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  • Biochemistry and Biotechnology  (16,218)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 1-10 
    ISSN: 0006-3592
    Keywords: pTRIDENT ; tricistronic expression vectors ; gene expression ; mammalian cells ; tetracycline-responsive promoter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We constructed tricistronic expression vectors for the simultaneous and coordinated expression of three independent genes in mammalian cells. One single promoter allows high level and, in some vectors, adjustable transcription of all three cistrons. Whereas the first cistron is translated in a cap-dependent manner, the subsequent ones utilize intercistronic regions of viral origin such as the internal ribosomal entry site of poliovirus or the cap-independent translation enhancer of encephalomyocarditis virus for enhanced translation. Three multiple cloning sites with a total of up to 18 unique restriction sites allow sequential cloning of the genes of interest. The modular structure of this pBluescript®-based high copy number vector system allows straightforward movement of individual cistrons among members of the pTRIDENT family, and facilitates their combination with existing expression vectors. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 1-10, 1998.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 22-25 
    ISSN: 0006-3592
    Keywords: eCG ; pregnant mare serum gonadotrophin ; batch sorption ; hormone purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Equine chorionic gonadotrophin (eCG) is a hormone of practical value in veterinary medicine and animal production. Here we report a novel preparation procedure based on its direct adsorption onto anionic-exchange resins in a batch-wise mode. The active plasma is previously conditioned to reduce pH and ionic strength to required levels. After the adsorption stage, a 90% recovery of the initial eCG is achieved, with a concentration factor of about 50 and an enrichment factor around 500, with high preservation of biological activity. Further purification is carried out by cation-exchange column chromatography. The recovery for the whole process is higher than 70%, and the final potency of the preparation is close to 4000 IU/mg. The process is well suited for its application to the industrial scale. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 22-25, 1998.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 121-125 
    ISSN: 0006-3592
    Keywords: sucrose monoester synthesis ; lipase-catalyzed acylation ; water activity (a w) ; regioselectivity ; salt hydrate pair ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Sucrose monoesters of a fatty acid were synthesized by using lipase in a solvent-free system. When lipase from Mucor miehei was used as a catalyst with capric acid as the donor and sugar as the acceptor, sucrose 6-monocaprate was predominantly produced in a yield of 25.3%. The yield of product was significantly increased by the direct addition of a suitable pair of solid salt hydrates to the reaction mixture to control the water activity (aw). Among the salt hydrate pairs investigated, the barium hydroxide, 8/1H2O pair resulted in the highest yield of the product. This salt addition method was also successfully employed for acylation of primary hydroxyl groups in various unprotected mono- and disaccharides such as glucose, galactose, fructose, trehalose, mannose, maltose, and lactose. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 121-125, 1998.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 216-219 
    ISSN: 0006-3592
    Keywords: liposomes ; vesicles ; microreactor ; permeability ; chymotrypsin ; enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase α-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide) - for which the liposome bilayer was permeable - were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor. © 1998 John Wiley & Sons, Inc.Biotechnol. Bioeng. 57: 216-219, 1998.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 220-227 
    ISSN: 0006-3592
    Keywords: PAH degradation ; white rot fungus ; Bjerkandera sp. ; surfactant ; toxicity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of nonionic surfactants on the polycyclic aromatic hydrocarbon (PAH) oxidation rates by the extracellular ligninolytic enzyme system of the white-rot fungus Bjerkandera sp. strain BOS55 was investigated. Various surfactants increased the rate of anthracene, pyrene, and benzo[a]pyrene oxidation by two to fivefold. The stimulating effect of surfactants was found to be solely due to the increased bioavailability of PAH, indicating that the oxidation of PAH by the extracellular ligninolytic enzymes is limited by low compound bioavailability. The surfactants were shown to improve PAH dissolution rates by increasing their aqueous solubility and by decreasing the PAH precipitate particle size. The surfactant Tween 80 was mineralized by Bjerkandera sp. strain BOS55; as a result both the PAH solubilizing activity of Tween 80 and its stimulatory effect on anthracene and pyrene oxidation rates were lost within 24 h after addition to 6-day-old cultures. It was observed that the surfactant dispersed anthracene precipitates recrystallized into larger particles after Tween 80 was metabolized. However, benzo[a]pyrene precipitates remained dispersed, accounting for a prolonged enhancement of the benzo[a]pyrene oxidation rates. Because the endogenous production of H2O2 is also known to be rate limiting for PAH oxidation, the combined effect of adding surfactants and glucose oxidase was studied. The combined treatment resulted in anthracene and benzo[a]pyrene oxidation rates as high as 1450 and 450 mg L-1 d-1, respectively, by the extracellular fluid of 6-day-old fungal cultures. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 220-227, 1998.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 172-186 
    ISSN: 0006-3592
    Keywords: hybridoma ; futile cycling ; hollow fiber bioreactor ; glutamine ; NMR ; C-13 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of changes in extracellular glutamine level on metabolism of a murine hybridoma was examined with in vivo nuclear magnetic resonance (NMR) spectroscopy. Cells were cultured in a hollow-fiber bioreactor at high cell density to allow intracellular metabolite levels to be determined on a metabolically relevant time scale. Steady infusions of [1-13C] glucose were used to label glycolytic and tricarboxylic acid cycle intermediates, which permitted continuous monitoring with NMR spectroscopy during changes in environmental glutamine level. Samples of the extracellular medium were also analyzed to determine the effect of glutamine on other metabolites associated with primary and secondary metabolism. The changes in glutamine concentration had several effects on primary and secondary metabolism, depending on the rate the changes were made. For a brief reduction in feed glutamine concentration from 4 to 0 mM (which produced a rapid change from 0.67 to ∼0 mM in residual glutamine), large changes were observed in the rate of consumption of metabolites normally associated with energy production. Antibody synthesis was strongly stimulated and nitrogen metabolism was significantly altered. For a more prolonged reduction from 2.4 to 1.2 mM (which produced a slower reduction from 0.30 to 0.08 mM in residual glutamine), much smaller changes were observed even though the concentration of glutamine at the reduced feed level was very low. Energy metabolism did not appear to be limited by glutamine at 0.08 mM, which suggests that significant futile cycling may occur in energy producing pathways when excess glucose and glutamine are available. However, this concentration of extracellular glutamine appeared to affect some anabolic pathways, which require amino groups from glutamine. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 172-186, 1998.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 198-210 
    ISSN: 0006-3592
    Keywords: Xanthan fermentation ; agitator speed ; caverns ; dissolved oxygen ; specific oxygen uptake rate ; specific Xanthan production rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Agitation speed affects both the extent of motion in Xanthan fermentation broths because of their rheological complexity and the rate of oxygen transfer. The combination of these two effects causes the dissolved oxygen concentration and its spatial uniformity also to change with agitator speed. Separating these complex interactions has been achieved in this study in the following way. First, the influence of agitation speeds of 500 and 1000 rpm has been investigated at a constant nonlimiting dissolved oxygen concentration of 20% of air saturation using gas blending. Under these controlled dissolved oxygen conditions, the results demonstrate that the biological performance of the culture was independent of agitation speed as long as broth homogeneity could be ensured. With the development of increasing rheological complexity lending to stagnant regions at Xanthan concentrations 〉20 g/L, it is shown that the superior bulk mixing achieved at 1000 rpm, compared with 500 rpm, leading to an increased proportion of the cells in the fermentor to be metabolically active and hence higher microbial oxygen uptake rates, was responsible for the enhanced performance. Second, the effects of varying dissolved oxygen are compared with a control in each case with an agitator speed of 1000 rpm to ensure full motion, but with a fixed, nonlimiting dissolved oxygen of 20% air saturation. The specific oxygen uptake rate of the culture in the exponential phase, determined using steady-state gas analysis data, was found to be independent of dissolved oxygen above 6% air saturation, whereas the specific growth rate of the culture was not influenced by dissolved oxygen, even at levels as low as 3%, although a decrease in Xanthan production rate could be measured. In the production phase, the critical oxygen level was determined to be 6% to 10%, so that, below this value, both specific Xanthan production rate as well as specific oxygen uptake rate decreased significantly. In addition, it is shown that the dynamic method of oxygen uptake determination is unsuitable even for moderately viscous Xanthan broths. © 1998 John Wiley & Sons, Inc. Biotechnol. Bioeng. 57: 198-210, 1998.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 251-261 
    ISSN: 0006-3592
    Keywords: continuous culture ; metabolic overflow ; multiplicity ; stability analysis ; dynamics ; growth inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 251-261, 1998.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 211-215 
    ISSN: 0006-3592
    Keywords: protein ; conformational memory ; organic solvent ; molecular imprinting ; enzyme ; catalysis ; transition state analogue ; bovine serum albumin ; β-lactoglobulin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The induction of catalytic activity in proteins by lyophilization in the presence of a transition state analogue (biomolecular imprinting) has been attempted. It was shown that proteins which were freeze-dried with n-isopropyl-4-nitrobenzyl-amine (a transition state analogue for the reaction of dehydrofluorination of 4-fluoro-4-[p-nitrophenyl] butan-2-one) displayed higher β-elimination activity as compared to their-non-imprinted counterparts. It was also found that native bovine serum albumin has a high dehydrofluorination activity towards the above substrate with kinetic parameters rather similar to those of a catalytic antibody prepared by Shokat et al. (1989). A comparison of the kinetic parameters determined in this study with those obtained for analogous catalytic antibodies and imprinted polymers was made. © 1998 John Wiley & Sons, Inc. Biotechnol. Bioeng. 57: 211-215, 1998.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 545-551 
    ISSN: 0006-3592
    Keywords: enzyme array ; pulsed amperometric detection ; carbohydrate analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The introduction of an enzyme array-electrochemical detection method for carbohydrate analysis is demonstrated by using two complex and one high mannose N-linked oligosaccharides. Instead of measuring the remaining uncleaved oligosaccharides in enzymatic digestion, released monosaccharides are directly quantified by pulsed amperometric detection at a gold electrode. The measured monosaccharide concentrations in combination with the enzyme array analysis provide structural characterization of oligosaccharides. The enzyme array-electrochemical detection method does not require any separation procedure or any prior labeling of oligosaccharides. However, this method is limited by the use of purified oligosaccharide samples and the nature of the enzyme array. The development of more sophisticated enzyme arrays relies upon the introduction of a bank of highly specific (bond, arm, aglycon) exoglycosidases. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 545-551, 1998.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 557-570 
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; polyhydroxyalkanoates ; metabolic engineering ; mathematical modeling ; enzyme kinetics ; regulation of metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model describing intracellular polyhydroxybutyrate (PHB) synthesis in Alcaligenes eutrophus has been constructed. The model allows investigation of issues such as the existence of rate-limiting enzymatic steps, possible regulatory mechanisms in PHB synthesis, and the effects different types of rate expressions have on model behavior. Simulations with the model indicate that activities of all PHB pathway enzymes influence overall PHB flux and that no single enzymatic step can easily be identified as rate limiting. Simulations also support regulatory roles for both thiolase and reductase, mediated through AcCoA/CoASH and NADPH/NADP+ ratios, respectively. To make the model more realistic, complex rate expressions for enzyme-catalyzed reactions were used which reflect both the reversibility of the reactions and the reaction mechanisms. Use of the complex kinetic expressions dramatically changed the behavior of the system compared to a simple model containing only Michaelis-Menten kinetic expressions; the more complicated model displayed different responses to changes in enzyme activities as well as inhibition of flux by the reaction products CoASH and NADP+. These effects can be attributed to reversible rate expressions, which allow prediction of reaction rates under conditions both near and far from equilibrium. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 557-570, 1998.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 552-556 
    ISSN: 0006-3592
    Keywords: baroenzymology ; reversed micelles ; α-chymotrypsin ; catalytic activity and stability ; effect of pressure, temperature, and glycerol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Thermostability of α-chymotrypsin at normal pressure in reversed micelles depends on both an effective surfactant solvation degree and glycerol content in the system. The difference in α-chymotrypsin stability in reversed micelles at various glycerol concentrations [up to 60% (v/v)] was more pronounced at high surfactant degrees of solvation, R ≥ 16. After a 1-h incubation at 40°C in “aqueous” reversed micelles (in the absence of glycerol), α-chymotrypsin retained only 1% of initial catalytic activity and 10, 22, 59, and 48% residual activity in glycerol-solvated micelles with 20, 30, 50, and 60% (v/v) glycerol, respectively. The explanation of the observed effects is given in the frames of micellar matrix structural order increasing in the presence of glycerol as a water-miscible cosolvent that leads to the decreasing mobility of the α-chymotrypsin molecule and, thus the increase of its stability. It was found that glycerol or hydrostatic pressure could be used to stabilize α-chymotrypsin in reversed micelles; a lower pressure is necessary to reach a given level of enzyme stability in the presence of glycerol. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 552-556, 1998.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 571-582 
    ISSN: 0006-3592
    Keywords: TGFα ; autocrine ; modeling ; cell density ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed an experimental system for testing mathematical model predictions concerning escape of autocrine ligands into the extracellular bulk medium. This system employs anti-receptor blocking antibodies against the epidermal growth factor receptor (EGFR)/transforming growth factor alpha (TGFα) receptor/ligand pair. TGFα was expressed under the control of a tetracycline-repressed promoter, together with a constitutively expressed human EGFR in B82 mouse fibroblast cells. This expression system allowed us to vary TGFα synthesis rates over a roughly 300-fold range by adjusting tetracycline concentration. TGFα accumulation in the extracellular bulk medium was then measured as a function of cell density, TGFα synthesis rate, and anti-EGFR blocking antibody concentration. Consistent with model predictions, amounts of ligand in the medium on a per cell basis were found to diminish as cell density was increased but with reduced dependence on cell density at higher ligand synthesis rates. Similarly consistent with model predictions, higher ligand synthesis rates also decreased the effect of anti-receptor blocking antibodies. Our investigation has established that we can successfully analyze and understand autocrine ligand secretion behavior from the basis of our theoretical model. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 571-582, 1998.
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  • 14
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 590-599 
    ISSN: 0006-3592
    Keywords: protein refolding ; hollow-fibre membrane ; dialysis ; carbonic anhydrase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have used a cellulose acetate, hollow-fibre (HF) ultrafiltration membrane to refold bovine carbonic anhydrase, loaded into the lumen space, by removing the denaturant through controlled dialysis via the shell side space. When challenged with GdnHCl-denatured carbonic anhydrase, 70% of the loaded protein reptated through the membrane into the circulating dialysis buffer. Reptation occurred because the protein, in its fully unfolded configuration, was able to pass through the pores. The loss of carbonic anhydrase through the membrane was controlled by the dialysis conditions. Dialysis against 0.05 M Tris-HCl for 30 min reduced the denaturant around the protein to a concentration that allowed the return of secondary structure, increasing the hydrodynamic radius, thus preventing protein transmission. Under these conditions a maximum of 42% of carbonic anhydrase was recovered (from a starting concentration of 5 mg/mL) with 94% activity. This is an improvement over refolding carbonic anhydrase by simple batch dilution, which gave a maximum reactivation of 85% with 35% soluble protein yield. The batch refolding of carbonic anhydrase is very sensitive to temperature; however, during HF refolding between 0 and 25°C the temperature sensitivity was considerably reduced. In order to reduce the convection forces that give rise to aggregation and promote refolding the dialyzate was slowly heated from 4 to 25°C. This slow, temperature-controlled refolding gave an improved soluble protein recovery of 55% with a reactivation yield of 90%. The effect of a number of additives on the refolding system performance were tested: the presence of PEG improved both the protein recovery and the recovered activity from the membrane, while the detergents Tween 20 and IGEPAL CA-630 increased only the refolding yield. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 590-599, 1998.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 583-589 
    ISSN: 0006-3592
    Keywords: animal cell ; cell adhesion ; fluorocarbon ; liquid/liquid interface ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In general, anchorage-dependent animal cells cultivated on a solid culture substrate, such as polystyrene, are collected by trypsin treatment. This treatment may have detrimental effects such as the proteolysis of the cell membrane proteins. To avoid these effects, cell cultivation using a liquid/liquid interface system has been investigated. In this cultivation method, the cells grow at the interface between a culture medium and a hydrophobic liquid. In this study, various fluorocarbons (FC-40, FC-70, KPF-91, KPF-102, and KPF-142) were used as substrates for the interface, and the cultivation of fibroblast cells (L-929; the mouse-derived cell line) at the interfaces was investigated. Early in the cultivation period, the growth of L-929 cells depended on the substrate type. Although cell cultivation at the interfaces was possible, it was slower than that at the polystyrene surface. Cell spreading at the interfaces was relatively small, which indicates that cell adhesion at the interfaces may be weak. In particular, the cells at the MEM/FC-70 interface anchored with one another and formed multicellular hemispherical aggregations shaped like spheroids. The difference in the adhesions to the interfaces appears to be dependent on the contaminants contained in the fluorocarbons because the physical properties of the fluorocarbon did not affect the cell growth and adhesion. Moreover, subcultivation from the interfaces to the same interface was possible without trypsin treatment. In this case, the delay of the growth at the interfaces did not occur because the cells were not affected by trypsin treatment. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 583-589, 1998.
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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 600-609 
    ISSN: 0006-3592
    Keywords: rhG-CSF ; fusion protein ; secretion efficiency ; glycosylation ; multimer ; conformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The synthesis and secretion of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are investigated in fed-batch cultures at high cell concentration of recombinant Saccharomyces cerevisiae, and some important characteristics of the secreted rhG-CSF are demonstrated. Transcription of the recombinant gene is regulated by a GAL1-10 upstream activating sequence (UASG), and the rhG-CSF is expressed in a hybrid fusion protein consisting of signal sequence of Kluyveromyces lactis killer toxin and N-terminal 24 amino acids of human interleukin 1β. The intracellular KEX2 cleavage leads to excretion of mature rhG-CSF into extracellular culture broth, and the cleavage process seems to be highly efficient. In spite of relatively low copy number the plasmid propagation is stably maintained even at nonselective culture conditions. The rhG-CSF synthesis does not depend on galactose level, whereas the production of extracellular rhG-CSF was significantly enhanced by increasing the inducer concentration above a certain level and also by supplementing the nonionic surfactant to the culture medium, which is notably due to the enhanced secretion efficiency. Various immunoblotting analyses demonstrate that none of the rhG-CSF is accumulated in the cell wall fraction and that a significant amount of intracellular rhG-CSF antibody-specific immunoreactive proteins is located in the ER. A core N-glycosylation at fused IL-1β fragment is likely to play a critical role in directing the high-level secretion of rhG-CSF, and the O-glycosylation of secreted rhG-CSF seems nearly negligible. Also the extracellular rhG-CSF is observed to exist as various multimers, and the nature of molecular interaction is evidently not the covalent disulfide bridges. The CD spectra of purified rhG-CSF and Escherichia coli-derived standard show that the conformations of both are similar and are almost identical to that reported for natural hG-CSF. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 600-609, 1998.
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  • 17
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 610-619 
    ISSN: 0006-3592
    Keywords: dynamic model ; Saccharomyces cerevisiae ; oxidative capacity ; feedback control ; calorimetry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this study was to characterize the dynamic adaptation of the oxidative capacity of Saccharomyces cerevisiae to an increase in the glucose supply rate and its implications for the control of a continuous culture designed to produce biomass without allowing glucose to be diverted into the reductive metabolism. Continuous cultures subjected to a sudden shift-up in the dilution rate showed that the glucose uptake rate increased immediately to the new feeding rate but that the oxygen consumption could not follow fast enough to ensure a completely oxidative metabolism. Thus, part of the glucose assimilated was degraded by the reductive metabolism, resulting in a temporary decrease of biomass concentration, even if the final dilution rate was below Dcrit. The dynamic increase of the specific oxygen consumption rate, qO2, was characterized by an initial immediate jump followed by a first-order increase to the maximum value. It could be modeled using three parameters denoted qjumpO2, qmaxO2, and a time constant τ. The values for the first two of the parameters varied considerably from one shift to another, even when they were performed under identical conditions. On the basis of this model, a time-dependent feed flow rate function was derived that should permit an increase in the dilution rate from one value to another without provoking the appearance of reductive metabolism. The idea was to increase the glucose supply in parallel with the dynamic increase of the oxidative capacity of the culture, so that all of the assimilated glucose could always be oxidized. Nevertheless, corresponding feed-profile experiments showed that deviations in the reductive metabolism could not be completely suppressed due to variability in the model parameters. Therefore, a proportional feedback controller using heat evolution rate measurements was implemented. Calorimetry provides an excellent and rapid estimate of the metabolic activity. Satisfactory control was achieved and led to constant biomass yields. Ethanol accumulated only up to 0.49 g L-1 as compared to an accumulation of 1.82 g L-1 without on-line control in the shift-up experiment to the same final dilution rate. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 610-619, 1998.
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  • 18
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 620-623 
    ISSN: 0006-3592
    Keywords: protein refolding ; reversed micelles ; solid-liquid extraction ; RNase A ; DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article reports that a reversed micellar solution is useful for refolding proteins directly from a solid source. The solubilization of denatured RNase A, which had been prepared by reprecipitation from the denaturant protein solution, into reversed micelles formulated with sodium di-2-ethylhexyl sulfosuccinate (AOT) has been investigated by a solid-liquid extraction system. This method is an alternative to the ordinary protein extraction in reversed micelles based on the liquid-liquid extraction. The solid-liquid extraction method was found to facilitate the solubilization of denatured proteins more efficiently in the reversed micellar media than the ordinary phase transfer method of liquid extraction. The refolding of denatured RNase A entrapped in reversed micelles was attained by adding a redox reagent (reduced and oxidized glutathion). Enzymatic activity of RNase A was gradually recovered with time in the reversed micelles. The denatured RNase A was completely refolded within 30 h. In addition, the efficiency of protein refolding was enhanced when reversed micelles were applied to denatured RNase A containing a higher protein concentration that, in the case of aqueous media, would lead to protein aggregation. The solid-liquid extraction technique using reversed micelles affords better scale-up advantages in the direct refolding process of insoluble protein aggregates. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 620-623, 1998.
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  • 19
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    Biotechnology and Bioengineering 57 (1998), S. 624-629 
    ISSN: 0006-3592
    Keywords: thermostable esterase ; hyperthermophilic archaeon ; Pyrococcus furiosus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A genomic library of the hyperthermophilic archaeon Pyrococcus furiosus was constructed in Escherichia coli using pBluescript II SK(+) as a cloning vector. One positive clone exhibiting thermophilic ester-hydrolyzing activity was directly detected by an in situ plate assay using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-acetate. The plasmid isolated from the clone contained a 3.8 kb HindIII fragment from P. furiosus. Expression of active thermostable esterase in E. coli was independent of isopropyl-β-d-thiogalactopyranoside, suggesting that the archaeal esterase gene was heterologously controlled by its own promoter sequence, not by the vector-located lac promoter. Assays of esterase activity in heat-treated extract of the recombinant E. coli showed the highest temperature optimum (100°C) and thermostability (a half-life of 50 min at 126°C) among esterases reported to date. ©1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 624-629, 1998.
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  • 20
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    Biotechnology and Bioengineering 57 (1998), S. 631-641 
    ISSN: 0006-3592
    Keywords: ceramic membrane ; water-oil ; shear rate ; transmembrane pressure ; pore size ; lumen diameter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recovery of an aqueous bioconversion product from complex, two-phase Pseudomonas putida broths containing 20% (v/v) soybean oil presents a significant challenge for downstream processing. Although not used before in multiple-phase separation for complex biotech products, crossflow filtration employing ceramic filters is one of the most attractive options which allow the design of integrated, continuous bioconversion processes. As a first attempt, we studied multichannel, monolithic ceramic membranes of different nominal pore sizes and lumen diameters under steady-state conditions. The best performance was obtained with 0.2-μm-pore/3-mm-lumen membrane, which completely rejected both cells and oil droplets from the permeate, creating a clear aqueous product stream. Although the same separation was achieved, the 50K molecular weight cut-off (MWCO) ultrafilter showed greater irreversible but similar reversible resistance, in addition to an order-of-magnitude higher membrane resistance. Larger nominal pore microfilters, such as 0.45 and 1.0 μm, experienced both cell and oil leakage even at low transmembrane pressure (10 psig). Attributed to greater shear at the same recirculation rate, smaller lumen filters did provide greater permeate flux. However, for practical purposes, the 0.2-μm-pore/4-mm-lumen ceramic membrane was chosen for further evaluation. Transmembrane pressures up to 50 psig provided only marginal gains in filtration performance, whereas increasing shear rate resulted in linear increases in steady-state flux, presumably due to formation of shear-sensitive, complex gel/oil/cell layer near the membrane surface. A nominal shear rate of 9200 s-1 and 20 psig transmembrane pressure were chosen as optimal operating conditions. Additional studies in a clean system revealed that as low as 5% (v/v) soybean oil in deionized (DI) water resulted in an order-of-magnitude decline in steady-state permeate flux. Breakthrough of oil droplets occurred at 35 psig transmembrane pressure. The severe fouling and breakthrough phenomena disappeared in the presence of washed cells for transmembrane pressure up to 43 psig, implying an oil/cell layer coating the membrane surface, thus preventing oil penetration. Serious membrane fouling was also experienced in microfiltration of oil-free, cell-free supernatant and oil-free whole broth. Consequently, soluble proteins/surfactants were suspected to be the major membrane foulants. Interestingly, soybean oil up to 30% (v/v) enhanced the flux, presumably through complicated interactions with the major foulants. Regeneration of membrane was best achieved with protease and hot caustic/bleach treatments, supporting the hypothesized fouling mechanisms mentioned above. This work provides process and system information for batch microfiltration runs in the future, to be reported elsewhere as Part II of this work. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 631-641, 1998
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  • 21
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    Biotechnology and Bioengineering 57 (1998), S. 642-654 
    ISSN: 0006-3592
    Keywords: animal cell culture ; growth ; cell death ; kinetics ; autoinhibitor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Experimental data from six hybridoma cell lines grown under diverse experimental conditions in both normal continuous and perfusion cultures are analyzed with respect to the significance of nutrients and products in determining the growth and death rates of cells and with respect to their mathematical modeling. It is shown that neither nutrients (glucose and glutamine) nor the common products lactic acid, ammonia, and monoclonal antibody can be generally assumed to be the clear-limiting or inhibiting factors for most of the cultures. Correspondingly, none of the unstructured models existing in the literature can be generally applied to describe the experimental data obtained over a relatively wide range of cultivation conditions as considered in this work. Surprisingly, for all cultures the specific growth rate (μ) almost linearly correlates with the ratio of the viable cell concentration (NV) to the dilution (perfusion) rate (D). Similarly, the specific death rate (kd) is a function of the ratio of the total cell concentration (Nt) to the dilution (perfusion) rate. These results strongly suggest the formation of not yet identified critical factors or autoinhibitors that determine both the growth and death rates of hybridoma cells. Based on these observations, simple kinetic models are developed for μ and kd which describe the experimental data satisfactorily. Analysis of the experimental data with the kinetic models reveals that under the current cultivation conditions the formation rate of the autoinhibitor(s) or the sensitivity of cell growth and death to the autoinhibitor(s) is mainly affected by the medium composition. Irrespective of the cell lines, cells grown on serum-containing media have almost the same model parameters, which are distinctively different from those of cells grown on serum-free media. Furthermore, in contrast to the prevailing view, kd is shown to positively correlate with μ if the effects of cell concentration and dilution (perfusion) rate are considered. Several important implications of these findings are discussed for the optimization and control of animal cell culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 642-654, 1998
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  • 22
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    Biotechnology and Bioengineering 58 (1998), S. 85-91 
    ISSN: 0006-3592
    Keywords: furin ; proprotein convertases ; transforming growth factor beta ; baculovirus ; precursor proteins ; overexpression systems ; growth factors ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: One important limitation of the widely used insect baculovirus overexpression system is its inefficiency to properly process heterologous proteins which are initially biosynthesized as larger inactive precursor proteins. One example is transforming growth factor beta 1 (TGFβ1), a 25-kDa homodimeric protein with pleiotropic functions. As many growth factors, the inactive TGFβ1 precursor molecule needs to be proteolytically cleaved C-terminal to a basic sequence to yield the mature and active homodimer. In insect cells, a large proportion of overexpressed TGFβ1 was found in an inactive precursor form suggesting that the levels of endogenous convertases are limiting for the production of mature and bioactive TGFβ1 in this system. We have demonstrated that furin, a member of a novel family of mammalian prohormone convertases (PCs) can efficiently process TGFβ1 precursor resulting in the production of the mature and active growth factor. Taking advantage of this observation, we have developed an improved overproduction system for TGFβ1 by coexpressing prohTGFβ1 and human furin convertase in High Five cells. Using this system, the production of mature active TGFβ1 increased in a dose-dependent fashion reaching up to 7.8-fold the amount obtained with the growth factor only. Thus, eliminating the rate-limiting step in recombinant TGFβ1 production maximizes its processing efficiency and the yield of the mature active growth factor. Such simple and efficient technology could be useful for large scale production of other proproteins which undergo similar maturation processes and share furin recognition sequences at the junction between the proregion and the mature polypeptide. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:85-91, 1998.
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  • 23
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    Biotechnology and Bioengineering 58 (1998), S. 92-100 
    ISSN: 0006-3592
    Keywords: E. coli HB101[pGEc47] ; defined medium ; medium development ; yield coefficients ; critical dilution rate ; batch and continuous cultivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101[pGEc47] can be related to yeast extract-enriched medium rather than plasmid properties. An optimal medium for growth of E. coli HB101[pGEc47] was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1). The yield coefficient for l-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for l-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation). Growth on defined medium after medium optimization is as rapid as on complex medium (0.42-0.45 h-1). The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:92-100, 1998.
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  • 24
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    Biotechnology and Bioengineering 58 (1998), S. 117-117 
    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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  • 25
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    Biotechnology and Bioengineering 58 (1998), S. 101-116 
    ISSN: 0006-3592
    Keywords: biofilm ; structure ; shape ; surface ; cellular automata ; discrete ; modeling ; roughness ; fractal ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A hybrid differential-discrete mathematical model has been used to simulate biofilm structures (surface shape, roughness, porosity) as a result of microbial growth in different environmental conditions. In this study, quantitative two- and three-dimensional models were evaluated by introducing statistical measures to characterize the complete biofilm structure, both the surface structure and volume structure. The surface enlargement, coefficient of roughness, fractal dimension of surface, biofilm compactness, and solids hold-up were found to be good measures of biofilm structure complexity. Among many possible factors affecting the biofilm structure, the influence of biomass growth in relation to the diffusive substrate transport was investigated. Porous biofilms, with many channels and voids between the “finger-like” or “mushroom” outgrowth, were obtained in a substrate-transport-limited regime. Conversely, compact and dense biofilms occurred in systems limited by the biomass growth rate and not by the substrate transfer rate. The surface complexity measures (enlargement, roughness, fractal dimension) all increased with increased transport limitation, whereas the volume measures (compactness, solid hold-up) decreased, showing the change from a compact and dense to a highly porous and open biofilm. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:101-116, 1998.
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  • 26
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    Biotechnology and Bioengineering 58 (1998), S. 345-355 
    ISSN: 0006-3592
    Keywords: cyclodextrin ; polychlorobiphenyl ; chlorobenzoic acid ; soil ; bioremediation ; biodegradation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The possibility of enhancing the intrinsic ex-situ bioremediation of a chronically polychlorinated biphenyl-contaminated soil by using cyclodextrins was studied in this work. The soil, contaminated with a large array of polychlorinated biphenyls and deriving from a dump site where it has been stored for about 10 years, was found to contain indigenous cultivable aerobic bacteria capable of utilising biphenyl and chlorobenzoic acids. The soil was amended with inorganic nutrients and biphenyl, saturated with water, and treated in aerobic batch slurry- and fixed-phase reactors. Hydroxypropyl-β-cyclodextrin and γ-cyclodextrin, added to both reactor systems at the concentration of 10 g/L at the 39th and 100th days of treatment, were found to generally enhance the depletion rate and extent of the soil polychlorobiphenyls. Despite some abiotic losses could have affected the depletion data, experimental evidence, such as the production of metabolites tentatively characterized as chlorobenzoic acids and chloride ion accumulation in the reactors, indicated that cyclodextrins significantly enhanced the biological degradation of the soil polychlorobiphenyls. This result has been ascribed to the capability of cyclodextrins of enhancing the availability of polychlorobiphenyls in the hydrophilic soil environment populated by immobilised and suspended indigenous soil microorganisms. Both cyclodextrins were metabolised by the indigenous soil microorganisms at the concentration at which they were used. Therefore, cyclodextrins, both for their capability of enhancing the biodegradation of soil polychlorobiphenyls and for their biodegradability, can have the potential of being successfully used in the bioremediation of chronically polychlorinated biphenyl-contaminated soils. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:345-355, 1998.
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  • 27
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    Biotechnology and Bioengineering 58 (1998), S. 366-373 
    ISSN: 0006-3592
    Keywords: trypsin ; stabilization ; peptide synthesis ; organic solvents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30° and 70°C: T50 values were 59°C and 46°C, respective. EG trypsin's half-life of 25 min at 55°C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-l-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-l-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:366-373, 1998.
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  • 28
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    Biotechnology and Bioengineering 58 (1998), S. 374-379 
    ISSN: 0006-3592
    Keywords: reversed micelles ; ribonuclease A ; activity ; recovery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have investigated the effect of two simple sugars, glucose and sucrose, on the extraction of ribonuclease A by AOT-isooctane reversed micelles. Including the sugars at concentrations up to 0.75 M in the feed solution resulted in moderate improvements in the forward transfer efficiency. The greatest effects were seen observed in the backward transfer step where both the protein recovery yield and the activity of the protein were greatly increased. Protein transfer and activity yields were also dependent on the AOT concentration. We suggest that the presence of sucrose, which was solubilized into the reversed micelles, results in preferential hydration of ribonuclease A, reducing the protein-surfactant interactions. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:374-379, 1998.
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  • 29
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    Biotechnology and Bioengineering 58 (1998), S. 356-365 
    ISSN: 0006-3592
    Keywords: Escherichia coli HB101[pGEc47] ; defined medium ; batch and continuous cultivation ; transient experiments ; bioconversion ; octanoic acid ; linear inhibition kinetics ; model simulation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: E. coli HB101[pGEc47], which is able to convert octane to octanoate, but cannot oxidize octanoate further, was grown on defined medium with glucose as carbon source in batch and continuous culture. The biomass yield on glucose decreased from 0.32 ± 0.02 g g-1 in aqueous cultivations to 0.25 ± 0.02 g g-1 in the presence of octane. Maximal octanoate productivities of 0.6 g L-1 h-1 were the same as found in cultivations on complex medium. The glucose-based carbon recovery in these experiments was 99 ± 4% (in extreme, between 90% and 105%). An increase of the octane feed from 1% to 2% (v/v) or more led to washout of cells. This effect was reversible when the octane feed was decreased to its initial value of 1%. Analysis of experimental data by model simulation strongly suggested that washout was due to inhibition by octanoate only. Pulses of octanoate to a continuous culture grown on aqueous media were applied to analyze the inhibition further. Inhibition by acetate was not significant, but its presence in the medium reflected a physiological state that made the cells more sensitive to octanoate inhibition. Model simulation with linear inhibition kinetics could perfectly predict glucose consumption and the resulting glucose concentration. The linear type of inhibition was confirmed by a variety of batch experiments in the presence of different concentrations of octanoate. The glucose-based specific growth rate, μ, decreased linearly with increasing concentrations of octanoate and became zero at a threshold concentration pmax of 5.25 ± 0.25 g L-1. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:356-365, 1998.
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  • 30
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    Biotechnology and Bioengineering 58 (1998), S. 387-399 
    ISSN: 0006-3592
    Keywords: population balance ; cell cycle ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A cell cycle population model based on the transition probability model of Smith and Martin (1973) has been extended to include product synthesis and export. The model handles two probable mechanisms. In the direct production model, the product is the protein. In the transcription model, the product is the specific mRNA. The protein is synthesized by translation of the specific mRNA and subsequently exported. In either case, the cell density is jointly distributed in the primary product and maturity age in the cell cycle. This extended model also is capable of describing a large range of conditions, including substrate dependent batch and continuous cultures. With the use of unity maturity-velocity (but the transition rate a function of limiting substrate), the model is shown to exhibit a negative growth association between the specific productivity of monoclonal antibodies from hybridomas and the dilution rates of a chemostat. Possibilities of maturity age dependent transcription and translation are considered, and the results show that these features can amplify the specific productivity negative association with specific growth rate. While this model may provide a partial elucidation of monoclonal antibody productivity in a chemostat, the present work provides a proper framework with which probable cell cycle dependent product formation can be analyzed rigorously with a comprehensive computational model. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:387-399, 1998.
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  • 31
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    Biotechnology and Bioengineering 58 (1998), S. 408-415 
    ISSN: 0006-3592
    Keywords: nitric oxide ; NOx ; flue gas ; denitrification ; aerobic ; biofilter ; aerosol ; biomass control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The presence of significant denitrification activity in an aerobic toluene-treating biofilter was demonstrated under batch and flow-through conditions. N2O concentrations of 9.2 ppmv were produced by denitrifying bacteria in the presence of 15% acetylene, in a flow-through system with a bulk gas phase O2 concentration of 〉17%. The carbon source for denitrification was not toluene but a byproduct or metabolite of toluene catabolism. Denitrification conditions were successfully used for the reduction of 60 ppmv nitric oxide to 15 ppmv at a flow rate of 3 L min-1 (EBRT of 3 min) in a fully aerated, 17% v/v O2 (superficially aerobic) biofilter. Higher NO removal efficiency (97%) was obtained by increasing the toluene supply to the biofilter. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:408-415, 1998.
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  • 32
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    Biotechnology and Bioengineering 58 (1998), S. 494-501 
    ISSN: 0006-3592
    Keywords: mixture optimization ; cellulase ; experimental design ; synergism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A factorial experimental design approach was used to optimize mixtures of six cellulases (five Thermomonospora fusca cellulases and plus/minus Trichoderma reesei CBHI along with β-glucosidase) so as to maximize the glucose produced from filter paper. Optimized mixture A and mixture B produced glucose at 25 and 8.3 μmol glucose/μmol enzyme/min, respectively, which are 8 and 1.5 times higher than the sum of the activity of the individual cellulases. In both mixtures, the glucose yield depended on the ratio and the cellulases used. Most enzymes showed synergistic interactions that increased the glucose yield. The yield of glucose with the optimum mixtures depended on the total enzyme concentration. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 494-501, 1998.
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  • 33
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    Biotechnology and Bioengineering 58 (1998), S. 250-253 
    ISSN: 0006-3592
    Keywords: in vivo 13C-NMR ; Rhizobium meliloti ; polymer biosynthesis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The use of in vivo 13C-NMR approach for the monitoring of the synthesis of various polymers within cells of Rhizobium meliloti (M5N1 strain) is reported. Significant differences in polymer biosynthesis have been shown as a function of the metabolic state of the cells and the labeled carbon source used. Consumption of carbon source and produced glycogen was complete with mid-exponential phase harvested cells. This was not the case with stationary phase harvested cells, for which polyhydroxybutyrate synthesis was higher and gluconate synthesis was lower than the former. [1-13C]fructose-grown cells produced more exopolysaccharide and polyhydroxybutyrate, but less β-(1,2) glucan and gluconate than [1-13C]glucose-grown cells. This approach offers a suitable tool to examine the kinetics of polymer biosynthesis by Rhizobia. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:250-253, 1998.
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  • 34
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    Biotechnology and Bioengineering 58 (1998), S. 154-161 
    ISSN: 0006-3592
    Keywords: central carbon pathways ; metabolic optimization ; ethanol production ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Many attempts to engineer cellular metabolism have failed due to the complexity of cellular functions. Mathematical and computational methods are needed that can organize the available experimental information, and provide insight and guidance for successful metabolic engineering. Two such methods are reviewed here. Both methods employ a (log)linear kinetic model of metabolism that is constructed based on enzyme kinetics characteristics. The first method allows the description of the dynamic responses of metabolic systems subject to spatiotemporal variations in their parameters. The second method considers the product-oriented, constrained optimization of metabolic reaction networks using mixed-integer linear programming methods. The optimization framework is used in order to identify the combinations of the metabolic characteristics of the glycolytic enzymes from yeast and bacteria that will maximize ethanol production. The methods are also applied to the design of microbial ethanol production metabolism. The results of the calculations are in qualitative agreement with experimental data presented here. Experiments and calculations suggest that, in resting Escherichia coli cells, ethanol production and glucose uptake rates can be increased by 30% and 20%, respectively, by overexpression of a deregulated pyruvate kinase, while increase in phosphofructokinase expression levels has no effect on ethanol production and glucose uptake rates. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:154-161, 1998.
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  • 35
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    Biotechnology and Bioengineering 58 (1998), S. 263-266 
    ISSN: 0006-3592
    Keywords: Streptomyces lividans ; simple structured modeling ; cybernetic modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The growth of Streptomyces lividans in defined media was modeled using a simple structured growth model. Conventional unstructured models like Monod kinetics, substrate inhibition kinetics, and the logistic equation were also used in an attempt to fit the data, but the results were all unsatisfactory. The main reason for failure in applying simple unstructured models is that they cannot describe the long lag phases sometimes observed during growth of S. lividans. The simple structured growth model was derived along similar principles to cybernetic growth models. This model quite accurately describes the growth of S. lividans. It assumes that the rate of assimilation of a substrate depends on the concentration of a specific key enzyme. This key enzyme is only produced in the presence of the substrate, and it is broken down at a steady rate. An enzyme synthesis allocation variable, w, similar to the cybernetic variable, u, described in cybernetic growth models, is proposed to control enzyme synthesis. Until the key enzyme concentration approaches its maximum level, very little substrate is consumed. And consequently, the lag phase is sustained. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:263-266, 1998.
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  • 36
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    Biotechnology and Bioengineering 58 (1998), S. 162-169 
    ISSN: 0006-3592
    Keywords: bioinformatics ; metabolic engineering ; genetic engineering ; mathematical analysis ; stoichiometry ; enzyme kinetics ; modal analysis ; genetic circuits ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Ten microbial genomes have been fully sequenced to date, and the sequencing of many more genomes is expected to be completed before the end of the century. The assignment of function to open reading frames (ORFs) is progressing, and for some genomes over 70% of functional assignments have been made. The majority of the assigned ORFs relate to metabolic functions. Thus, the complete genetic and biochemical functions of a number of microbial cells may be soon available. From a metabolic engineering standpoint, these developments open a new realm of possibilities. Metabolic analysis and engineering strategies can now be built on a sound genomic basis. An important question that now arises; how should these tasks be approached? Flux-balance analysis (FBA) has the potential to play an important role. It is based on the fundamental principle of mass conservation. It requires only the stoichiometric matrix, the metabolic demands, and some strain specific parameters. Importantly, no enzymatic kinetic data is required. In this article, we show how the genomically defined microbial metabolic genotypes can be analyzed by FBA. Fundamental concepts of metabolic genotype, metabolic phenotype, metabolic redundancy and robustness are defined and examples of their use given. We discuss the advantage of this approach, and how FBA is expected to find uses in the near future. FBA is likely to become an important analysis tool for genomically based approaches to metabolic engineering, strain design, and development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:162-169, 1998.
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  • 37
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    Biotechnology and Bioengineering 58 (1998), S. 191-195 
    ISSN: 0006-3592
    Keywords: control analysis ; Lactococcus lactis ; gene expression ; flux ; oligonucleotide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we review some of the expression systems that are available for Metabolic Control Analysis and Metabolic Engineering, and examine their advantages and disadvantages in different contexts. In a recent approach, artificial promoters for modulating gene expression in micro-organisms were constructed using synthetic degenerated oligonucleotides. From this work, a promoter library was obtained for Lactococcus lactis, containing numerous individual promoters and covering a wide range of promoter activities. Importantly, the range of promoter activities was covered in small steps of activity change. Promoter libraries generated by this approach allow for optimization of gene expression and for experimental control analysis in a wide range of biological systems by choosing from the promoter library promoters giving, e.g., 25%, 50%, 200%, and 400% of the normal expression level of the gene in question. If the relevant variable (e.g., the flux or yield) is then measured with each of these constructs, then one can calculate the control coefficient and determine the optimal expression level. One advantage of the method is that the construct which is found to have the optimal expression level is then, in principle, ready for use in the industrial fermentation process; another advantage is that the system can be used to optimize the expression of different enzymes within the same cell. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:191-195, 1998.
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  • 38
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    Biotechnology and Bioengineering 58 (1998), S. 175-190 
    ISSN: 0006-3592
    Keywords: protein-based polymers ; inverse temperature transitions ; hydrophobic-induced pKa shifts ; waters of hydrophobic hydration ; five axioms for protein engineering; microwave dielectric relaxation ; a universal mechanism for biological energy conversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolism is the conversion of available energy sources to those energy forms required for sustaining and propagating living organisms; this is simply biological energy conversion. Proteins are the machines of metabolism; they are the engines of motility and the other machines that interconvert energy forms not involving motion. Accordingly, metabolic engineering becomes the use of natural protein-based machines for the good of society. In addition, metabolic engineering can utilize the principles, whereby proteins function, to design new protein-based machines to fulfill roles for society that proteins have never been called upon throughout evolution to fulfill.This article presents arguments for a universal mechanism whereby proteins perform their diverse energy conversions; it begins with background information, and then asserts a set of five axioms for protein folding, assembly, and function and for protein engineering. The key process is the hydrophobic folding and assembly transition exhibited by properly balanced amphiphilic protein sequences. The fundamental molecular process is the competition for hydration between hydrophobic and polar, e.g., charged, residues. This competition determines Tt, the onset temperature for the hydrophobic folding and assembly transition, Nhh, the numbers of waters of hydrophobic hydration, and the pKa of ionizable functions.Reported acid-base titrations and pH dependence of microwave dielectric relaxation data simultaneously demonstrate the interdependence of Tt, Nhh and the pKa using a series of microbially prepared protein-based poly(30mers) with one glutamic acid residue per 30mer and with an increasing number of more hydrophobic phenylalanine residues replacing valine residues. Also, reduction of nicotinamides and flavins is shown to lower Tt, i.e., to increase hydrophobicity.Furthermore, the argument is presented, and related to an extended Henderson-Hasselbalch equation, wherein reduction of nicotinamides represents an increase in hydrophobicity and resulting hydrophobic-induced pKa shifts become the basis for understanding a primary energy conversion (proton transport) process of mitochondria. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:175-190, 1998.
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  • 39
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    Biotechnology and Bioengineering 58 (1998), S. 196-203 
    ISSN: 0006-3592
    Keywords: baculovirus ; chaperone ; hsp70 ; insect cell ; immunoglobulin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The secretion of heterologous IgG proteins in the baculovirus-insect cell expression system is accompanied by substantial insoluble immunoglobulin in the infected cells. The accumulation of these insoluble forms suggests a limitation in the processing and secretory pathway of the infected cells. As a result, cytosolic hsp70 chaperones, which are known to associate and prevent aggregation of polypeptides in vitro, have been coexpressed in the infected cells. The hsp70 protein coprecipitated with the immunoglobulin to indicate the formation of a specific hsp70-immunoglobulin complex in vivo. Immunoblot and pulse chase studies indicated that coexpression of hsp70 increased intracellular immunoglobulin solubility. Metabolic labeling experiments revealed that hsp70 increased secreted immunoglobulin levels after several days infection as compared to infection with control baculoviruses. Pulse chase studies indicated that hsp70 increases the solubility of immunoglobulin precursors that are then processed and assembled into the complete antibody oligomer. A comparison of the action of cytosolic hsp70 chaperone to the endoplasmic reticulum chaperone BiP suggests sequential action in which hsp70 increases the solubility of preprocessed immunoglobulin, while BiP enhances the solubility of processed immunoglobulin chains. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 196-203, 1998.
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  • 40
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    Biotechnology and Bioengineering 58 (1998), S. 215-221 
    ISSN: 0006-3592
    Keywords: butanol ; butyrate ; vector ; complementation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The genes coding for enzymes involved in butanol or butyrate formation were subcloned into a novel Escherichia coli-Clostridium acetobutylicum shuttle vector constructed from pIMP1 and a chloramphenicol acetyl transferase gene. The resulting replicative plasmids, referred to as pTHAAD (aldehyde/alcohol dehydrogenase) and pTHBUT (butyrate operon), were used to complement C. acetobutylicum mutant strains, in which genes encoding aldehyde/alcohol dehydrogenase (aad) or butyrate kinase (buk) had been inactivated by recombination with Emr constructs. Complementation of strain PJC4BK (buk mutant) with pTHBUT restored butyrate kinase activity and butyrate production during exponential growth. Complementation of strain PJC4AAD (aad mutant) with pTHAAD restored NAD(H)-dependent butanol dehydrogenase activity, NAD(H)-dependent butyraldehyde dehydrogenase activity and butanol production during solventogenic growth. The development of an alternative selectable marker makes it is possible to overexpress genes, via replicative plasmids, in mutant strains that lack specific enzyme activities, thereby expanding the number of possible genetic manipulations that can be performed in C. acetobutylicum. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:215-221, 1998.
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  • 41
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    Biotechnology and Bioengineering 58 (1998), S. 231-239 
    ISSN: 0006-3592
    Keywords: polyphosphate metabolism ; metabolic engineering ; Escherichia coli ; bioremediation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyphosphate metabolism plays an important role in the bioremediation of phosphate contamination in municiple wastewater, and may play a key role in heavy metal tolerance and bioremediation. However, little is known about the regulation of polyphosphate metabolism in microorganisms and its role in heavy metal toxicity. We have manipulated polyphosphate metabolism in Escherichia coli by overexpressing the genes for polyphosphate kinase (ppk) and for polyphosphatase (ppx) under control of their native promoters and inducible promoters. Overexpression of ppk results in high levels of intracellular polyphosphate, improved phosphate uptake, but no increase in tolerance to heavy metals. Overexpression of both ppk and ppx results in lower levels of intracellular polyphosphate, secretion of phosphate from the cell, and increased tolerance to heavy metals. Metabolic flux analysis indicates that the cell responds to increased flux through the PPK-PPX pathway by altering flux through the TCA cycle. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:231-239, 1998.
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  • 42
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    Biotechnology and Bioengineering 58 (1998), S. 222-230 
    ISSN: 0006-3592
    Keywords: stress mediators ; hepatocyte sandwich culture ; factorial design ; hepatocyte redox ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Stress mediators play a major role in inducing the hypermetabolic stress state in the liver after major injuries. The majority of studies on the effect of mediators on hepatocytes have focused on single factor effects or on the effect of very complex additives (e.g., serum), and there are no reports which have rigorously identified specific interactions between stress mediators. We used a factorial design experimental approach to evaluate the effects of a four to five day exposure to hormone (glucagon, hydrocortisone, and epinephrine) and cytokine [tumor necrosis factor-α (TNF-α) interleukin-1β (IL-1β) and interleukin-6 (IL-6)] stress mediators on stable cultures of rat hepatocytes. Both individual-factor effects and two factor interactions on the metabolism of urea, glucose, lactate, ketone bodies, albumin, and fibrinogen were evaluated. The cultured hepatocyte model exhibited physiologic responses to the applied stress mediators. While hydrocortisone and epinephrine had no effect, glucagon induced an increase in glucose and urea synthesis. Interleukin-6 increased fibrinogen and decreased albumin production. Furthermore, IL-6 and glucagon caused an increase in the ketone-body ratio (KBR = [acetoacetate]/[β-hydroxybutyrate]), which is in equilibrium with the intramitochondrial NAD+/NADH. Tumor necrosis factor-α and IL-1β, on the other hand, decreased the KBR. An important two-factor interaction between IL-1β and IL-6 was identified, namely that IL-1β effectively negates the positive effect of IL-6 on the KBR when both are present. These results provide further understanding of the effect of stress mediators on hepatic function and metabolism. These effects may have important implications in the pathogenesis of progressive organ dysfunction which often follows prolonged inflammatory states triggered by major injuries. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:222-230, 1998.
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  • 43
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    Biotechnology and Bioengineering 58 (1998), S. 309-315 
    ISSN: 0006-3592
    Keywords: l-ascorbic acid ; vitamin C ; 2-keto-l-gulonic acid ; l-sorbose dehydrogenase ; l-sorbosone dehydrogenase ; Gluconobacter ; chemical mutation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from d-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of l-sorbosone and 2-KLGA, l-sorbose dehydrogenase (SDH) and l-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine resulted in improvement of the production level. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:309-315, 1998.
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  • 44
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    Biotechnology and Bioengineering 58 (1998), S. 321-324 
    ISSN: 0006-3592
    Keywords: yeast cell wall porosity and permeability ; β-1,3-glucanase ; selective protein release ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:321-324, 1998.
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  • 45
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    Biotechnology and Bioengineering 58 (1998), S. 316-320 
    ISSN: 0006-3592
    Keywords: ATP allocation ; celluloytic microorganisms ; consolidated bioprocessing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Under anaerobic, carbon limited conditions, celluloytic fermentative microorganisms face a metabolic choice with respect to the allocation of relatively scarce ATP: to invest it in cells or in hydrolytic enzymes. A model is proposed that defines an allocation parameter reflecting the fractional expenditure of ATP on cell synthesis relative to the total ATP available (gross ATP synthesized less maintenance). This parameter is then incorporated into an ATP-centered model of anaerobic cellulose fermentation based on the ethanol fermentation of yeast and the cellulase system of Trichoderma reesei. Results indicate that high processing rates are possible via a consolidated bioprocessing strategy, especially at high cellulase specific activities, and that cell/cellulase allocation represents an interesting system in which to study, and perhaps exploit, microbial evolution and metabolic control. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:316-320, 1998.
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  • 46
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    Biotechnology and Bioengineering 58 (1998), S. 325-328 
    ISSN: 0006-3592
    Keywords: poly(3-hydroxybutyrate) ; Escherichia coli ; filamentation suppression ; defined medium ; high cell density culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E. coli that underwent filamentation. Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E. coli. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:325-328, 1998.
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  • 47
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    Biotechnology and Bioengineering 58 (1998), S. 329-332 
    ISSN: 0006-3592
    Keywords: tobacco cultured cells ; heat-shock promoter of Arabidopsis thaliana ; strong promoter from tobacco cell ; β-glucuronidase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Construction of a gene expression system in tobacco cultured cells (BY2) was studied. A 925 bp promoter fragment of a heat-shock protein gene (HSP18.2) of Arabidopsis thaliana showed clear heat-shock response of expression of the β-glucuronidase (GUS) reporter gene in BY2 cells. Similar results were observed in a 500 mL flask and 3-L jar fermentor.Isolation of strong promoters in BY2 cells was tried. cDNA clones, in which the mRNA level is high in log-phase cells and the copy number in the genome is low, were isolated. These clones showed high homology with F1-ATPase (mitochondria type), elongation factor 1-α, and a gene with an unknown function of A. thaliana (clone 27), respectively. A 5′-flanking region of clone 27 showed 6.2 times the promoter activity of the CaMV35S promoter in BY2 cells.Three cDNA clones, which are expressed in the stationary growth phase of BY2 cells, were isolated by a differential screening. These clones showed high sequence homologies to alcohol dehydrogenase, pectin esterase, and extensin. Promoters of these genes will be useful in gene expression in high cell-density culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:329-332, 1998.
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  • 48
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    Biotechnology and Bioengineering 59 (1998), S. 1-1 
    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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  • 49
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    Biotechnology and Bioengineering 58 (1998), S. 333-338 
    ISSN: 0006-3592
    Keywords: Catharanthus roseus ; hairy roots ; indole alkaloids ; tabersonine ; elicitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we present a review of the current state of metabolic engineering in Catharanthus roseus. A significant amount of research has contributed to characterization of several individual steps in the biosynthetic pathway of medicinally valuable alkaloids. However, knowledge of the regulation of these pathways is still sparse. Using hairy root cultures, we studied the responses of alkaloid metabolism to environmental stimulation such as light and elicitation. Through precursor feeding studies, the putative rate-limiting steps of the terpenoid pathway in hairy root cultures also have been examined. Relating this knowledge to specific events at the molecular level, and the cloning of corresponding genes are the next key steps in metabolic engineering of the C. roseus alkaloids. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:333-338, 1998.
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  • 50
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    Biotechnology and Bioengineering 58 (1998), S. 339-343 
    ISSN: 0006-3592
    Keywords: L-DOPA(3,4-dihydroxyphenyl-L-alanine) ; hybrid pathway ; tyrosine phenol lyase ; toluene dioxygenase ; benzene ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: As an alternative approach to the production of L-DOPA from a cheap raw material, we constructed a hybrid pathway consisting of toluene dioxygenase, toluene cis-glycol dehydrogenase, and tyrosine phenol-lyase. In this pathway, catechol is formed from benzene through the sequential action of toluene dioxygenase and toluene cis-glycol dehydrogenase, and L-DOPA is synthesized from the resulting catechol in the presence of pyruvate and ammonia by tyrosine phenol-lyase cloned from Citrobacter freundii. When the hybrid pathway was expressed in E. coli, production of L-DOPA was as low as 3 mM in 4 h due to the toxic effect of benzene on the cells. In order to reduce lysis of cells, Pseudomonas aeruginosa was employed as an alternative, which resulted in accumulation of about 14 mM L-DOPA in 9 h, showing a stronger resistance to benzene. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:339-343, 1998.
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  • 51
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    Biotechnology and Bioengineering 59 (1998), S. 10-20 
    ISSN: 0006-3592
    Keywords: surface marker density ; receptor density ; immunomagnetic cell separation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A theoretical analysis was performed to determine the number of fractions a multidisperse, immunomagnetically labeled cell population can be separated into based on the surface marker (antigen) density. A number of assumptions were made in this analysis: that there is a proportionality between the number of surface markers on the cell surface and the number of immunomagnetic labels bound; that this surface marker density is independent of the cell diameter; and that there is only the presence of magnetic and drag forces acting on the cell. Due to the normal distribution of cell diameters, a “randomizing” effect enters into the analysis, and an analogy between the “theoretical plate” analysis of distillation, adsorption, and chromatography can be made. Using the experimentally determined, normal distribution of cell diameters for human lymphocytes and a breast cancer cell line, and fluorescent activated cell screening data of specific surface marker distributions, examples of theoretical plate calculations were made and discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:10-20, 1998.
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  • 52
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    Biotechnology and Bioengineering 59 (1998), S. 2-9 
    ISSN: 0006-3592
    Keywords: polyethyleneglycol ; murine macrophages ; fibroblasts ; cell adhesion ; peptide immobilization ; multinucleated giant cell formation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyethyleneglycol-based networks were employed as substrates to graft bioactive peptides to study macrophage interactions with materials. Our overall objective was to utilize biologically active factors to stimulate certain macrophage function on materials suitable for implantation in connective tissues. In this study, we sought to explore the bioactivity of several peptides derived from extracellular matrix adhesion proteins and macrophage-active proteins that are normally soluble. The candidate peptides examined corresponded to residues 63 to 77 of complement component C3a (C3a(63-77)), residues 178 to 207 of interleukin-1 beta (IL1β(178-207)), residues 1615 to 1624 of fibronectin (FN(1615-1624)), endothelial-macrophage activating polypeptide II, complement component C5a inhibitory sequence, macrophage inhibitory peptide, and YRGDG; materials lacking peptides were used as negative controls. An established murine cell-line IC-21 was employed as a macrophage model, and human dermal fibroblasts were used for comparison. Our results showed that the substrates without grafted peptides were free from artifactual cell adhesion associated with the adsorption of serum or cellularly secreted proteins for long duration of culture. Of all grafted samples, IL1β(178-207)- and C3a(63-77)-grafted surfaces supported higher adherent macrophage densities. C3a(63-77)- and FN(1615-1624)-grafted surfaces supported higher adherent fibroblast densities. From competitive inhibition studies, cell adhesion was determined to occur in a receptor-peptide specific manner. The presence of grafted YRGDG in addition to IL1β(178-207), C3a(63-77), or FN(1615-1624) synergistically increased macrophage and fibroblast adhesion. Materials grafted with IL1β(178-207) or C3a(63-77) co-grafted with or without YRGDG did not support the formation of multinucleated giant cells from the fusion of adherent macrophages in vitro. © 1998 John Wiley & Sons, Inc., Biotechnol Bioeng 59:2-9, 1998.
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  • 53
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    Biotechnology and Bioengineering 59 (1998), S. 90-98 
    ISSN: 0006-3592
    Keywords: apoptosis ; necrosis ; bcl-2 ; amino acids ; cell culture ; cell death ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90-98, 1998.
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  • 54
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    Biotechnology and Bioengineering 59 (1998), S. 144-155 
    ISSN: 0006-3592
    Keywords: lysozyme ; protein precipitation ; thiocyanate ; hydrogen exchange ; nuclear magnetic resonance ; protein unfolding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have employed nuclear magnetic resonance (NMR) measurements of hydrogen exchange to identify residue-level conformational changes in hen egg white lysozyme (HEWL) as induced by salt precipitation. Deuterated HEWL was dissolved into a phosphate (H2O) buffer and precipitated at pH 2.1 upon addition of solid KSCN or (ND4)2SO4, allowing isotope labeling of unfolded regions. After 1 h, each precipitate was then dissolved at pH 3.8 to initiate refolding and preserve labeling and subsequently purified for NMR analysis. HEWL precipitated by 1.0 M KSCN exhibited increased hydrogen exchange at 14 residues out of 42 normally well-protected in the native state. Of the affected residues, 9 were situated in the β-sheet/loop domain. A similar, though less extensive, effect was observed at 0.2 M KSCN. Precipitation by 1.2 M (ND4)2SO4 resulted in none of the changes detected with KSCN. The popularity of ammonium sulfate as a precipitant is thus supported by this observed preservation of structural integrity. KSCN, in comparison, produced partial unfolding of specific regions in HEWL due most likely to known preferential interactions between -SCN and proteins. The severity of unfolding increased with KSCN concentration such that, at 1.0 M KSCN, almost the entire β-sheet/loop domain of HEWL was disrupted. Even so, a portion of the HEWL core encompassed by three α-helices remained intact, possibly facilitating precipitate dissolution. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 144-155, 1998.
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    Biotechnology and Bioengineering 59 (1998), S. 163-170 
    ISSN: 0006-3592
    Keywords: enzymes ; organic solvents ; alcohol inhibition ; activity coefficients ; substrate specificity ; rate-limiting step ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Alcohol inhibition of the lipase B from Candida antarctica has been studied through two different approaches: using the same inhibitor (1-butanol) in different organic solvents and using different inhibitors (differing in chain length) in the same solvent. The competitive inhibition constant values obtained in each case correlate with the calculated activity coefficients of the substrate, suggesting that desolvation of the alcohol is the major force changed. Data dispersion observed using the second approach has been interpreted to come from contributions of enzyme-inhibitor interactions to the binding energy. On the other hand, deacylation has been found to be much less influenced by the solvent variation than the acylation step, despite of the fact that solvation of the substrate involved in this step (the alcohol) is expected to change more than for the ester. Concerning the specificity behavior of the enzyme, a bimodal pattern was observed for the deacylation rate dependence on the alcohol chain length, with the highest values for hexanol (C6) and decanol (C10). With regard to the ester specificity, ethyl caproate (C6) is the preferred one. These results have been confronted with those reported for the lipase from Candida rugosa. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 163-170, 1998.
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  • 56
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    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 321-329 
    ISSN: 0006-3592
    Keywords: structured model ; morphology ; DiOC6 ; image analysis ; Aspergillus oryzae ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A morphologically structured model is well suited for obtaining a good description of growth and product formation of filamentous fungi for use in a process model, for example. This article describes a new morphologically structured model and its application to an α-amylase producing strain of Aspergillus oryzae. The model is based on a division of the fungal hyphae into three different regions: an extension zone, representing the tips of the hyphae; an active region, which is responsible for growth and product formation; and an inactive hyphal region. Two metamorphosis reactions describing branching and inactivation are included in the model, and the kinetics of branching and tip extension are based on known experimentally verified models of fungal microscopic morphology. To verify the structure of the model a double-staining method, based on a combination of fluorescence microscopy and automated image analysis, has been developed for measuring the fraction of active cells. The method employs the fluorescent dye 3,3′-dihexyloxocarbocyanin to stain organelles inside the hyphae and Calcoflour White to stain the cell wall. The ratio between the projected areas of the organelles and of the entire hyphal element is then taken to be proportional to the fraction of active cells. When applied to chemostat and fed-batch experiments, the double-staining method seemed to confirm the basic morphological structure of the model. The model is able to produce accurate simulations of steady-state and transient conditions in chemostats, of batch cultivations, and even the formation of a single hyphal element from a spore, all with the same values of the model parameters. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 321-329, 1998.
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  • 57
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    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 57 (1998), S. 330-341