferon-γ (IFN-γ) derived from Chinese hamster ovary (CHO) cell culture in both batch and fed-batch modes. In batch cultures, decreased sialylation was observed at each of the glycosylation sites (i.e., Asn25 and Asn97) of IFN-γ with the use of elevated concentrations of the peptone. Although poorest sialylation was obtained with the use of a growth-inhibiting concentration of Primatone RL, diminished sialylation was observed at the optimal peptone concentration for cell growth and product yield. Since incubation of the product in Primatone RL-supplemented acellular medium did not result in decreased sialylation, the negative effect of Primatone RL could not be attributed to extracellular desialylation of IFN-γ by components of the peptone. In the fed-batch mode, a culture utilizing a serum-free feeding medium supplemented with Primatone RL demonstrated poorer sialylation than a similar culture not fed the peptone. The results of both the batch and fed-batch experiments indicate that the adverse effect of the peptone was not due solely to ammonia accumulation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 353-360, 1997.
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  • 29
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    Biotechnology and Bioengineering 54 (1997), S. 535-542 
    ISSN: 0006-3592
    Keywords: fermentor monitoring ; mass spectrometer ; Pichia stipitis ; carbon dioxide ; yeast ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An in situ sterilizable plug-in membrane inlet mass spectrometer for monitoring dissolved gases and volatiles in fermentors was constructed and tested. The design ensured a minimal distance to be traveled by analyte molecules from the bulk of the fermentation broth to the ionization chamber of the mass spectrometer. Apart from the specific cross talk due to overlapping mass peaks from different compounds, we found that carbon dioxide interfered unspecifically with all the mass peaks of other substances, changing them by the same factor. The interference changed slowly with time and could be positive or negative depending on the history of the mass spectrometer. Also, the general sensitivity of the instrument changed slowly with time. These effects can be neglected or corrected for empirically in short-term measurements. When the fermentor was aerated with a three-component gas mixture including carbon dioxide, a rapid change in the partial pressure of carbon dioxide in the gas mixture gave rise to a transient in the signal of a gas whose partial pressure was kept constant. This effect revealed a transient change in the composition of the gas mixture in the bubbles caused by net import or export of carbon dioxide during equilibration with the new gas mixture. An experimental method to determine the effective partial pressures of gases in the bubbles during steady-state transport of carbon dioxide was designed. The plug-in membrane inlet mass spectrometer was tried as a probe for oxygen and ethanol in an oxystatic culture of the yeast Pichia stipitis. We found that it was possible to keep a steady-state concentration of as little as 0.5 μM throughout the lifetime of the culture. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 535-542, 1997.
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    Biotechnology and Bioengineering 54 (1997), S. 543-548 
    ISSN: 0006-3592
    Keywords: biofilm reactor ; fluidized bed reactor ; dentrification ; nitrate ; nitrite ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A uniquely simple model is developed to describe the NO3- and NO2- concentration profiles within a dentrification fluidized bed biological reactor. This simple model is compared to experimental data, and to a more complex model similar to those previously proposed in the literature. The simple model fits the experimental data at least as well as the more complex model. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 543-548, 1997.
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  • 31
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    Biotechnology and Bioengineering 54 (1997), S. 577-582 
    ISSN: 0006-3592
    Keywords: Streptomyces coelicolor A3(2) ; actinorhodin ; chemostat culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Streptomyces coelicolor was grown in variously limited chemostat cultures and the specific rate of extracellular actinorhodin production (qactinorhodin) was measured. The highest qactinorhodin values were observed in glucose- or ammonia-limited cultures, whereas almost no actinorhodin was produced in sulfate-, phosphate-, potassium-, or magnesium-limited cultures. The effect of the dilution rate on actinorhodin production was studied in glucose-limited cultures. It was found that qactinorhodin was highest at D = 0.06h-1, which was well below the maximal D value tested (0.14 h-1). This explains why, in batch cultures, actinorhodin production starts at the onset of the stationary phase. It was also found that the use of nitrilotriacetate instead of citrate as a chelating agent had a negative effect on actinorhodin production. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 577-582, 1997.
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  • 32
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    Biotechnology and Bioengineering 54 (1997), S. 567-576 
    ISSN: 0006-3592
    Keywords: continuous cultivation ; unstable steady state ; substrate inhibition ; phenol degradation ; Pseudomonas cepacia G4 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Multiplicity of steady states of a continuous culture with an inhibitory substrate was used to estimate kinetic parameters under steady-state conditions. A continuous culture of Pseudomonas cepacia G4, using phenol as the sole source of carbon and energy, was overloaded by increasing the dilution rate above the critical dilution rate. The culture was then stabilized in the inhibitory branch by a proportional controller using the carbon dioxide concentration in the reactor exhaust gas as the controlled variable and the dilution rate as the manipulated variable. By variation of the set point, several unstable steady states in the inhibitory branch were investigated and the specific phenol conversion rates calculated. In addition, phenol degradation was investigated under substrate limitation (chemostat operation).The results show that the phenol degradation by P. cepacia can be described by the same set of inhibition parameters under substrate limitation and under high substrate concentrations in the inhibitory branch. Biomass yield and maintenance coefficients were identical. Fitting of the data to various inhibition models resulted in the best fit for the Yano and Koga equation. The well-known Haldane model, which is most often used to describe substrate inhibition by phenol, gave the poorest fit. The described method allows a precise data estimation under steady-state conditions from the maximum of the biological reaction rate up to high substrate concentrations in the inhibitory branch. Inhibition parameter estimation by controlling unstable steady states may thus be useful in avoiding discrepancies between data generated by batch runs and their application to continuous cultures which have been often described in the literature. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 567-576, 1997.
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  • 33
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    Biotechnology and Bioengineering 54 (1997), S. 549-566 
    ISSN: 0006-3592
    Keywords: hybrid models ; neural networks ; penicillin G ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the serial gray box modeling strategy, generally available knowledge, represented in the macroscopic balance, is combined naturally with neural networks, which are powerful and convenient tools to model the inaccurately known terms in the macroscopic balance. This article shows, for a typical biochemical conversion, that in the serial gray box modeling strategy the identification data only have to cover the input-output space of the inaccurately known term in the macroscopic balances and that the accurately known terms can be used to achieve reliable extrapolation. The strategy is demonstrated successfully on the modeling of the enzymatic (repeated) batch conversion of penicillin G, for which real-time results are presented. Compared with a more data-driven black box strategy, the serial gray box strategy leads to models with reliable extrapolation properties, so that with the same number of identification experiments the model can be applied to a much wider range of different conditions. Compared to a more knowledge-driven white box strategy, the serial gray box model structure is only based on readily available or easily obtainable knowledge, so that the development time of serial gray box models still may be short in a situation where there is no detailed knowledge of the system available. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 549-566, 1997.
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  • 34
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    Biotechnology and Bioengineering 55 (1997), S. 11-15 
    ISSN: 0006-3592
    Keywords: NIR spectroscopy ; bioreactor monitoring ; insect cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The purpose of this study was to develop non-invasive techniques to monitor the composition of cell culture media in insect cell bioreactors. Such a monitor could be used in conjunction with a fed-batch feeding scheme to ensure that cells are maintained in an optimal environment for growth and protein production. Glucose and glutamine concentrations in an insect cell culture bioreactor were determined off-line with near-infrared (NIR) absorption spectroscopy. Spectra were collected from 5000 to 4000 cm-1 with a 1.5-mm optical path length. Partial least squares (PLS) regression was applied to correlate the collected spectra with the concentration of the desired analytes. Under the culture conditions evaluated here, glucose and glutamine concentrations ranged from 38 to 55 mM and from 3 to 13 mM, respectively. Accurate measurements of glucose and glutamine in insect cell culture samples were possible over these entire ranges. The standard error of prediction (SEP) and mean percent error (MPE) for glutamine were 0.52 mM and 5.3%, respectively. Glucose could be measured with an SEP of 1.30 mM and an MPE of 2.3%. These levels of error are quite low considering the changing complexity of the growth media due to the shifting levels of amino acids, carbohydrates, yeastolate, proteins, and cell debris. This study represents an important step in the development of noninvasive on-line monitoring devices for cell culture bioreactors. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 11-15, 1997.
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  • 35
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    Biotechnology and Bioengineering 55 (1997), S. 33-40 
    ISSN: 0006-3592
    Keywords: anaerobic process control ; overload ; toxicity ; kinetic constant determination ; biosensor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Process control of anaerobic reactors is difficult due to the complexity of the methabolic pathways in the microbial consortium and to the difficulty of detecting and monitoring process instability in short time, before the biomass is poisoned by incoming toxicants. Process control based on the Rantox biosensor is based on the following principle: the wastewater that can potentially induce an overload or contains a toxicant is first tested on a small “upstream” digester (the Rantox). This reactor makes possible to detect the potential instability and, if necessary, to divert the concentrated and/or contaminated wastewater to a buffer tank and consequently to protect the active biomass of the full-scale reactor. It is generally accepted that methanogens are the most sensitive microorganisms in anaerobic digestion. Among these bacteria, the acetoclastic methanogens are of primary importance because some 70% of the converted chemical oxyen demand (COD) mass flow passes through acetic acid. Therefore the first objective in the development of the Rantox biosensor has been to monitor the metabolism of acetoclastic methanogens in the presence of toxicants. This article presents the theoretical background required to evaluate the toxicity effects by determining the kinetic constants of the considered microorganisms from experimental data. The results of two series of calibration tests, performed in order to obtain a preliminary evaluation of the biosensor response to overload and toxicity conditions, are reported. In a second article, calibration tests will be described which refer to two prototypes of the biosensor tested in different operating conditions. The crucial point related to the Rantox, i.e., its comparison with a “normal” laboratory-scale digester to simulate a full-scale plant, will be the subject of the third (and last) article, which is planned to describe the development of this instrument. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 33-40, 1997.
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  • 36
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    Biotechnology and Bioengineering 55 (1997), S. 54-64 
    ISSN: 0006-3592
    Keywords: fluidized-bed adsorption ; dispersion ; particle diameter ; bed height ; frontal adsorption ; mass transport ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of matrix properties and operating conditions on the performance in fluidized-bed adsorption has been studied using Streamline diethyl-aminoethyl (DEAE), an ion exchange matrix based on quartz-weighted agarose, and bovine serum albumin (BSA) as a model protein. Three different particle size fractions (120-160 μm, 120-300 μm, and 250-300 μm) were investigated. Dispersion in the liquid phase was reduced when particles with a wide size distribution were fluidized compared to narrow particle size distributions. When the mean particle diameter was reduced, the breakthrough capacities during frontal adsorption were enlarged due to a shorter diffusion path length within the matrix. At small particle diameters the effect of film mass transfer became more relevant to the adsorption performance in comparison to larger particles. Therefore matrices designed for fluidized-bed adsorption should have small particle diameter and increased mean particle density to ensure small diffusion path length in the particle and a high interstitial velocity to improve film mass transfer. Studies on the influence of sedimented matrix height on axial mixing showed an increased Bodenstein number with increasing bed length. Higher breakthrough capacities were also found for longer adsorbent beds due to reduced dispersion and improved fluid and particle side mass transfer. With increasing bed height the influence of flow rate on breakthrough capacity was reduced. For a settled bed height of 50 cm breakthrough capacities of 80% of the equilibrium capacity for flow rates varying from 3 to 9 cm/min could be achieved. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 54-64, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 82-90 
    ISSN: 0006-3592
    Keywords: toluene degradation ; xylene degradation ; Azoarcus tolulyticus ; nitrate reduction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Groundwater from a xylene-contaminated acquifer was enriched in the laboratory in the presence of toluene, xylenes, ethylbenzene, and benzene. A pure culture that degrades toluene and m-xylene under nitrate-reducing conditions was isolated. Fatty acid analysis, 16S rRNA sequencing, and morphological traits indicate that the isolate was a strain of Azoarcus tolulyticus. The kinetics of toluene degradation under nitrate-reducing conditions by this isolate was determined. Nitrate reduction does not proceed beyond nitrite. Nitrate and toluene are substrate limiting at low concentrations, whereas toluene, nitrate, and nitrite are inhibitory at high concentrations. Several inhibition models were compared to experimental data to represent inhibition by these substrates. A kinetic model for toluene and nitrate degradation as well as for cell growth and nitrite production was developed and compared to experimental data. The results of this work may find important application in the remediation of groundwater aquifers contaminated with aromatic hydrocarbons. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 82-90, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 91-100 
    ISSN: 0006-3592
    Keywords: microfiltration ; membrane ; protein ; fouling ; filtration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recent studies of protein fouling have provided considerable insight into both the underlying fouling mechanisms and the mathematical description of the flux decline. However, most of the data have been obtained with a single model protein, making it difficult to generalize the results to commercially relevant process streams. Experiments were thus performed using a range of proteins with different physicochemical characteristics to determine the relationship between the protein structure and fouling behavior. Fouling in these systems occurred by two distinct mechanisms: deposition of large protein aggregates and chemical attachment of native proteins to the growing deposit. The chemical attachment generally occurred via the formation of intermolecular disulfide linkages involving a free sulfhydryl group in the native protein. Proteins without a free sulfhydryl group were typically unable to form these intermolecular linkages. The quasi-steady flux for the different proteins was proportional to the square of the protein surface charge density, consistent with a model in which protein deposition occurs when the drag force on the proteins associated with the convective filtrate flow is sufficient to overcome electrostatic repulsive interactions. These results clearly demonstrate the importance of the protein structure, charge, and reactivity in determining the rate and extent of protein fouling during microfiltration. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 91-100, 1997.
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  • 39
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    Biotechnology and Bioengineering 54 (1997), S. 142-152 
    ISSN: 0006-3592
    Keywords: insect cells ; baculovirus ; protein expression ; high cell density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Per cell protein expression in virally-infected insect cells declines significantly at high cell density resulting in a decrease in volumetric productivity. Specific protein expression levels in Spodoptera frugiperda (Sf-21) cells could be increased at high cell densities by increasing the oxygen supply and by supplementing the medium with glutamine post-infection. β-Galactosidase yield was increased from 411 to 855 IU/ml by increasing the glutamine concentration in the medium by 46% and increasing the gas phase oxygen concentration from 21 to 80%. Similarly, the yield of a secreted alkaline phosphatase was increased from 14.2 to 26.2 IU/mL using the same conditions. Part of the increase in production with Sf-21 culture was due to increased release to the extra-cellular compartment at the higher oxygen concentrations. Increasing the gas phase oxygen concentration to 95% in conjunction with a 100% increase in glutamine and glucose concentrations did not improve the yield any further. Peak production under elevated oxygen and nutrient conditions occurred at 72 h about 24-48 h earlier than under normal conditions. In a Trichoplusia ni cell line (BTI-TN-5B1-4), the maximum secreted alkaline phosphatase activity was increased from 10 to 27.2 IU/mL by similarly manipulating the oxygen supply. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 142-152, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 136-147 
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosyl-phosphatidylinositol p97 concentration ; protein harvest ; controlled release ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chinese hamster ovary (CHO) cells expressing the human melanoma tumour antigen, p97, were used to develop a controlled release process for the production of recombinant glycosyl-phosphatidylinositol (GPI) anchored proteins. The cells were cultured either in suspension or immobilized on porous microcarriers and p97 was selectively cleaved from the cell surface by the bacterial enzyme, phosphatidylinositol-phospholipase C (PI-PLC). The kinetics of p97 cleavage from the cell surface by PI-PLC was shown to be approximated by Michaelis-Menten kinetics. The recovered p97 concentrations were increased by reusing the PI-PLC enzyme solution to harvest multiple batches of cells. A convenient PI-PLC assay was developed to monitor the harvesting process and to determine the stability of PI-PLC under harvesting conditions. Although the Pl-PLC was stable under harvesting conditions, it rapidly adsorbed to the cell surface and was depleted from the reused enzyme solution. In order to maintain PI-PLC activity, it was necessary to add fresh PI-PLC to the reused enzyme solution before harvesting a fresh batch of cells. The maximum p97 concentration that could be obtained from harvesting CHO cells cultured on porous microcarriers was limited by the dilution effects of sample removal, adding fresh PI-PLC and liquid associated with settled microcarriers. A model was developed that adequately predicted the p97 concentration after each harvest and the maximum p97 concentration that could be achieved by this harvesting method. The dilution effects were minimized by harvesting from centrifuged suspension culture cells and the harvested p97 concentration was increased by over sixfold to 0.64 mg/mL. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 136-147, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 170-181 
    ISSN: 0006-3592
    Keywords: metabolic reaction model ; physiological state recognition ; Corynebacterium glutamicum ; lysine fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A metabolic reaction model was developed for the lysine fermentation process by Corynebacterium glutamicum AJ-3462 to estimate the physiological state of the cells - that is, the growth and production activity, and the flux distribution of metabolites - from on-line measurable rates only. First, the extended Kalman filter was applied to eliminate noise in the measured rates. Then, using the metabolic reaction model, the lysine production rate and flux distribution were calculated. The estimation results allowed the physiological state of lysine production to be recognized, and an appropriate measure corresponding to the estimated state, such as intermittent addition of glucose and/or leucine, to be taken to maintain a high level of lysine productivity in batch culture. Finally, application of the recognition system enabled lysine to be produced from glucose at a higher yield than that from glucose- or leucine-limited exponential fed-batch cultures. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 170-181, 1997.
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  • 42
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    Biotechnology and Bioengineering 55 (1997), S. 399-407 
    ISSN: 0006-3592
    Keywords: lipase ; chiral kinetics ; organic solvent ; micelle ; emulsion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lipase from Pseudomonas cepacia was used for asymmetric hydrolysis of the substrate (±)1-chloro-2-acetoxy-3-(1-naphthyloxy)-propane, which is a precursor for (S)-(-)-β-blocker synthesis. Because this substrate is insoluble in water and partially soluble in hydrophobic solvents such as hexane and octane, a mixture of hydrophilic organic solvents and aqueous buffer was used to study the initial reaction rates. Because of the amphipathic nature of the substrate, it can remain in three different forms: (1) monomeric (solution); (2) micellar; and (3) emulsion, depending on the acetone and substrate concentrations in the medium. This behavior is presented in a phase diagram. The enzyme was found to be active with micelle as well as emulsion form of the substrate, whereas it showed negligible activity with the monomeric form. Michaelis-Menten constants were determined experimentally for the emulsion and micellar part of the substrate. The initial rate of hydrolysis (v0) goes through a maximum with respect to the acetone content of the mixture. It is due to the combined effect of various factors occurring simultaneously with the increase in acetone content in the solvent. These phenomena are discussed based on the interfacial activation of lipase, deactivation of the enzyme at very high acetone concentration, and increase in critical micelle concentration (CMC) and critical emulsion concentration (CEC) with the increase in acetone content in the solvent. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 399-407, 1997.
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  • 43
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    Biotechnology and Bioengineering 55 (1997), S. 390-398 
    ISSN: 0006-3592
    Keywords: γ-IFN ; sialylation ; glycosylation ; sialidase ; CHO cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Since sialic acid content is known to be a critical determinant of the biological properties of glycoproteins, it is essential to characterize and monitor sialylation patterns of recombinant glycoproteins intended for therapeutic use. This study reports site- and branch-specific differences in sialylation of human interferon-γ (IFN-γ) derived from Chinese hamster ovary (CHO) cell culture. Sialylation profiles were quantitated by reversed-phase HPLC separations of the site-specific pools of tryptic glycopeptides representing IFN-γ's two potential N-linked glycosylation sites (i.e., Asn25 and Asn97). Although sialylation at each glycosylation site was found to be incomplete, glycans of Asn25 were more heavily sialylated than those of Asn97. Furthermore, Man(α1-3) arms of the predominant complex biantennary structures were more favorably sialylated than Man(α1-6) branches at each glycosylation site. When the sialylation profile was analyzed throughout a suspension batch culture, sialic acid content at each site and branch was found to be relatively constant until a steady decrease in sialylation was observed coincident with loss of cell viability. The introduction of a competitive inhibitor of sialidase into the culture supernatant prevented the loss of sialic acid after the onset of cell death but did not affect sialylation prior to cell death. This finding indicated that incomplete sialylation prior to loss of cell viability could be attributed to incomplete intracellular sialylation while the reduction in sialylation following loss of cell viability was due to extracellular sialidase activity resulting from cell lysis. Thus, both intracellular and extracellular processes defined the sialic acid content of the final product. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 390-398, 1977.
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    Biotechnology and Bioengineering 55 (1997), S. 419-426 
    ISSN: 0006-3592
    Keywords: metabolic engineering ; glycogen synthesis ; glycogen degradation ; carbon metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In metabolic engineering, systems which allow coordinated control of two metabolic pathways can be useful. We designed two expression systems and demonstrated their application by coordinating glycogen synthesis and degradation. The first expression vector pMSW2 expressed the glycogen synthesis genes in one operon and the glycogen degradation gene in a separate, coordinately regulated operon. The plasmid was designed to switch off expression of the first operon and activate expression of the second operon on addition of IPTG. As an alternative means to control glycogen synthesis and degradation pathways, we constructed expression vector pGTSD100, which contains the native Escherichia coli glycogen synthesis and degradation operon under control of the tac promoter. Both expression vectors work successfully to control the net synthesis and degradation of glycogen. In cultures of the E. coli strain TA3476 carrying the plasmid pMSW2, before the addition of IPTG, glycogen continued to accumulate in the culture. About three hours after IPTG was added, glycogen levels began to decrease. When no IPTG was added to cultures of TA3476:pMSW2, glycogen accumulated in the cells as before but the rate of degradation of glycogen was much lower. When IPTG was added to TA3476:pMSW2, the total cell protein at the end of batch cultivation was approximately 15% higher compared to cultures without IPTG addition. The extra biomass was formed during the glycogen degradation phase. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 419-426, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 439-446 
    ISSN: 0006-3592
    Keywords: cell culture viability ; apoptosis ; IL-6 ; hybridomas ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Sp2/0 hybridoma cells die principally by apoptosis in batch culture. We have found that cultures of the Sp2/0 hybridoma exhibit increased viability in response to interleukin 6 (IL-6) supplementation relative to control cultures during serum shiftdown experiments. When shifted from a medium containing 10% fetal bovine serum (FBS) to a medium with 1% FBS, IL-6 supplemented cultures displayed viabilities and viable cell densities similar to control cultures containing 10% FBS. The degree of the survival response induced varied in accordance with the severity of the shiftdown, as cells resuspended in a high serum medium showed little observable enhancement in viability. The extension in culture viability was not accompanied by an observable decrease in growth relative to control cultures, indicating that the effect was not a consequence of growth inhibition. These results suggest the existence of serum components with behavior functionally similar to IL-6, with respect to enhancing cell survival, and that under certain experimental conditions IL-6 serves as a survival factor. In contrast to the extended viability displayed by cultures supplemented with IL-6, Sp2/0 cultures transfected with IL-6 cDNA expression vectors displayed a growth inhibitory response relative to control cultures. This inhibitory response was characterized by an extended lag phase following inoculation, and a decrease in batch culture cell yield. The depression in cell yield varied with serum concentration, with the largest depression occurring at high serum concentrations. We conclude that interactions between components in serum, presumably growth factors, and cytokines play an important role in altering the behavior of industrially relevant cell lines in culture. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 439-446, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 283-288 
    ISSN: 0006-3592
    Keywords: chloroperoxidase ; Caldariomyces fumago ; indole ; total turnover number ; space-time yield ; membrane reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chloroperoxidase from Caldariomyces fumago was applied for the oxidation of indole to oxindole using hydrogen peroxide as the oxidant in aqueous t-butyl alcohol medium. Different ways of adding the oxidant, various reactor types, and the use of a hydrogen peroxide-stat were compared, resulting in a 20-fold increase of the total turnover number (ttn) and space-time yield (sty). The highest ttn of 〉860,000 was obtained in a fed-batch reactor, whereas the highest sty of 120 g/(L · d) was reached in a continuously operated enzyme membrane reactor. The results were compared to other enzyme systems already established for the synthesis of amino acids and carbohydrates. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 283-288, 1997.
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  • 47
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    Biotechnology and Bioengineering 55 (1997), S. 480-489 
    ISSN: 0006-3592
    Keywords: pentachlorophenol ; Mycobacterium chlorophenolicum ; adsorption and desorption ; isotherm model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The sorption behavior of pentachlorophenol (PCP) by the Gram-positive bacterium Mycobacterium chlorophenolicum PCP-1 was quantitatively characterized in this work, with emphasis on the effects of biomass and pH and on the reversibility of PCP adsorption. Both the adsorption and desorption of PCP showed a fast kinetic, reaching an equilibrium in less than 1.5-min mixing under the experimental conditions. For PCP concentrations up to 600 μmol/L no saturation of the adsorption was observed and the adsorption isotherms can be adequately described by the Freundlich equation. The adsorption capacity (qads) of M. chlorophenolicum PCP-1 increased significantly with decreasing biomass in the low concentration range (below 0.5 g/L). The biomass concentration merely affected the capacity constant K of the Freundlich model while the intensity parameter n remained constant. The qads also increased with decreasing pH, particularly at acidic pH values. Again, the pH effect was mainly reflected by the change of K. Based on these results a correlation for qads, in which K is a function of both biomass concentration and pH, was obtained to describe the adsorption isotherms at different biomass concentrations and pH values. The desorption of PCP was also found to be strongly affected by pH. At pH 5.4 the adsorption was almost completely irreversible, while a nearly complete desorption was obtained at pH 7. The effect of pH on the sorption behavior was found to be related to the ionization of PCP. The irreversibly adsorbed PCP is a strict function of concentration of undissociated PCP, while the reversibly adsorbed PCP correlates well with the concentration of ionic PCP. The irreversible adsorption has a much higher adsorption capacity than the reversible adsorption. These findings led to the derivation of a semimechanistic model that satisfactorily describes the sorption of PCP by M. chlorophenolicum. The results obtained also give clues to the patterns and mechanism(s) of PCP adsorption by microbial cells. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 480-489, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 497-504 
    ISSN: 0006-3592
    Keywords: glucose ; glucose oxidase ; biosensor ; on-line monitoring ; 1,1′-dimethylferrocene ; mammalian cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A flow injection analysis (FIA) biosensor system has been developed for on-line determination of glucose during mammalian cell cultivation. The culture sample was peristaltically withdrawn from the bioreactor and after cell separation by a steam sterilizable ceramic microfilter, the filtrate was continuously fed to the FIA mediated-biosensor system at 4 mLh-1, whereas the cell-containing retentate was recirculated to the bioreactor. In the amperometric biosensor system, glucose oxidase was covalently immobilized onto a preactivated nylon membrane and attached to the sensing area of a platinum working electrode. The enzyme reaction was coupled with the mediator 1,1′-dimethylferricinium (DMFe+)-cyclodextrin inclusion complex to recycle the reduced glucose oxidase to its original active state. 1,1′-Dimethylferrocene (DMFe) was then reoxidized to DMFe+ at the surface of the platinum electrode poised at + 0.15 V vs silver/silver chloride. The FIA mediated-biosensor was linear up to 6 mM glucose, with a detection limit of 0.1 mM, and possessed excellent reproducibility (± 0.4 %, 95 % confidence interval) over 123 repeated analyses during a 62 h continuous operation. The immobilized glucose oxidase was stable for up to 7 days when applied to glucose measurement during 5-10 day fed-batch cultivation of 293S mammalian cells. The results obtained from the mediated-biosensor system compared well with the hexokinase and HPLC data. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 497-504, 1997
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  • 49
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    Biotechnology and Bioengineering 56 (1997), S. 361-371 
    ISSN: 0006-3592
    Keywords: biofilms ; bioremediation ; toluene ; vapor phase bioreactors ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A Pseudomonas putida 54G biofilm was grown on toluene vapor supplied as the sole external carbon and energy source in a flat plate biofilm reactor. Enumerations of cells in the biofilm were made using culture techniques (selective and nonselective for toluene) and microscopic techniques (total and respiring cells), and an analysis of the progression of the state of the culture was made by examination of various fractions of the populations. Long-term exposure to higher levels of toluene produced the following trends: (i) lower fraction of total cells that respired; (ii) lower fraction of culturable cells that also grew on toluene; (iii) higher fraction of respiring cells that could not grow on toluene plates; and (iv) a relatively constant fraction of total cells that could not be cultured on toluene. Respiration rate was determined using oxygen microsensors, and the fraction of the total respiration that was not associated with toluene uptake increased with higher toluene exposure. A combination of cryosectioning and respiration rate data was used to demonstrate that more respiring cells and a higher respiration rate both occurred at the base of the film, suggesting a deterioration in physiological state with continued exposure to toluene. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 361-371, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 380-390 
    ISSN: 0006-3592
    Keywords: insect cells ; baculovirus ; bcl-2 ; recombinant proteins ; cell viability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of bcl-2 expression on cell viability and recombinant protein synthesis was investigated in the Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five™) insect cell lines. It was found that coinfection with a baculovirus expressing bcl-2 [Autographa californica nuclear polyhedrosis virus (AcNPV)-bcl2] extended the life span of High Five™ cells but not Sf-9 cells when compared to infection with recombinant baculoviruses expressing either human tissue plasminogen activator (AcNPV-tPA) or Escherichia coli β-galactosidase (AcNPV-βgal). Similar results were obtained in coinfection experiments; i.e., AcNPV-bcl2 coinfection increased the life span of High Five™ cells over that of cells infected with either AcNPV-tPA or AcNPV-βgal alone, but they did not affect the life span of coinfected Sf-9 cells. Coinfection of Sf-9 cells with AcNPV-bcl2 and AcNPV-βgal resulted in a decrease in the maximum β-gal expression levels of over 90% when compared to infection with AcNPV-βgal alone. A similar trend was found in the β-gal mRNA levels. Coinfection also resulted in a reduced β-gal expression level in High Five™ cells, but the reduction was consistent with what would be expected when two recombinant viruses compete for use of the cellular machinery. In contrast to the inhibitory effect of AcNPV-bcl2 coinfection on βgal expression, t-PA expression levels were either not affected (Sf-9 cells) or were increased 50% (High Five™ cells) over those obtained by infection with AcNPV-tPA alone. These results support the hypotheses that bcl-2 can inhibit transcription of genes under polyhedrin promoter control and that β-gal expression levels, but not t-PA expression levels, are controlled at the transcriptional level. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 380-390, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 398-421 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; metabolism ; flux ; linear optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A stoichiometric model of metabolism was developed to describe the balance of metabolic reactions during steady-state growth of Escherichia coli on glucose (or metabolic intermediates) and mineral salts. The model incorporates 153 reversible and 147 irreversible reactions and 289 metabolites from several metabolic data bases for the biosynthesis of the macromolecular precursors, coenzymes, and prosthetic groups necessary for synthesis of all cellular macromolecules. Correlations describing how the cellular composition changes with growth rate were developed from experimental data and were used to calculate the drain of precursors to macromolecules, coenzymes, and prosthetic groups from the metabolic network for the synthesis of those macromolecules at a specific growth rate. Energy requirements for macromolecular polymerization and proofreading, transport of metabolites, and maintenance of transmembrane gradients were included in the model rather than a lumped maintenance energy term. The underdetermined set of equations was solved using the Simplex algorithm, employing realistic objective functions and constraints; the drain of precursors, coenzymes, and prosthetic groups and the energy requirements for the synthesis of macromolecules served as the primary set of constraints. The model accurately predicted experimentally determined metabolic fluxes for aerobic growth on acetate or acetate plus glucose. In addition, the model predicted the genetic and metabolic regulation that must occur for growth under different conditions, such as the opening of the glyoxylate shunt during growth on acetate and the branching of the tricarboxylic acid cycle under anaerobic growth. Sensitivity analyses were performed to determine the flexibility of pathways and the effects of different rates and growth conditions on the distribution of fluxes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 398-421, 1997.
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    Biotechnology and Bioengineering 56 (1997), S. 433-440 
    ISSN: 0006-3592
    Keywords: simple dissolution-reaction model ; enzymatic conversion ; solid substrate suspension ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Although reactions in substrate suspension are employed in industry for several bioconversion processes, there appears to be no quantitative model available in the literature to rationalize the optimization of these processes. We present a simple model that incorporates the kinetics of substrate dissolution and a simultaneous enzymatic reaction. The model was tested in the α-chymotrypsin-catalyzed hydrolysis of an aqueous suspension of dimethyl benzylmethylmalonate to a homogeneous solution of enantiomerically pure monoester. This reaction occurs in the bulk phase, so catalysis by enzyme absorbed at the solid-liquid interface plays no role. The value of the parameters in the model (i.e., the mass transfer coefficient of substrate dissolution (kL), the substrate solubility, and the rate constant for the enzymatic reaction) were determined in separate experiments. Using these parameter values, the model gave a good quantitative prediction of the rate of the overall dissolution-reaction process. When the particle size distribution is known, kL may also be calculated instead. The model seems to be applicable also for other poorly soluble substrates, other enzymes, and other solvents. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 433-440, 1997.
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  • 53
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    Biotechnology and Bioengineering 55 (1997), S. 831-840 
    ISSN: 0006-3592
    Keywords: isotopomer mapping matrix ; isotopomer modeling ; metabolic flux analysis ; 13C NMR ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Within the last decades NMR spectroscopy has undergone tremendous development and has become a powerful analytical tool for the investigation of intracellular flux distributions in biochemical networks using 13C-labeled substrates. Not only are the experiments much easier to conduct than experiments employing radioactive tracer elements, but NMR spectroscopy also provides additional information on the labeling pattern of the metabolites. Whereas the maximum amount of information obtainable with 14C-labeled substrates is the fractional enrichment in the individual carbon atom positions, NMR spectroscopy can also provide information on the degree of labeling at neighboring carbon atom positions by analyzing multiplet patterns in NMR spectra or using 2-dimensional NMR spectra. It is possible to quantify the mole fractions of molecules that show a specific labeling pattern, i.e., information of the isotopomer distribution in metabolite pools can be obtained. The isotopomer distribution is the maximum amount of information that in theory can be obtained from 13C-tracer studies. The wealth of information contained in NMR spectra frequently leads to overdetermined algebraic systems. Consequently, fluxes must be estimated by nonlinear least squares analysis, in which experimental labeling data is compared with simulated steady state isotopomer distributions. Hence, mathematical models are required to compute the steady state isotopomer distribution as a function of a given set of steady state fluxes. Because 2n possible labeling patterns exist in a molecule of n carbon atoms, and each pattern corresponds to a separate state in the isotopomer model, these models are inherently complex. Model complexity, so far, has restricted usage of isotopomer information to relatively small metabolic networks. A general methodology for the formulation of isotopomer models is described. The model complexity of isotopomer models is reduced to that of classical metabolic models by expressing the 2n isotopomer mass balances of a metabolite pool in a single matrix equation. Using this approach an isotopomer model has been implemented that describes label distribution in primary carbon metabolism, i.e., in a metabolic network including the Embden-Meyerhof-Parnas and pentose phosphate pathway, the tricarboxylic acid cycle, and selected anaplerotic reaction sequences. The model calculates the steady state label distribution in all metabolite pools as a function of the steady state fluxes and is applied to demonstrate the effect of selected anaplerotic fluxes on the labeling pattern of the pathway intermediates. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:831-840, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 864-879 
    ISSN: 0006-3592
    Keywords: Corynebacterium glutamicum mutants ; transconjugation ; intracellular flux analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The physiology and central carbon metabolism of Corynebacterium glutamicum was investigated through the study of specific disruption mutants. Mutants deficient in phosphoenolpyruvate carboxylase (PPC) and/or pyruvate kinase (PK) activity were constructed by disrupting the corresponding gene(s) via transconjugation. Standard batch fermentations were carried out with these mutants and results were evaluated in the context of intracellular flux analysis. The following were determined. (a) There is a significant reduction in the glycolytic pathway flux in the pyruvate kinase deficient mutants during growth on glucose, also evidenced by secretion of dihydroxyacetone and glyceraldehyde. The resulting metabolic overflow is accommodated by the pentose phosphate pathway (PPP) acting as mechanism for dissimilating, in the form of CO2, large amounts of accumulated intermediates. (b) The high activity through the PPP causes an overproduction of reducing power in the form of NADPH. The overproduction of biosynthetic reducing power, as well as the shortage of NADPH produced via the tricarboxylic acid cycle (as evidenced by a reduced citrate synthase flux), are compensated by an increased activity of the transhydrogenase (THD) enzyme catalyzing the reaction NADPH + NAD+↔NADP+ + NADH. The presence of active THD was also confirmed directly by enzymatic assays. (c) Specific glucose uptake rates declined during the course of fermentation and this decline was more pronounced in the case of a double mutant strain deficient in both PPC and PK. Specific ATP consumption rates similarly declined during the course of the batch. However, they were approximately the same for all strains, indicating that energetic requirements for biosynthesis and maintenance are independent of the specific genetic background of a strain. The above results underline the importance of intracellular flux analysis, not only for producing a static set of intracellular flux estimates, but also for uncovering changes occurring in the course of a batch fermentation or as result of specific genetic modifications. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:864-879, 1997.
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  • 55
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    Biotechnology and Bioengineering 55 (1997), S. 890-908 
    ISSN: 0006-3592
    Keywords: metabolic engineering ; N-linked glycosylation ; mathematical model ; CHO cells ; glycoform ; oligosaccharides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic engineering of N-linked oligosaccharide biosynthesis to produce novel glycoforms or glycoform distributions of a recombinant glycoprotein can potentially lead to an improved therapeutic performance of the glycoprotein product. Effective engineering of this pathway to maximize the fractions of beneficial glycoforms within the glycoform population of a target glycoprotein can be aided by a mathematical model of the N-linked glycosylation process. A mathematical model is presented here, whose main function is to calculate the expected qualitative trends in the N-linked oligosaccharide distribution resulting from changes in the levels of one or more enzymes involved in the network of enzyme-catalyzed reactions that accomplish N-linked oligosaccharide biosynthesis. It consists of mass balances for 33 different oligosaccharide species N-linked to a specified protein that is being transported through the different compartments of the Golgi complex. Values of the model parameters describing Chinese hamster ovary (CHO) cells were estimated from literature information. A basal set of kinetic parameters for the enzyme-catalyzed reactions acting on free oligosaccharide substrates was also obtained from the literature. The solution of the system for this basal set of parameters gave a glycoform distribution consisting mainly of complex-galactosylated oligosaccharides distributed in structures with different numbers of antennae in a fashion similar to that observed for various recombinant proteins produced in CHO cells. Other simulations indicate that changes in the oligosaccharide distribution could easily result from alteration in glycoprotein productivity within the range currently attainable in industry. The overexpression of N-acetylglucosaminyltransferase III in CHO cells was simulated under different conditions to test the main function of the model. These simulations allow a comparison of different strategies, such as simultaneous overexpression of several enzymes or spatial relocation of enzymes, when trying to optimize a particular glycoform distribution. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:890-908, 1997.
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  • 56
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    Biotechnology and Bioengineering 55 (1997), S. 940-940 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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  • 57
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    Biotechnology and Bioengineering 55 (1997), S. 927-939 
    ISSN: 0006-3592
    Keywords: CHO cells ; human secreted alkaline phosphatase ; tumor suppressor genes ; green fluorescent protein ; cell cycle ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have established a novel production process which allows up to fourfold higher production of a model secreted protein, the human secreted alkaline phosphatase (SEAP), in Chinese hamster ovary (CHO) cells. A cytostatic production phase is established in which cell proliferation is inhibited or completely abolished. Such a cytostatic production phase is established by overexpression of the tumor suppressor genes p21, p27, or p53175P (a p53 mutant showing specific loss of apoptotic function) under transcriptional control of a tetracycline-repressible promoter (PhCMV*-1). In order to minimize complications due to possible clonal variation of selected, stable cell lines, our investigations are based on transiently transfected subpopulations, that have become a useful tool in industrial R&D. These subpopulations have been selected by flow cytometry for the expression of genes encoded on a dicistronic expression vector. These vectors contain a dicistronic expression unit consisting of the genes encoding the green fluorescent protein (GFP) or SEAP, followed by one of the cytostatic genes p21, p27 or p53175P encoded by the second cistron. p21, p27 as well as p53175P block the cell cycle of CHO cells in the G1-phase for a prolonged period. However, these G1-arrested cells remain viable and proliferation proficient upon repression of expression of the cytostatic gene. All three of the cytostatic genes studied provided similar regulation of proliferation, and also similar enhancements in SEAP production, suggesting that higher productivity may be a general and intrinsic feature of G1-phase arrested CHO cells. Overall productivity is most likely enhanced because growth-arrested cells do not need to devote cellular resources to biomass production. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:927-939, 1997.
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  • 58
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    Biotechnology and Bioengineering 56 (1997), S. 32-44 
    ISSN: 0006-3592
    Keywords: insect cells ; baculovirus expression vector system ; multiplicity of infection ; time of infection ; substrate limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The inability to infect insect cell cultures at the highest achievable cell densities has imposed major limitations to both the fundamental understanding of the Baculovirus Expression Vector System (BEVS) as well as full exploitation of its potential productive capacity for recombinant (β-galAcNPV) products. The current literature does not characterize and identify the exact nature of the observed limitations, which therefore has become the major objective and contribution of the following study. Critical densities for infection of Spodoptera frugiperda (Sf9) cells with nuclear polyhedrosis virus expressing β-galactosidase (Autographa californica) grown in media both containing fetal calf serum (FCS) and free of serum were found to be at 2 × 106 and 5 × 106 cells/ml respectively. Medium exchange was found to completely reverse the effect if renewed up to 24 hours post-infection (HPI). The inevitable arrest of uninfected cell growth and decreased production of recombinant products at high cell densities of infection were both correlated to nutrient depletion. Cystine was found to be depleted in uninfected insect cell cultures at the onset of the stationary phase and in serum-free insect cell cultures infected with baculovirus above a cell density of 5 × 106 cells/ml. Neither glucose depletion nor accumulation of possible inhibitory metabolites such as alanine, ammonia, or lactate could be correlated to growth arrest or decreased recombinant product yields. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 32-44, 1997.
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  • 59
    ISSN: 0006-3592
    Keywords: fed-batch ; gamma-Interferon ; CHO cells ; glycosylation ; glucose starvation ; protein quality ; nutritional control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The application of a stoichiometric medium design approach was studied in fed-batch cultivation of Chinese hamster ovary (CHO) cells. A serum-free medium containing a very low protein concentration (2 mg/L insulin) was developed. A supplemental medium was formulated according to the stoichiometric equation governing cell growth using cell composition obtained from hybridoma cells. Fed-batch culture was conducted in spinner flasks using the supplemental medium for feeding. Significant improvement in cell growth, by-product reduction, and Gamma-Interferon (IFN-γ) production was achieved as compared to a typical batch culture. Results indicate that the stoichiometric approach, originally developed for hybridoma cultures, is a fast and effective method for cell culture process design and improvement. The glycosylation of IFN-γ was monitored off-line during the culture process. The accumulative IFN-γ glycosylation efficiency was slightly improved as compared to that of the batch culture, due to the nutritional control through the stoichiometric feeding. Periodic glucose starvation was observed during the fed-batch culture as a result of the manual feeding. Pulse-chase radiolabeling assay shows that glucose starvation leads to a deteriorated IFN-γ glycosylation efficiency. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 577-582, 1997.
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  • 60
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    Biotechnology and Bioengineering 54 (1997), S. 291-302 
    ISSN: 0006-3592
    Keywords: nanofiltration ; selectivity ; amino acid ; charged membranes ; physicochemical parameters ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A charged organic-inorganic nanofiltration (NF) membrane prototype was used to separate a mixture of nine amino acids (AA) on the basis of differential electrostatic interactions with the membrane because, for a given pH, some of them were positively charged, some were negative, and some were zwitterions. Effect of pH, amino acid concentration (Cr), and added ionic strength ([NaCI]) on the process selectivity was studied. A global statistical study revealed that pH was the dominant parameter regarding fractionation. Cr and [NaCI] had a weaker effect, but the ratio Cr/[NaCI] demonstrated a pronounced effect on system selectivity. Two split-ups of the mixture were obtained at pH 2 and at pH 12, for a 1-g/L total AA concentration and a Cr/[NaCI] ratio of 0.16. Under these conditions, the differences in transmissions between basic and acid AA were higher than 70%. Interpretation of the results according to the Donnan theory allows us to foresee the potentialities of charged nanofiltration membranes for the fractionation of a complex mixture, such as peptidic hydrolysate to streams containing peptides and amino acids having different isoelectric points. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 291-302, 1997.
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  • 61
    ISSN: 0006-3592
    Keywords: lactic acid ; ageous two-phase system ; polymer analysis ; Lactococcus lactis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new chromatographic system for the simultaneous analysis of polyethylene glycol, dextran, sugars, and low-molecular-weight fatty acids was developed. The system is based on a gel exclusion column which allows a first separation between high- and low-molecular-weight compounds, and a cationic exchange column used to further separate the low-molecular-weight compounds. Two applications of the system were demonstrated: (i) after optimizing eluent conditions the gel exclusion column was used to determine the influence of lactic acid, phosphate buffer, and lactic acid bacteria on the ethylene oxide propylene oxide-dextran T40 phase diagram by HPLC; (ii) the ion exchange column was coupled in series with the gel exclusion column and the concentration of polyethylene glycol, dextran, glucose, lactate, acetate, and formate was determined in samples from the fermentative production of lactic acid in a polyethylene glycol 8000-dextran T40 aqueous two-phase system. The fermentation was operated without pH control in a repeated extractive batch mode, where the cell-free top phase was replaced four times, whereas the cell-containing bottom phase was reused repeatedly. The yield was 1.1 mol of lactic acid formed per mole of glucose added and the productivity was 4.7 mM·h-1. The polymeric composition of the fermentation system was monitored during the five repeated extractive batches, and it showed a progressive depletion in polyethylene glycol and a progressive enrichment in dextran. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 303-311, 1997.
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  • 62
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    Biotechnology and Bioengineering 54 (1997), S. 312-318 
    ISSN: 0006-3592
    Keywords: xylanase ; kraft pulp ; peroxide bleaching ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of multiple xylanase treatments were assessed during the peroxide bleaching of three pulps: Douglas-fir (kraft); Western hemlock (oxygen delignified kraft); and trembling Aspen (kraft). The addition of a xylanase treatment stage, either before or after the peroxide bleaching stage(s), resulted in the enhanced brightening of all pulps. A higher brightness was achieved using two enzyme treatments, one before and one after the peroxide stage(s). Both bleach boosting and direct brightening seemed to contribute to the enhancement of the peroxide bleaching. Compared to xylanase prebleaching, xylanase posttreatment of peroxide bleached pulps solubilized less lignin and chromophores and made smaller amounts of these materials alkaline soluble. Nevertheless, the final brightness achieved by xylanase posttreatment was similar or superior to that achieved with xylanase prebleaching of the corresponding unbleached pulps. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 312-318, 1997.
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  • 63
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    Biotechnology and Bioengineering 54 (1997), S. 329-332 
    ISSN: 0006-3592
    Keywords: reversed micelle ; urease ; urea sensor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new improvised urea sensor is described. The new sensor makes use of entrapment inside reversed micelles as a method for enzyme immobilization and glass electrode for the purpose of sensing. Urea content in clinical blood samples has been estimated using this new sensor. Agreement of the results thus obtained with those from clinical methods gives credence to the new sensor. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 329-332, 1997.
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  • 64
    ISSN: 0006-3592
    Keywords: Ni2+ removal ; Citrobacter sp. ; hydrogen uranyl phosphate ; citrate complex ; intercalation ; ion-exchange ; interlayer ; XRD ; PIXE ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ni2+ was removed quantitatively from aqueous flows by columns loaded with polycrystalline hydrogen uranyl phosphate (HUP) bound to immobilized cells of Citrobacter sp. The columns functioned effectively in Ni uptake/regeneration cycles; five cycles were completed without significant decrease in the Ni-removing capacity of the column. The influence of pH, temperature, and flow rate on the Ni-removing capacity of the columns was examined. The composition of the Ni/HUP cell-bound deposits was confirmed by X-ray diffraction analysis (XRD) and proton-induced X-ray emission (PIXE) spectroscopy following several consecutive metal challenges and is discussed in relation to the mechanism of Ni2+ removal from solution via ion-exchange intercalation into the interlayer space of HUP. Ni was selectively recovered from the columns using citrate or tartrate. The regenerated columns functioned effectively in Ni removal throughout repeated Ni challenge and desorption cycles. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 319-328, 1997.
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  • 65
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    Biotechnology and Bioengineering 54 (1997), S. 333-343 
    ISSN: 0006-3592
    Keywords: aggregation ; folding intermediates ; inclusion body ; polymerization ; protein folding ; protein-protein interactions ; self-assembly ; P22 tailspike protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The misfolding of polypeptide chains and aggregation into the insoluble inclusion body state is a serious problem for biotechnology and biomedical research. Developing a rational strategy to control aggregation requires understanding the mechanism of polymerization. We investigated the in vitro aggregation of P22 tailspike polypeptide chains by classical light scattering, nondenaturing gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis (PAGE), and computer simulations. The aggregation of polypeptide chains during refolding occurred by multimeric polymerization, in which two multimers of any size could associate to form a larger aggregate and did not require a sequential addition of monomeric subunits. The cluster-cluster polymerization mechanism of aggregation is an important determinant in the kinetic competition between productive folding and inclusion body formation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 333-343, 1997.
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  • 66
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    Biotechnology and Bioengineering 54 (1997), S. 344-350 
    ISSN: 0006-3592
    Keywords: biosorption ; brown algae ; cadmium ; iron ; metal uptake ; Sargassum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nonliving biomass of Sargassum, a brown marine alga, is capable of binding more than 10% of its dry weight in toxic cadmium ions. Although ubiquitous iron interferes with Cd uptake, only approximately 4.5% of it is sequestered (biomass dry weight). Biosorption of both metals at ph 4.5 could be described by Langmuir-type isotherms with b, the affinity-related coefficient (Cd: b = 0.015; Fe: b = 0.027). The interference of Fe with Cd uptake, and vice versa, was assessed by deriving three-dimensional equilibrium two-metal sorption isotherm surfaces, smoothed and “cut” to reveal the inhibition effect of Fe on biosorption of Cd: at the equilibrium concentration Cf[Cd] = 1.5 mM, the presence of Fe at 1.5 mM equilibrium concentration suppressed the Cd uptake to only 76% of the original value. For 50% Cd uptake reduction, a very high equilibrium Fe presence of 4.5 mM was required. The Cd presence affected the uptake of Fe very strongly. To obtain equal values of uptake for each metal in the biosorbent, the ratio of equilibrium concentrations of 0.42 Cd to 1 Fe is necessary in the liquid phase. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 344-350, 1997.
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  • 67
    ISSN: 0006-3592
    Keywords: xylitol production ; recombinant Saccharomyces cerevisiae ; protein burden ; plasmid stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Xylitol production with two recombinant Sacharomyces cerevisiae strains expressing the XYL1 gene, coding for xylose reductase (XR), at different levels, the ‘low XR strain’ at 0.51 U/mg and the ‘high XR strain’ at 10.8 U/mg, was compared in batch and fed-batch culture. Xylose was not consumed in the presence of high glucose concentrations, because both sugars are transported by the glucose transport system, which has a higher affinity for glucose than for xylose. When glucose was fed gradually to the culture, high concentrations were avoided, and xylose was converted to xylitol with a specific productivity of 0.10 g g-1 h-1 attained with the low XR strain and 0.19 g g-1 h-1 with the high XR strain, indicating that factors other than the XR-activity control the rate of xylose conversion.The overproduction of XR put a substantial protein burden on the high XR strain, contributing to a 50% decrease in specific growth rate and reduced biomass yield compared with the low XR strain. Despite the use of selective medium, the stability of the high XR strain was poor in long fed-batch and chemostat cultures, whereas the low XR strain was stable. The high XR strain lost its XR activity almost completely in some fed-batch cultures and in chemostat culture. In chemostat cultivation, part of the population lost the plasmid harboring the XR gene. This was due to the fact that leucine was released into the broth from plasmid containing cells, which enabled some cells to grow without the plasmid containing the LEU2 auxotrophic complementation selection marker. Furthermore, isolation and analysis of plasmids from a population that had lost its XR activity, showed that in addition to the original plasmid, a rearranged form of the plasmid, retaining the selection marker but not the expression of active XR, was present. However, these observations could only partly explain the decrease in XR activity. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 391-399, 1997.
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  • 68
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    Biotechnology and Bioengineering 54 (1997), S. 513-519 
    ISSN: 0006-3592
    Keywords: plant-microbial associations ; 2,4-D ; biodegradation ; plant protection ; Dolichos lablab ; cotton ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A significant “biosafening” protection of plants from the effect of 2,4-D in plant-microbial associations has been demonstrated in this study. The 2,4-D-degrading plasmid, pJP4 was transferred into Rhizobium sp. CB1024, which nodulates Dolichos lablab, and Azospirillum brasilense Sp7 carrying a nifA-lacZ gene marker, which can colonize cotton roots. Both transconjugants degraded 2,4-D in pure culture via cometabolism up to 50 μg mL-1. When the transconjugants were inoculated onto Dolichos lablab and cotton, respectively, such plants were resistant to this herbicide when the nutrient solution was treated with 2,4-D up to 10 μg mL-1 for Dolichos lablab and 0.5 μg mL-1 for cotton. Plants inoculated with wild-type strains were dead (Dolichos lablab) or dying (cotton). Because cotton is more sensitive to herbicides, only incomplete protection of plants was achieved with the transconjugant. Improving the effect of colonization of Azospirillum on cotton roots may be critical for a complete degradation and plant protection. The transconjugant of Rhizobium sp. CB1024 was still able to nodulate Dolichos lablab, N2-fixing activity was only slightly affected. Other pesticide-degrading capacities may also be inserted into those plant-associated bacterial strains for the degradation of these chemicals by plant-microbial associations. Whether such systems will be successful when applied in the field with competition from other bacteria is being examined. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 513-519, 1997.
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  • 69
    ISSN: 0006-3592
    Keywords: nitrifying bacteria ; Nitrosomonas europaea ; cometabolism ; ammonia monooxygenase ; biodegradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Pure cultures of ammonia-oxidizing bacteria, Nitrosomonas europaea, were exposed to trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), chloroform (CF), 1,2-dichloroethane (1,2-DCA), or carbon tetrachloride (CT), in the presence of ammonia, in a quasi-steady-state bioreactor. Estimates of enzyme kinetics constants, solvent inactivation constants, and culture recovery constants were obtained by simultaneously fitting three model curves to experimental data using nonlinear optimization techniques and an enzyme kinetics model, referred to as the inhibition, inactivation, and recovery (IIR) model, that accounts for inhibition of ammonia oxidation by the solvent, enzyme inactivation by solvent product toxicity, and respondent synthesis of new enzyme (recovery). Results showed relative enzyme affinities for ammonia monooxygenase (AMO) of 1,1-DCE ≈ TCE 〉 CT 〉 NH3 〉 CF 〉 1,2-DCA. Relative maximum specific substrate transformation rates were NH3 〉 1,2-DCA 〉 CF 〉 TCE ≈ 1,1-DCE 〉 CT (=0). The TCE, CF, and 1,1-DCE inactivated the cells, with 1,1-DCE being about three times more potent than TCE or CF. Under the conditions of these experiments, inactivating injuries caused by TCE and 1,1-DCE appeared limited primarily to the AMO enzyme, but injuries caused by CF appeared to be more generalized. The CT was not oxidized by N. europaea while 1,2-DCA was oxidized quite readily and showed no inactivation effects. Recovery capabilities were demonstrated with all solvents except CF. A method for estimating protein yield, the relationship between the transformation capacity model and the IIR model, and a condition necessary for sustainable cometabolic treatment of inactivating substrates are presented. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 520-534, 1997.
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    Biotechnology and Bioengineering 54 (1997), S. 583-594 
    ISSN: 0006-3592
    Keywords: trickle-bed biofilter ; mathematical model ; volatile organic compound (VOC) ; waste gas treatment ; biofiltration ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this article is to define and validate a mathematical model that desribes the physical and biological processes occurring in a trickle-bed air biofilter for waste gas treatment. This model considers a two-phase system, quasi-steady-state processes, uniform bacterial population, and one limiting substrate. The variation of the specific surface area with bacterial growth is included in the model, and its effect on the biofilter performance is analyzed. This analysis leads to the conclusion that excessive accumulation of biomass in the reactor has a negative effect on contaminant removal efficiency. To solve this problem, excess biomass is removed via full media fluidization and backwashing of the biofilter. The backwashing technique is also incorporated in the model as a process variable. Experimental data from the biodegradation of toluene in a pilot system with four packed-bed reactors are used to validate the model. Once the model is calibrated with the estimation of the unknown parameters of the system, it is used to simulate the biofilter performance for different operating conditions. Model predictions are found to be in agreement with experimental data. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 583-594, 1997.
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  • 71
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    Biotechnology and Bioengineering 55 (1997), S. 41-53 
    ISSN: 0006-3592
    Keywords: baculovirus ; insect cell ; p97 ; glycosylation ; GPI anchor ; protein expression ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/Spodoptera frugiperda Sf9 cells) was used to express the GPI-anchored human melanoma tumor antigen, melanotransferrin or p97. This system served to study the expression and productivity of recombinant GPI-anchored p97 by insect cells. The Sf9 cells expressed a cell surface GPI-anchored form of p97 as well as a soluble form of p97 that did not appear to be derived from the GPI-anchored form of p97. Both recombinant forms, although Endo H resistant, migrated slightly faster (∼88 kDa) than the native p97 (∼95-97 kDa). The insect GPI-anchored p97 was sensitive to PI-PLC, which exposed a detectable cross-reacting determinant. The Sf9 cell surface p97 expression was similar to that of human melanoma (SK-MEL-28) cells, whereas the Sf9 cell specific secretion rate was 10-fold higher. Also Sf9 cells retained considerably higher levels of p97 within the cell. The Sf9 cell surface expression of p97 varied with time after infection, with the maximum expression, which appeared independent of multiplicities of infection greater than 1, occurring at 48 h. After 48 h, levels of cell surface and secreted p97 fell whereas p97 retained within the cell increased, which possibly reflected the lytic nature of the expression system. The successful expression of GPI-anchored human p97 by the baculovirus/insect cell system not only provides a source of p97 for further research but also is the basis of an alternative method for the commercial production of GPI-anchored proteins. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 41-53, 1997.
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  • 72
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    Biotechnology and Bioengineering 55 (1997), S. 65-71 
    ISSN: 0006-3592
    Keywords: protein ; osmotic pressure ; activity coefficient ; solubility ; virial expansion ; UNIQUAC model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Modeling of the properties of biochemical components is gaining increasing interest due to its potential for further application within the area of biochemical process development. Generally protein solution properties such as protein solubility are expressed through component activity coefficients which are studied here. The original UNIQUAC model is chosen for the representation of protein activity coefficients and, to the best of our knowledge, this is the first time it has been directly applied to protein solutions. Ten different protein-salt-water systems with four different proteins, serum albumin, alphacymotrypsin, beta-lactoglobulin and ovalbumin, are investigated. A root-mean-squared deviation of 0.54% is obtained for the model by comparing calculated protein activity coefficients and protein activity coefficients deduced from osmotic measurements through virial expansion. Model predictions are used to analyze the effect of salt concentrations, pH, salt types, and temperature on protein activity coefficients and also on protein solubility and demonstrate consistency with results from other references. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 65-71, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 72-81 
    ISSN: 0006-3592
    Keywords: antisense ; receptor targeted delivery ; acute phase response ; IL-6 ; gp 130 ; HepG2 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Antisense technology is potentially a powerful means by which to selectively control gene expression. We have used antisense oligonucleotides to modulate the response of the hepatoma cell line, HepG2, to the inflammatory cytokine, IL-6, by inhibiting the expression of its multifunctional signal transducer, gp130. HepG2 cells respond to IL-6 by upregulating acute phase proteins, such as haptoglobin, by five- to tenfold. Gp130 is central to this response, as the upregulation of haptoglobin is almost completely blocked by the addition of high concentrations (∼100 μg/ml) of a monoclonal antibody to gp 130. Antisense oligodeoxynucleotides complementary to the mRNA encoding gp 130 inhibited the upregulation of haptoglobin by IL-6-stimulated HepG2 cells by about 50%. However, a nonsense sequence also inhibited haptoglobin secretion by about 20%. To improve the specificity and efficiency of action, we targeted the antisense oligonucleotides to HepG2 cells using a conjugate of asialoglycoprotein-poly-L-lysine. The targeted antisense reduced the binding of IL-6 to HepG2 cells, virtually eliminating high affinity binding. In addition, it inhibited haptoglobin upregulation by over 70%. Furthermore, the dose of targeted antisense required for biological effect was reduced by about an order of magnitude as compared with unconjugated antisense. These results demonstrate the potential of antisense oligonucleotides as a means to control the acute phase response as well as the need for a greater understanding of the mechanism and dynamics of antisense molecules as they are developed toward therapeutic application. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 72-81, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 118-135 
    ISSN: 0006-3592
    Keywords: stationary flux estimation ; sensitivity analysis ; covariance analysis ; nonlinear statistics ; Corynebacterium glutamicum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic carbon labelling experiments enable a large amount of extracellular fluxes and intracellular carbon isotope enrichments to be measured. Since the relation between the measured quantities and the unknown intracellular metabolic fluxes is given by bilinear balance equations, flux determination from this data set requires the numerical solution of a nonlinear inverse problem. To this end, a general algorithm for flux estimation from metabolic carbon labelling experiments based on the least squares approach is developed in this contribution and complemented by appropriate tools for statistical analysis. The linearization technique usually applied for the computation of nonlinear confidence regions is shown to be inappropriate in the case of large exchange fluxes. For this reason a sophisticated compactification transformation technique for nonlinear statistical analysis is developed. Statistical analysis is then performed by computing appropriate statistical quality measures like output sensitivities, parameter sensitivities and the parameter covariance matrix. This allows one to determine the order of magnitude of exchange fluxes in most practical situations. An application study with a large data set from lysine-producing Corynebacterium glutamicum demonstrates the power and limitations of the carbon-labelling technique. It is shown that all intracellular fluxes in central metabolism can be quantitated without assumptions on intracellular energy yields. At the same time several exchange fluxes are determined which is invaluable information for metabolic engineering. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 118-135, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 317-327 
    ISSN: 0006-3592
    Keywords: ethanol production ; fed-batch fermentations ; Markov decision processes ; real time optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article describes a methodology that implements a Markov decision process (MDP) optimization technique in a real time fed-batch experiment. Biological systems can be better modeled under the stochastic framework and MDP is shown to be a suitable technique for their optimization. A nonlinear input/output model is used to calculate the probability transitions. All elements of the MDP are identified according to physical parameters. Finally, this study compares the results obtained when optimizing ethanol production using the infinite horizon problem, with total expected discount policy, to previous experimental results aimed at optimizing ethanol production using a recombinant Escherichia coli fed-batch cultivation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 317-327, 1997.
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  • 76
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    Keywords: animal cell fermentation ; perfusion culture ; temperature control ; fermentor optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Temperature is a key environmental variable whose potential in animal cell fermentor optimization is not yet fully utilized. The scarce literature data suggests that reduced fermentor temperature results in an improved viability and shear resistance, higher cell density and titer in batch cultures, and reduction in glucose/lactate metabolism. Due to the arrest of the cells in the G1 phase, the specific growth rate was found to decrease at temperatures below 37.0°C. The response of the specific production rate was cell line dependent: in some cases it increased 2-to-3-fold, but decreased in other cases. The controlable slowdown of cell metabolism at lower temperature can be used in optimization of perfusion mammalian cell cultures with several potential advantages, including higher cell density in oxygen limited reactors, lower perfusion rate, improved product quality, simplified pH control, and others. To evaluate this strategy, a series of long-term experiments in 15 L perfusion bioreactors culturing recombinant hamster cells at 20.0 × 106 cells/mL were conducted. The temperature was changed over a range of set points, and maintained at each of these for a long period of time. Steady state process data was collected and analyzed. The effect of temperature on the following characteristics of the perfusion process was studied: cell growth, glucose/lactate metabolism, glutamine/ammonia metabolism, cell respiration, cell density at constant oxygen transfer rate, proteolytic activity, and product quality (glycosylation and molecule fragmentation). The results suggest that temperature is a variable with a significant potential in optimization of perfusion cultures. Properly selected temperature set point will contribute to the overall improvement of process performance. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 328-338, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 348-358 
    ISSN: 0006-3592
    Keywords: enzymatic resolution ; ο-transaminase ; biphasic system ; product inhibition ; α-methylbenzylamine ; biocompatibility ; extracting capacity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two microorganisms showing high ο-transaminase activity (Klebsiella pneumoniae JS2F and Bacillus thuringiensis JS64) were screened by the enrichment method using (S)-α-methylbenzylamine (α-MBA) as a sole nitrogen source. Optimal carbon and nitrogen sources for enzyme induction and the properties of ο-transaminases were investigated. ο-Transaminase from B. thuringiensis JS64 was highly enantioselective (E = 75.3) for (S)-enantiomer of α-MBA and showed remarkable stability. However, ο-transaminase showed severe product inhibition by acetophenone. An aqueous/organic two-phase system was introduced to overcome this problem. Through solvent screening, cyclohexanone and ethyl acetate were selected as the best organic phases. The acetophenone-extracting capacity of the solvent and the biocompatibility of the solvent to the cell were important determinants in the reaction rate at high concentrations of α-MBA. The reaction rate of ο-transamination was strongly influenced by the volume ratio of organic phase to aqueous phase as well as agitation speed in the biphasic mixture. Using the optimal volume ratio (Vorg:Vaq = 1:4) in the biphasic system with cyclohexanone, the reaction rate of ο-transaminase under vigorous mixing conditions increased ninefold compared with that in the monophasic aqueous system. At the same optimal conditions, using whole cells, 500 mM α-MBA could be resolved successfully to above 95% enantiomeric excess of (R)-α-MBA with ca. 51% conversion. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 348-358, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 375-389 
    ISSN: 0006-3592
    Keywords: bacteriophage T7 ; kinetic simulation ; intracellular growth ; gene expression ; antiviral strategies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Viruses have evolved to efficiently direct the resources of their hosts toward their own reproduction. A quantitative understanding of viral growth will help researchers develop antiviral strategies, design metabolic pathways, construct vectors for gene therapy, and engineer molecular systems that self-assemble. As a model system we examine here the growth of bacteriophage T7 in Escherichia coli using a chemical-kinetic framework. Data published over the last three decades on the genetics, physiology, and biophysics of phage T7 are incorporated into a genetically structured simulation that accounts for entry of the T7 genome into its host, expression of T7 genes, replication of T7 DNA, assembly of T7 procapsids, and packaging of T7 DNA to finally produce intact T7 progeny. Good agreement is found between the simulated behavior and experimental observations for the shift in transcription capacity from the host to the phage, the initiation times of phage protein synthesis, and the intracellular assembly of both wild-type phage and a fast-growing deletion mutant. The simulation is utilized to predict the effect of antisense molecules targeted to different T7 mRNA. Further, a postulated mechanism for the down regulation of T7 transcription in vivo is quantitatively examined and shown to agree with available data. The simulation is found to be a useful tool for exploring and understanding the dynamics of virus growth at the molecular level. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 375-389, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 408-418 
    ISSN: 0006-3592
    Keywords: lead absorption ; microorganisms ; modeling ; Anabaena cylindrica ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The ability of microorganisms to selectively adsorb various heavy metal ions has been recognized for over a decade. We have investigated the biosorption of lead by an active culture of the cyanobacterium Anabaena cylindrica. Energy dispersive X-ray microanalysis was used to evaluate the different uptake mechanisms in the various subcellular regions. Three were identified: a very fast adsorption mechanism in the cell envelope; a time-dependent deposition reaction on the cell surface; and an adsorption mechanism, also time dependent, on the polyphosphate body inside the cell. Atomic absorption spectrometry was then used to quantify the changes with time of bulk fluid concentrations of lead solutions exposed to cyanobacteria. A mass transfer kinetic model was developed which quantitatively predicts the concentration of lead in cells of Anabaena cylindrica as a function of spatial dimensions and time. The model predictions are consistent with a pattern, documented in literature and confirmed by our own experimental evidence, of a very fast uptake in the cell envelope and then a longer uptake period inside the cell. Our experimental evidence also revealed a time-dependent uptake mechanism on the surface of the cells, which is included in the model. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 408-418, 1997.
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    Biotechnology and Bioengineering 55 (1997), S. 427-438 
    ISSN: 0006-3592
    Keywords: multisensor array ; baker's yeast ; sensor fusion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of a multisensor array for measuring the emission from a production-scale baker's yeast manufacturing process is reported. The sensor array, containing 14 different gas-sensitive semiconductor devices and an infrared gas sensor, was used to monitor the gas emission from a yeast culture bioreactor during fed-batch operation. The signal pattern from the sensors was evaluated in relation to two key process variables, the cell mass and the ethanol concentrations. Fusion with the on-line sensor signals for reactor weight and aeration rate made it possible to estimate cell mass and ethanol concentration using computation with backpropagating artificial neural nets. Identification of process states with the same fusion of sensor signals was realized using principal component analysis. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 427-438, 1997.
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    Biotechnology and Bioengineering 53 (1997), S. 88-99 
    ISSN: 0006-3592
    Keywords: biofilm structure ; detachment ; abrasion ; collisions ; airlift-reactor ; hydrodynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The detachment of biomass from suspended biofilm pellets in three-phase internal loop airlift reactors was investigated under nongrowth conditions and in the presence of bare carrier particles. In different sets of experiments, the concentrations of biofilm pellets and bare carrier particles were varied independently. Gas hold-up, bubble size, and general flow pattern were strongly influenced by changes in volume fractions of biofilm pellets and bare carrier particles. In spite of this, the rate of biomass detachment was found to be linear with both the concentration of biofilm pellets and the bare carrier concentration up to a solids hold-up of 30%. This implies that the detachment rate was dominated by collisions between biofilm pellets and bare carrier particles. These collisions caused an on-going abrasion of the biofilm pellets, leading to a reduction in pellet volume. Breakage of the biofilm pellets was negligible. The biofilm pellets were essentially ellipsoidal, which made three-dimensional size determination necessary. Calculating particle volumes from two-dimensional image analysis measurements and assuming a spherical shape led to serious errors. The abrasion rate was not equal on all sides of the biofilm pellets, resulting in an increasing flattening of the pellets. This flattening was oriented with the basalt carrier inside the biofilm and independent of the absolute abrasion rate. These observations suggest that the collisions causing abrasion are somehow oriented. The internal structure of the biofilms showed two layers, a cell-dense outer layer and an interior with a low biomass density. Taking this density gradient into account, the washout of detached biomass matched observed changes in volume of the biofilm pellets. No gradient in biofilm strength with biofilm depth was indicated. © 1997 John Wiley & Sons, Inc.
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  • 82
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    Keywords: citrobacter sp. ; immobilized cells ; low pH ; phosphatase ; uranium ; wastewater ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A biotechnological process for the removal of heavy metals from aqueous solution utilizes enzymatically liberated phosphate ligand which precipitates with heavy metals (M) as cell-bound MHPO4. The enzyme, a phosphatase, obeys Michaelis-Menten kinetics in resting and immobilized cells; an integrated form of the Michaelis-Menten equation was used to calculate the apparent Km (Km app.) as operating in immobilized cells in flow-through columns by a ratio method based on the use of two enzyme loadings (Eo1, Eo2) or two input substrate concentrations (So1, So2). The calculated Km app. (4.08 mM) was substituted into an equation to describe the removal of metals by immobilized cells. In operation the activity of the bioreactor was in accordance with that predicted mathematically, within 10%. The initial tests were done at neutral pH, whereas the pH of industrial wastewaters is often low; an increase in the Km app. at low pH was found in previous studies. Immobilized cells were challenged with acidic mine drainage wastewaters, where the limiting factors were chemical and not biochemical. Bioreactors initially lost activity in this water, but recovered to remove uranyl ion with more than 70% efficiency under steady-state conditions in the presence of competing cations and anions. Possible reasons for the bioreactor recovery are chemical crystallization factors. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 121-131 
    ISSN: 0006-3592
    Keywords: biocalalysis ; microemulsion ; gelatin ; organogel ; lipase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chromobacterium viscosum (CV) lipase was immobilized in gelatin-containing Aerosol-OT (AOT) microemulsion-based organogels (MBGs). The behavior of this novel, predominantly hydrophobic matrix as an esterification catalyst has been examined. The biocatalyst was most effective when the MBG was granulated to yield gel particles of ∼500 μm diameter, providing a total surface area of ca. 106 mm2 per 10 cm3 of gel. The gel was generally contacted with a solution of the substrate(s) in a hydrocarbon oil. Under most conditions reaction was not diffusion limited. Apparent lipase activity was influenced by certain compositional changes in the MBG, but most significantly when the R value, the mole ratio of water to surfactant, was altered. Higher activities were observed at lower R values. Although gels of lowest R value expressed the highest condensation activity, such formulations were physically unsuitable as immobilization matrices due to their proximity to the gel-solution phase boundary. MBGs of intermediate R values (between 60 and 80) were considered most suitable because they offer relatively high condensation activity and good physical stability. The gelatin concentration also exerted a small but measurable influence on the observed condensation rates. Apparent lipase activity was also influenced to some extent by the nature of the parent hydrocarbon used to prepare the MBG. Higher activities were obtained using formulations derived from isooctane and cyclohexane rather than the n-alkanes. Condensation activities expressed by CV lipase in the MBGs were broadly comparable to those expressed in the analogous parent water-in-oil (w/o) microemulsions. The MBGs functioned effectively in neat substrate solutions, but the condensation activity expressed by the MBGs in a series of successive batch syntheses was adversely affected by the formation and retention of the water coproduct. Selective removal of the water was achieved using a concentrated solution of dry reverse micelles, which resulted in recovery of lost activity. Pretreatment of lipase-containing MBGs resulted in the formation of MBGs with enhanced catalytic properties and modified composing the conventional procedure. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 139-150 
    ISSN: 0006-3592
    Keywords: filamentous fungus ; tridimensional structure ; fractal dimension ; spore aggregates ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The tridimensional growth of a filamentous fungus was simulated, based on a model for the evolution of the microscopic morphology of Trichoderma reesei. When supplemented with a spatial representation of growth, the model correctly simulates the evolution from a single spore to a pellet. Diffusion of oxygen is included in the model. The simulated tridimensional structures have a fractal nature; and the fractal dimension, determined by a box-counting method, increases during growth. The fractal dimension only depends on the mass of the pellet and is not affected by model parameters such as tip extension rate and branching frequency. Realistic pictures are obtained and the radius of the pellet increases at a constant rate. The influence of model parameters (tip extension rate, branching frequency, minimum porosity) on dissolved oxygen concentration profiles, biomass concentration profiles, rate at which the pellet diameter increases, and the evolution of the fractal dimension was determined. The dissolved oxygen profiles were found to be very different from the profiles, obtained by assuming a homogenous biomass distribution within the pellet. Finally, the formation of pellets from spore aggregates is calculated and the size of the spore aggregate is found to only influence the time needed before the appearance of a pellet and not its morphology. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 159-167 
    ISSN: 0006-3592
    Keywords: amino acid ; flor yeast ; L-proline ; urea ; wine aging ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Urea, ammonium, and free amino acid contents were quantified in biological aging of a young wine under two flor film forming yeast strains, Saccharomyces cerevisiae race capensis and S. cerevisiae race bayanus, and compared. Cell viability in the film was different for the two yeast strains. Thus, capensis maintained a much greater number of viable cells per surface area than bayanus and hence used greater amount of nitrogen compounds. The main source of nitrogen for the yeasts during the biological aging process was L-proline. The two yeast strains also differed in the amounts of assimilable nitrogen they utilized, in their preferences for amino acid consumption, and kinetics. To accelerate the aging process, the effect of controlled monthly aeration of the wine aged with capensis strain was investigated. The results revealed that short aeration did not appreciably increase the overall consumption of assimilable nitrogen, but consumption of some nitrogen compounds was accelerated (particularly L-proline, L-tryptophan, L-glutamic acid, ammonium ion, L-lysine, and L-arginine); the use of L-ornithine was inhibited; and GABA, L-methionine, and urea were depletes. Probably the aeration increases the aroma compounds, thereby producing wines with improved sensory properties. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 168-178 
    ISSN: 0006-3592
    Keywords: airlift reactor ; BAS reactor ; biofilm ; nitrification ; nitrite ; oxygen transfer ; residence time ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biofilm airlift suspension (BAS) reactor can treat wastewater at a high volumetric loading rate combined with a low sludge loading. Two BAS reactors were operated, with an ammonium load of 5 kg N/(m3 d), in order to study the influence of biomass and oxygen concentration on the nitrification process. After start-up the nitrifying biomass in the reactors gradually increased up to 30 g VSS/L. Due to this increased biomass concentration the gas-liquid mass transfer coefficient was negatively influenced. The resulting gradual decrease in dissolved oxygen concentration (over a 2-month period) was associated with a concomitantly nitrite build-up. Short term experiments showed a similar relation between dissolved oxygen concentration (DO) and nitrite accumulation. It was possible to obtain full ammonium conversion with approximately 50% nitrate and 50% nitrite in the effluent. The facts that (i) nitrite build up occurred only when DO dropped, (ii) the nitrite formation was stable over long periods, and (iii) fully depending on DO levels in short term experiments, led to the conclusion that it was not affected by microbial adaptations but associated with intrinsic characteristics of the microbial growth system. A simple biofilm model based on the often reported difference of oxygen affinity between ammonium and nitrite oxydizers was capable of adequately describing the phenomena.Measurements of biomass density and concentration are critical for the interpretation of the results, but highly sensitive to sampling procedures. Therefore we have developed an independent method, based on the residence time of Dextran Blue, to check the experimental methods. There was a good agreement between procedures.The relation between biomass concentration, oxygen mass transfer rate and nitrification in a BAS reactor is discussed. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 202-206 
    ISSN: 0006-3592
    Keywords: salting-out precipitation ; initial protein concentration ; protein solubility ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of initial protein concentration on the performance of salting-out precipitation is examined. In the precipitation of bovine albumin by ammonium sulfate, a peak occurs in the plot of protein solubility versus initial protein concentration; that is, the solubility first increases and then falls with increasing initial protein concentration. In addition, the dependence of solubility on the initial protein concentration is less significant if using higher salt concentrations. The solubility behavior of bovine albumin may be representative, because it covers all possible alternatives; namely, the solubility is independent of, increases with, decreases with, or first increases and then decreases with the initial protein concentration. The appearance of a solubility peak can be explained based on the occurrence of a primary particle during the precipitation process. However, inclusion of the influence of initial protein concentration into the Cohn equation is not feasible with the use of a logarithmic scale, which does not sensitively reflect the change in protein solubility. Increasing initial protein concentration favors protein recovery because it reduces the resultant volume of the supernatant phase. © l997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 214-219 
    ISSN: 0006-3592
    Keywords: Taxol ; plant cell culture ; continuous production ; immobilization ; Taxus cuspidata ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production characteristics for Taxol (paclitaxel) using free and immobilized cells of Taxus cuspidata were investigated in a perfusion culture bioreactor. Although the cell growth was inhibited by higher dilution rates, the specific production rate of Taxol was increased by perfusion compared with that using batch operation. Perfusion cultures using a nylon-mesh cell separator for free suspension cells showed similar production profiles to those obtained using immobilized cells. Continuous Taxol production was successfully obtained at an approximate specific production rate of 0.3 mg/g DCW (dry cell weight) per day for up to 40 days. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 207-213 
    ISSN: 0006-3592
    Keywords: mineral support ; cephamycin C ; soybean oil ; Streptomyces ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mineral support was used for cephamycin C production in a culture using soybean oil as the sole carbon source. When the support was added into an oil-water system, the soybean oil was emulsified as fine oil droplets, which was observed by a photomicroscope. Mycelia were also twined around the support, which was observed by a scanning electron microscope. That caused the formation of an oil-mycelia complex on or around the support, which provided a larger specific surface area of oil. When 15 g/L of the support was used in a batch culture of Streptomyces sp. P6621 with 50 g/L of soybean oil, the maximum cephamycin C concentration was 2.8 g/L, which was 2.2 times higher than that without the support. This indicates that the mineral support is useful for the culture system using vegetable oil as a carbon source. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 238-241 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; bacterial motility ; geometrical restriction ; capillary tube ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Motility of Escherichia coli K-12 cells was studied in a capillary tube with an inside diameter of 3 μm, which is comparable to the size of the cells' body. The extreme restriction, imposed by the capillary on the bacterial motility, caused a series of phenomena such as cell aggregation, swimming as a cluster, and break-up of aggregates, which were observable for the first time, and which are reported here. © 1997 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 53 (1997), S. 220-225 
    ISSN: 0006-3592
    Keywords: anaerobic processes ; immobilized anaerobic sludge ; intrinsic kinetic parameters ; fixed-bed reactors ; horizontal-flow anaerobic immobilized sludge reactor ; polyurethane foam ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (robs) under a diversity of external mass transfer resistance conditions. The robs curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters rmax, the maximum specific substrate utilization rate, and Ks, the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30°C resulted in intrinsic rmax and Ks, respectively, of 0.330 mg chemical oxygen demand (COD) · mg-1 volatile suspended solids (VSS) · h-1 and 72 mg COD · L-1. In comparison with the values found in the literature, intrinsic rmax is significantly high and intrinsic Ks is relatively low. © 1997 John Wiley & Sons, Inc.
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  • 92
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    Biotechnology and Bioengineering 53 (1997), S. 243-252 
    ISSN: 0006-3592
    Keywords: carbon dioxide evolution rate ; mass transfer ; modeling ; biodegradation ; pH ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Respirometry is a precious tool for determining the activity of microbial populations. The measurement of oxygen uptake rate is commonly used but cannot be applied in anoxic or anaerobic conditions or for insoluble substrate. Carbon dioxide production can be measured accurately by gas balance techniques, especially with an on-line infrared analyzer. Unfortunately, in dynamic systems, and hence in the case of short-term batch experiments, chemical and physical transfer limitations for carbon dioxide can be sufficient to make the observed carbon dioxide evolution rate (OCER) deduced from direct gas analysis very different from the biological carbon dioxide evolution rate (CER).To take these transfer phenomena into account and calculate the real CER, a mathematical model based on mass balance equations is proposed. In this work, the chemical equilibrium involving carbon dioxide and the measured pH evolution of the liquid medium are considered. The mass transfer from the liquid to the gas phase is described, and the response time of the analysis system is evaluated.Global mass transfer coefficients (KLa) for carbon dioxide and oxygen are determined and compared to one another, improving the choice of hydrodynamic hypotheses. The equations presented are found to give good predictions of the disturbance of gaseous responses during pH changes.Finally, the mathematical model developed associated with a laboratory-scale reactor, is used successfully to determine the CER in nonstationary conditions, during batch experiments performed with microorganisms coming from an activated sludge system. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 243-252, 1997.
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  • 93
    ISSN: 0006-3592
    Keywords: PHB ; poly(β-hydroxybutyrate) ; Paracoccus pantotrophus ; dynamic growth ; metabolic modeling ; polymers ; activated sludge process ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of the research was to obtain insights into the behavior of microorganisms under feast/famine conditions as often occur in wastewater treatment processes. The response of microorganisms to such conditions is the accumulation of storage polymers like poly(β-hydroxybutyrate). The research was performed using a pure culture of Paracoccus pantotrophus LMD 94.21. A steady-state C-limited chemostat culture was switched to batch mode and a pulse of acetate was added. As long as external substrate (acetic acid) was present, the organism grew and accumulated poly(β-hydroxybutyrate). After depletion of the external substrate, the stored poly(β-hydroxybutyrate) was used as growth substrate. Poly(β-hydroxybutyrate) accumulation was found to be strongly dependent on the growth rate of the organism before the pulse addition of acetate. Poly(β-hydroxybutyrate) accumulation was correlated to the difference in maximum acetate uptake rate and the acetate required for growth. Based on the interpretation of the experimental results, a metabolically structured model has been set up. This model adequately describes the observed kinetics of the poly(β-hydroxybutyrate) formation and consumption. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 773-782, 1997.
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  • 94
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    Biotechnology and Bioengineering 53 (1997), S. 290-295 
    ISSN: 0006-3592
    Keywords: yeast lysis ; Monte Carlo simulation ; cell rupture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The overall reaction in the enzymatic lysis of yeast takes place in three major steps: (i) the two-layer wall is digested, (ii) the cell bursts under the osmotic pressure difference to release its intracellular material, and (iii) the intracellular material is digested by the enzymes still present in the solution. The first and third steps are continuous processes, adequately described by Michaelis-Menten kinetic models. The second step is a discrete event, statistical in nature. A model of engineering value should effectively bridge the gap between the two continuous processes (first and third steps). In this work, Monte Carlo simulations are used to identify a suitable function that captures the statistical nature of cell rupture and represents the rate of release of intracellular material. It is shown that the two-parameter beta distribution function serves this purpose most effectively. Comparisons with experimental results indicate that the cell rupture ratio is a widely distributed statistical function. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 290-295, 1997.
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  • 95
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    Biotechnology and Bioengineering 53 (1997), S. 274-282 
    ISSN: 0006-3592
    Keywords: insulinoma cells ; regulated secretion ; insulin secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Controlled secretion of proteins from endocrine-derived cell lines has been proposed as a means to produce some classes of post-translationally modified proteins in bioreactors. Under the right biological and environmental conditions it may be possible to improve the product purity or quality relative to that obtained through steady (constitutive) secretion. The pancreatic-islet-derived cell line, βTC-3, was selected as a model system to explore the secretory dynamics of insulin under various combinations of stimulatory or inhibitory environmental conditions. The βTC-3 cells exhibited a glucose-mediated stimulus-response pattern which was saturated above 1 mM glucose and with an apparent “Kg” of 0.1 mM glucose. However, the kinetics of insulin synthesis were closely coupled to those of secretion such that βTC-3 cells cycled between saturating and basal levels of glucose were never perturbed far from an intracellular synthesis-secretion equilibrium. When more powerful and selective agents were used to control secretion, the system performance improved markedly. A combination of 1 mM isobytylmethylxanthine (IBMX) and 1 μM carbachol (with saturating levels of glucose) could discharge 75% of stored insulin in 2 h. When this treatment was followed by incubation in media adjusted to attenuate the influx of calcium into the cells, intracellular pools were efficiently replenished within 24 h. Calcium attenuating treatments included hyperpolarization with reduced potassium (1 mM), calcium channel blockade with the dihydropyridine verapamil (1 μM), and the direct mass-action effect of reduced environmental calcium (0.5 mM versus 1.8 mM). Other inhibitory treatments were explored, but these tended to reduce both insulin synthesis and secretion. The best recharging treatment found was a combination of verapamil (1 μM) with reduced calcium level (0.5 mM).To demonstrate the feasibility of a controlled secretion process, βTC-3 T-flask cultures were grown to confluence, then cycled through two periods of discharging (2 h) and recharging (20 h) with the best combinations of secretagogues and calcium attenuators. The overall process was quite efficient: Only 15% of the overall insulin secretion took place during the recharging episodes, and this residual secretion represented only 10% of the net insulin synthesis during these episodes. Discharging was very effective in the first episode (80% recovery of stored insulin), but slightly less efficient in subsequent discharging episodes, possibly due to a desensitization effect of the calcium attenuating media. Nevertheless, the regulated secretory pathway of βTC-3 cells could be successfully harnessed to a controlled secretion process for the selective recovery of stored insulin. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 274-282, 1997.
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  • 96
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    Biotechnology and Bioengineering 53 (1997), S. 310-319 
    ISSN: 0006-3592
    Keywords: T. ferrooxidans ; iron oxidation ; energetics ; model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A structured model for Thiobacillus ferrooxidans growth dependence on ferrous and ferric iron, arsenic, oxygen, carbon dioxide, pH, and temperature is presented. A new kinetic mechanism for ferrous oxidation by T. ferrooxidans is introduced. Data from several earlier experimental studies of T. ferroaxidans growth are used for model development. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 310-319, 1997.
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  • 97
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    Biotechnology and Bioengineering 53 (1997), S. 01-09 
    ISSN: 0006-3592
    Keywords: transferrin ; conalbumin ; metalloprotein ; affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recently these laboratories have demonstrated that it is possible to use proteins as efficient, selective agents for heavy metal removal and recovery. In this study, transferrin was chemically bound to an insoluble support. The ability of immobilized transferrin to produce clean water was demonstrated. Copper loading was independent of feed concentration. The loaded copper could be readily eluted and concentrated into the gram per liter range. The mechanism of copper release was studied. It was shown that release was dependent on pH and the chelating ability of the stripping agent. Metal release occurred slowly at pH 〈 7. However, at low pH in the presence of a chelator, metal removal occurred much more efficiently. The binding constant of copper to immobilized transferrin was determined as a function of pH. This information was used to model metal binding and release to the protein/support matrix. © 1997 John Wiley & Sons, Inc.
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  • 98
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    Biotechnology and Bioengineering 53 (1997), S. 17-20 
    ISSN: 0006-3592
    Keywords: P. chrysogenum ; alginate oligosaccharides ; oligomannuronate ; oligoguluronate ; penicillin G ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 μg/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. © 1997 John Wiley & Sons, Inc.
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  • 99
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    Biotechnology and Bioengineering 53 (1997), S. 26-31 
    ISSN: 0006-3592
    Keywords: protease ; transesterification ; enantioselectivity ; organic solvent ; solvent effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30°C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN′ always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. © 1997 John Wiley & Sons, Inc.
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  • 100
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    Biotechnology and Bioengineering 53 (1997), S. 41-48 
    ISSN: 0006-3592
    Keywords: nicotinamide adenine dinucleotide ; isourea ; imidocarbonate ; agarose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stability of NAD(H) immobilized to a crosslinked agarose support (Sepharose®-4B) was examined in buffer solutions at a pH of 7.0 and 8.5. Specifically, this study investigated particle attrition and ligand leakage rates from a cyanogen bromide activated agarose support. Particle attrition did not occur under the experimental conditions. Ligand leakage rates were found to be first order in immobilized ligand concentration with two labile populations of ligand. The two-population model is consistent with the cyanogen bromide coupling chemistry, which results in both an isourea and imidocarbonate ligand linkage. The rate of ligand leakage was found to occur over a time scale of days, with first order rate constants ranging from 0.007 to 0.15 d-1, depending on solution pH. © 1997 John Wiley & Sons, Inc.
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