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  • 1990-1994  (2,776)
  • 1993  (2,776)
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  • 1990-1994  (2,776)
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  • 1
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), 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
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  • 2
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 3-13 
    ISSN: 0006-3592
    Keywords: recombinant protein ; Escherichia coli ; inclusion body ; renaturation ; disulfide bond ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies, In case of expression of eukaryotic proteins containing cysteine, which may form disulfide bonds in the native active protein, often nonnative inter- and intramolecular disulfide bonds exist in the inclusion bodies. Hence, several methods have been developed to isolate recombinant eukaryotic polypeptides from inclusion bodies, and to generate native disulfide bonds, to get active proteins. This article summarizes the different steps and methods of isolation and renaturation of eukaryotic proteins containing disulfide bonds, which have been expressed in E. coli as inclusion bodies, and shows which methods originally developed for studying the folding mechanism of naturally occurring proteins have been successfully adapted for reactivation of recombinant eukaryotic proteins. © 1993 John Wiley & Sons, Inc.
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  • 3
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 25-34 
    ISSN: 0006-3592
    Keywords: bioreactor, packed bed ; ceramic beads ; cell culture, mammalian ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A packed bed bioreactor was investigated as means for the cultivation of mammalian cells. The packed bed is comprised of porous ceramic particles with pores sufficiently large for cell immobilization as well as for intraparticle convective flow. In this way, the transport of limiting nutrients such as oxygen can be significantly enhanced, allowing maintenance of cell viability and productivity in an environment protective of adverse shear effects. The extent of intraparticle convective medium flow was experimentally quantified relative to the reactor operating conditions, and was found to be the dominant mechanism of nutrient transport to cells immobilized in the particle interior. An approximate linear relationship was obtained between overall reactor productivity and the extent of intraparticle convection. As the latter can be controlled at the single-particle level through total flow rate control, this relationship is a useful scale-up tool for the design of bioreactors. The high cell densities and the high volumetric productivities achieved by using small lab-scale reactors underline the potential of this simple bioreactor configuration for large-scale cell culture applications. © 1993 John Wiley & Sons, Inc.
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  • 4
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 14-24 
    ISSN: 0006-3592
    Keywords: penicillin G amidase ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, we constructed various recombinant E. coli HB101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic and (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the Hindlll fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene. © 1993 John Wiley & Sons, Inc.
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  • 5
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 35-42 
    ISSN: 0006-3592
    Keywords: pattern recognition ; machine vision ; tissue cultures ; Betula pendula Roth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article deals with the automation of the process of somatic embryogenesis for the propagation of plants. An important problem is the monitoring of the embryo production process in order to decide the time to start harvesting embryos for further processing. The classification algorithm development for somatic embryos of birch (Betula pendula Roth) showed that automated recognition of embryos at different developmental stages is possible. No globular stage embryos were classified to be heart or torpedo stage and no heart or torpedo stage embryos were classified to be at globular stage. Heart and torpedo stage embryos were classified into three developmental classes by a new index that describes the relation of embryo breadth to the length of the root. The probability of classifying a nonembryo as an embryo was less than 1%, and 14% of the object classified as embryos by a human expert were discarded by the algorithm. A computer vision system suitable for automated monitoring of samples from the bioreactor was constructed. © 1993 John Wiley & Sons, Inc.
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  • 6
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 43-54 
    ISSN: 0006-3592
    Keywords: acetic acid ; alkaline protease ; Bacilus firmus ; continuous culture ; extracellular enzymes ; carbon/nitrogen/phosphorus limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. © 1993 John Wiley & Sons, Inc.
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  • 7
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    Biotechnology and Bioengineering 41 (1993), S. 72-78 
    ISSN: 0006-3592
    Keywords: aphrons ; aqueous two phase ; enzyme ; extraction ; intensification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel technique which intensifies conventional aqueous two-phase extraction by conversion of dispersed phase into colloidal gas aphrons (CGAs) has been developed for extraction of an enzyme. In the present work, amyloglucosidase (1,4-α-D-glucan glucohydrolase) was extracted using a polyethylene glycol-sodium sulfate-water system. The lighter phase, i.e., polyethylene glycol (PEG) rich phase, was converted into CGAs which were then dispersed into a salt rich phase. The effect of type of surfactant and its concentration, dispersed phase velocity, phase composition, and type of sparger on the dispersed phase mass transfer coefficient was investigated. The results suggests 9-16 times higher values of mass transfer coefficient compared to spray column. The multiorifice sparger at concentrations of 0.33 g/L of cetyl trimethyl ammonium chloride yielded best results. © 1993 John Wiley & Sons, Inc.
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  • 8
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    Biotechnology and Bioengineering 41 (1993), S. 55-66 
    ISSN: 0006-3592
    Keywords: estimation of state variables ; recombinant Saccharomyces carevisiae ; HBsAg expression ; extended kalman filter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model was formulated to examine the competitive growth of two phenotypes (Leu+ and Leu-) and the product formation with recombinant Saccharomyces cerevisiae strain DBY-745, which contains the shuttle vector pYGH3-16-s with the foreign gene HBsAg (hepatitis B virus surface antigen) as well as experimental fedbatch fermentation data. The important state variables and the process parameters evaluated include (1) the ratio of the plasmid-free cell concentration to the plasmid-containing cell concentration (ρ = X-X+), (2) the expression of human hepatitis B surface antigen g (CH), (3) the glucose consumption (S), (4) the ethanol production (/), (5) the change of working volume (V) in the fermentor, (6) the different specific growth rates of two phenotype cells, and (7) the plasmid loss frequency coefficient (α ). These variables and other parameters were carefully defined, their correlations were studied, and a mathematical model using a set of nonlinear ordinary differential equations (ODEs) for fed-batch fermentation was then obtained based on the theoretical considerations and the experimental results. The extended Kalman filter (EKF) methods was applied for the best estimate of these variables based on the experimentally observable variables: ρV, and g (CH). Each of these variable was affected by random measuring errors under the different operating conditions. Simulation results presented for verification of the model agreed with our observations and provided useful information relevant to the operation and the control of the fedbatch recombinant yeast fermentation. The method of predicting an optimal profile of the cell growth was also demonstrated under the different dissolved oxygen concentrations. © 1993 John Wiley & Sons, Inc.
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  • 9
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    Biotechnology and Bioengineering 41 (1993), S. 179-187 
    ISSN: 0006-3592
    Keywords: animal cell bioreactor ; confocal microscopy ; aggregates ; mammalian cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant human kidney epithelial 293 cells were cultivated as aggregates in suspension. The concentration calcium ion, in the range of 100 μM to 1mM, affected the rate of aggregate formation. During the course of cultivation the size distribution of aggregates shifted and the fraction of larger aggregates increased. This effect was more profound in cultures with a high calcium concentration. Scanning and transmission microscopic examination of the aggregates revealed that cell packing was greater in the high calcium cultures and that ultrastructural integrity was retained in aggregates from both low and high calcium cultures. Confocal microscopy was applied to examine the viability of cells in the interior of the aggregates. High viability was observed in the aggregates obtained from exponentially growing cultures. Aggregates from the high calcium culture in the stationary phase exhibited lower viability in the interior. With its ease of retention in a perfusion bioreactor, aggregate cultures offer an alternative choice for large-scale operation. © 1993 John Wiley & Sons, Inc.
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  • 10
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    Biotechnology and Bioengineering 41 (1993), S. 303-315 
    ISSN: 0006-3592
    Keywords: biological acid transformation ; sulfuric acid conversion ; sulfuric acid disposal ; sulfate-reducing bacteria ; dihydrogensulfide toxicity ; fixed-bed reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: As an alternative to the current disposal technologies for waste sulfuric acid, a new combination of recycling processes was developed. The strong acid (H2SO4) is biologically converted with the weak acid (CH3COOH) into two volatile weak acids (H2S, H2CO3) by sulfate-reducing bacteria. The transformation is possible without prior neutralization of the sulfuric acid. The microbially mediated transformation can be followed by physiochemical processes for the further conversion of the H2S.The reduction of sulfate to H2S is carried out under carbon-limited conditions at pH 7.5 to 8.5. A fixed-bed biofilm column reactor is used in conjunction with a separate gas-stripping column which was installed in the recycle stream. Sulfate, total sulfide, and the carbon substrate (in most cases acetate) were determined quantitatively. H2S and CO2 are continually removed by stripping with N2. Optimal removal is achieved under pH conditions which are adjusted to values below the pKa-values of the acids. The H2S concentration in the stripped gas was 2% to 8% (v/v) if H2SO4 and CH3COOH are fed to the recycle stream just before the stripping column.Microbiol conversion rates of 65 g of sulfate reduced per liter of bioreactor volume per day are achieved and bacterial conversion efficiencies for sulfate of more than 95% can be maintained if the concentration of undissociated H2S is kept below 40 to 50 mg/L. Porous glass spheres, lava beads, and polyurethane pellets are useful matrices for the attachment of the bacterial biomass. Theoretical aspects and the dependence of the overall conversion performance on selected process parameters are illustrated in the Appendix to this article. © 1993 John Wiley & Sons, Inc.
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  • 11
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    Biotechnology and Bioengineering 41 (1993), S. 330-340 
    ISSN: 0006-3592
    Keywords: hybridoma ; Immobilization ; monoclonal antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Immobilization offers several intrinsic advantages over free suspension cultures for the production of monoclonal antibodies. An important advantage of immobilization is the improved specific monoclonal antibody (MAb) productivity (qMAb) that can be obtained. However, there are conflicting reports in the literature on the enhancement of the qMAb with immobilization. The discrepancies between these reports can be attributed to the different to either the cultivation methods used for immobilized cell or to difference between the cell lines used in the various studies. We show that these differences may be attributed to the different cultivation methods used for one model hybridoma cell line. S3H5/ϒ2bA2 hybridoma cells entrapped in different sizes of calcium alginate beads were cultivated in both T- and spinner flasks in order to determine whether cultivation methods (T- and spinner flasks) and bead size influence the qMAb Free-suspended cell cultures inoculated with cells recovered from alginate beads were also carried out in order to determine whether changes in the qMab of the entrapped cells are reversible.The cultivation methods was found to influence significantly the qMAb of the entrapped cells. When the entrapped cells in 1-mn diameter beads were cultivated in T-flasks, the qMAb was not increased by 200% as previously observed in an entrapped cell culture using 1-mm-diameter alginate beads in spinner flasks. The qMAb of the entrapped cell was approximately 58% higher than that of the free-suspended cells in a control experiment. Unlike the cultivation method, the bead size in the range of 1- to 3-mm diameter did not significantly influence the qMAb, regardless of cultivations methods. The changes in qMAb of an entrapped cells were reversible. When the free-suspended cells recovered from the T- and spinner flasks were sub-cultured in T- and spinner flasks enhanced qMAb of the entrapped cells in both cases decreased to the level of the free-suspended cell in a control experiments. Taken together, these results shows that the method of cultivation of hybridoma cells immobilized in alginate beads determines the extent of enhancement of the qMAb. © 1993 John Wiley & Sons, Inc.
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  • 12
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    Biotechnology and Bioengineering 41 (1993), S. 354-360 
    ISSN: 0006-3592
    Keywords: glucose transport ; packed bed reactor ; stimulus-response tracer methods ; wood chips ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A stimulus-response tracer technique has been used to characterize packed beds of untreated, as well as acid prehydrolyzed, and enzymatically hydrolyzed aspen wood chips. Glucose was used as the tracer. Bulk liquid phase dispersion, interphase mass transfer, and intraparticle diffusion coefficients were determined for these materials as well as effective porosities and tortuosities. The untreated and prehydrolyzed aspen wood chips were found to have effective coid fractions of ca. 0.8, while the enzymatically hydrolyzed wood chips exhibited a void fraction of 0.37. Intraparticle diffusion was approximately twice as rapid in the prehydrolyzed and enzymatically hydrolyzed wood chips as in the untreated wood chips. Also, under the current experimental conditions, intraparticle diffusional transport resistance accounted for roughly half of the total tracer pulse dispersion. It is demonstrated that stimulus-response tracer techniques can be useful and convenient probes for beds of lignocellulosic, or other conversion and/or treatment. © 1993 John Wiley & Sons, Inc.
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  • 13
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    Biotechnology and Bioengineering 41 (1993), S. 459-464 
    ISSN: 0006-3592
    Keywords: Thiobacillus ferrooxidans ; carbon dioxide uptake ; carbon dioxide inhibition ; bacterial leaching ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of carbon dioxide concentration on the bacterial leaching of a pyrite-arsenopyrite ore concentrate was studied in continuous-flow reactors. Steady-state operation with two feed slurry densities, 6 wt% and 16 wt% solids, were tested for the effect of carbon dioxide concentration. Bacterial growth rates were estimated via the measurement of carbon dioxide consumption rates. Aqueous-phase carbon dioxide concentrations in excess of 10 mg/L were found to be inhibitory to bacterial growth. © 1993 John Wiley & Sons, Inc.
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  • 14
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    Biotechnology and Bioengineering 42 (1993), S. 1107-1112 
    ISSN: 0006-3592
    Keywords: Bacillus thuringiensis ; spore ; internal membrane filter ; two-stage continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two-stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 × 1010 CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 × 109 CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h-1 were used. © 1993 John Wiley & Sons, Inc.
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  • 15
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    Biotechnology and Bioengineering 42 (1993), S. 1164-1174 
    ISSN: 0006-3592
    Keywords: bio-oxidation ; high solids concentration ; rate limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bio-oxidation has proved to be a viable process for the oxidative pretreatment of refractory gold-bearing sulfides. Generally, the oxidation rate is maximal at 20% solids for high sulfide content materials [ca. 30% sulfur]. Low grade ores [1% sulfur] have been successfully oxidized at 55% solids, indicating a link between the sulfide grade of the material and the optimal solids concentration for operation. Concentrations of high solids have been reported to lower oxidation rates, increase lag times, and decrease the ultimate extent of oxidation. This review discusses the various factors that have been proposed as causes of these phenomena. The factors include oxygen and carbon dioxide availability, low bacteria-solids ratio; mechanical damage or inhibition of the bacteria, inhibition of bacterial attachment, and the buildup of toxic leach products or other detrimental substances such as some flotation reagents. © 1993 John Wiley & Sons, Inc.
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  • 16
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    Biotechnology and Bioengineering 42 (1993), S. 1190-1198 
    ISSN: 0006-3592
    Keywords: fermentation ; bioprocess monitoring ; bioluminescence ; inner filter effect ; Escherichia coli ; cell concentration monitoring ; fiber optic ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Bioluminescence has recently become a popular research tool in several fields, including medicine, pharmacology, biochemistry, bioprocessing, and environmental engineering. Beginning with purely qualitative goals, scientists are now targeting more demanding applications where accurate, quantitative interpretation of bioluminescence is necessary. Using the recent advances in fiber-optic technology, bioluminescence is easily monitored in vivo and in real time. However, the convenience of this measurement is often concealing an unsuspected problem: the bioluminescence signal might be corrupted by a large error caused by the extinction of light by biological cells. Since bioluminescent cultures not only emit light but also absorb and scatter it, the measured signal is related in a complex, nonlinear, and cell-concentration-dependent manner to the “true” bioluminescence. This light extinction effect, known as the “inner filter effect,” is significant in high-density cultures. Adequate interpretation of the bioluminescence signal can be difficult without its correction. Here, we propose a real-time algorithm for elimination of the inner filter effect in a bioreactor. The algorithm yields the bioluminescence which would be measured if the glowing culture was completely transparent. This technique has been successfully applied to batch and continuous cultivation of recombinant bioluminescent Escherichia coli. © 1993 John Wiley & Sons, Inc.
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  • 17
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    Biotechnology and Bioengineering 41 (1993), S. 541-549 
    ISSN: 0006-3592
    Keywords: microbial calorimetry ; heat of growth ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The possibility of continuously measuring the heat produced by microorganisms in an ordinary laboratory fermentor was studies. An inventory of the heat flows influencing the temperature of the culture was made. The magnitude and standard deviation in these heat flows were studied from theoretical and practical viewpoints. Calibration procedures were tested, and a model describing the heat flows in steady state and during dynamic conditions was made. Microbial heat production could be calculated accurately with the help of this model, appropriate temperature measurements, and equipment properties measured during the calibration procedures. It was found that the measurement of heat production could be done with an accuracy similar to that in the O2 uptake measurement. © 1993 John Wiley & Sons, Inc.
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  • 18
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    Biotechnology and Bioengineering 41 (1993), S. 557-565 
    ISSN: 0006-3592
    Keywords: amino acid addition ; protein stability ; stress response ; Escherichia coli ; chloramphenicol-acetyl-transferase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this work, effective feeding schemes that would minimize stress responses to cloned-protein overexpression are investigated. The cloned-protein (chloramphenicolacetyl-transferase, CAT) contains a high aromatic amino acid content, most notably a high phenylalanine content. Experiments performed on Escherichia coli RR1 [pBR329] (constitutive promoter) and E. coli JM105 [pSH101] (inducible promoter) demonstrated that phenylalanine addition increases the rate of synthesis and yield of CAT. A previous study correlating inducer strength with CAT expression in E. coli JM105 [pSH101] indicated that the highest expression rate was accompanied by the highest apparent rate of protein degradation. In this work, the combined addition of isopropyl-β-D-thiogalactopyranoside (IPTG) and phenylalanine at intermediate levels resulted in substantial increase of CAT synthesis and partial reduction of protein degradation. Furthermore, transmission electron micrographs verified the absence of inclusion bodies, which, along with proteases, were suspected to reduce protein activity. The research demonstrates that significant enhancement in production and stability of heterologous proteins is possible by designing feeding strategies that incorporate knowledge of the interaction between primary cellular metabolism and foreign protein expression. © 1993 John Wiley & Sons, Inc.
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  • 19
    ISSN: 0006-3592
    Keywords: adsorption-desorption ; purification ; recombinant HBsAg ; hepatitis B surface antigen ; P. pastoris ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recombinant hepatitis B surface antigen (r-HBsAg) produced in yeast is adsorbed on a diatomaceous earth matrix for purification purposes. A pH dependence in the adsorption-elution behavior was found. The capacity of celite (Hyflo Super Cei) for adsorbing r-HBsAg increased with decreasing pH. Nonspecific proteins were also adsorbed, but a low pH dependence was found. Elution from the matrix was performed using a basic pH buffer, in which r-HBsAg is more specifically adsorbed/desorbed than contaminant proteins, permitting the purification of the r-HBsAg. A pH of 4.0 was used for adsorption and pH 8.2 was used for desorption. The described protocol allows a purification factor between three- and fivefold with respect to contaminant proteins and sixfold with respect to contaminant DNA. Finally, the adsorption step was successfully scaled-up for production purposes. © 1993 John Wiley & Sons, Inc.
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  • 20
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    Biotechnology and Bioengineering 42 (1993) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 21
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    Biotechnology and Bioengineering 42 (1993), S. 1245-1251 
    ISSN: 0006-3592
    Keywords: enzyme denaturation ; thermal unfolding ; melting curve ; activity-temperature profile ; β-galactosidase ; hydrogenase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Measuring the reversible thermal unfolding of enzymes is valuable for quantifying the effects of environmental factors on the thermodynamic stability of proteins. The thermal unfolding behavior of enzymes is typically studied using calorimetry or optical techniques such as circular dichroism, fluorescence, or light scattering. These techniques often have practical limitations and usually require the protein to be electrophoretically pure. An alternative technique for analyzing the thermodynamic stability of enzymes is to estimate the melting curve from temperature-activity data. This technique does not require electrophoretically pure enzyme, provided the sample does not have competing enzymatic activities or proteins which can affect enzyme stability (e.g., proteases). Moreover, small amounts of contaminant proteins should not affect the results as long as enzymatic assays are performed at low protein concentrations where nonspecific protein-protein interactions are negligible. To illustrate this technique, the melting curve for β-galactosidase from Escherichia coli in the presence of 1 mM EDTA, and the shift caused by adding 1 mM Mg+2, were calculated from activity-temperature data. Melting temperatures predicted from activity-temperature data compared closely with those obtained using other techniques. Application of this analysis to multisubstrate enzymes is illustrated by estimating the melting profiles for partially purified hydrogenases from several thermophilic Methanococcii. Limitations and important considerations for estimating melting profiles from activity-temperature data are discussed. © 1993 John Wiley & Sons, Inc.
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  • 22
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    Biotechnology and Bioengineering 42 (1993), S. 1252-1262 
    ISSN: 0006-3592
    Keywords: biomedical reactor ; extracorporeal circuit ; hypercholesteremia New Zealand white rabbits ; immobilized phospholipase A2 ; plasma separator reactor (PSR) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The purpose of this study was to design a biomedical reactor that reduces plasma cholesterol when incorporated in an in vivo extracorporeal system. Phospholipase A2, immobilized onto Agarose beads and housed inside the bioreactor, modifies plasma low density lipoprotein (LDL) into a form that is rapidly removed from circulation. In a packed bed reactor, the enzymatic conversion of LDL to the modified form (with plasma taken from hypercholesterolemic New Zealand white rabbits) was relatively low, 25% ± 6 for a single pass of plasma through the reactor. An extended bed reactor, a hybrid of fluidized and packed bed reactors, was then developed to increase the conversion. This reactor displays a single pass conversion of 60% ± 5 under optimal flow conditions. An evaluation of the flow rate through the reactor indicates that the system is limited by external mass transfer when employed under in vivo conditions. In addition, this system requires blood separation before the enzyme modification, which complicates the circuit control. Therefore, a new system was designed for in vivo use with rabbits. The resulting design, called the plasma separator reactor (PSR), combines plasma separation and enzymatic conversion in a single chamber. The PSR has three advantages over other studied systems: improved external mass transfer conditions, easy controlability, and simple set-up procedures. Single pass conversion reached 52% ± 12 in suboptimal flow under simulated in vivo conditions. This reactor was also tested in vivo with hypercholesterolemic New Zealand white rabbits. A continuous conversion of up to 80% ± 6 of rabbit plasma phospholipids was observed during 90 min of blood circulation (5 mL/min). The decrease in total plasma cholesterol reached a level of 60% of the initial value and was observed to be a function of the bioreactor enzyme activity. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1263-1269 
    ISSN: 0006-3592
    Keywords: oxygen transfer control ; 2,3-butanediol ; oxygen-limited growth ; energetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Production of 2,3-butanediol by Klebsiella oxytoca is influenced by the degree of oxygen limitation. During batch culture studies, two phases of growth are observed: energy-coupled growth, during which cell growth and oxygen supply are coupled; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal 2,3-butanediol productivity occurs during the energy-coupled growth phase. In this article, a control system which maintains the batch culture at a constant level of oxygen limitation in the energy-coupled growth regime has been designed. Control, which involves feedback control on the oxygen transfer coefficient, is achieved by continually increasing the partial pressure of oxygen in the feed gas, which in turn continually increases the oxygen transfer rate. Control has resulted in a balanced state of growth, a repression of ethanol formation, and an increase in 2,3-butanediol productivity of 18%. © 1993 John Wiley & Sons, Inc.
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  • 24
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    Biotechnology and Bioengineering 42 (1993), S. 1270-1276 
    ISSN: 0006-3592
    Keywords: energetics ; oxygen-limited growth ; 2,3-butanediol fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Microbial production of 2,3-butanediol by Klebsiella oxytoca occurs under conditions of an oxygen limitation. The extent to which substrate is oxidized to 2,3-butanediol and its coproducts, (acetic acid, acetoin, and ethanol) and the relative flow rates of substrate to energetic and biosynthetic pathways are controlled by the degree of oxygen limitation. Two energetic relationships which describe the response to an oxygen limitation have been derived. The first relationship describes the coupling between growth and energy production observed under oxygen-limited conditions. This allows calculation of energetic parameters and modeling of the cell mass and substrate profiles in terms of the degree of oxygen limitation only. The second relationship describes the average degree of oxidation and the rate of the end-product flow. The model has been tested with both batch and continuous culture. During these kinetic studies, two phases of growth have been observed: energy-coupled growth, which was described above; and, energy-uncoupled growth, which arises when the degree of oxygen limitation reaches a critical value. Optimal culture performance with respect to 2,3-butanediol productivity occurs during energy-coupled growth. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 88-94 
    ISSN: 0006-3592
    Keywords: cyclodextrins ; cyclodextrin glycosyltransferase (CGTase) ; product inhibition ; ultrafiltration membrane bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cyclodextrin glycosyltransferase (CGTase) was found to be severely inhibited by cyclodextrins. In order to increase the conversion yield by reducing product inhibition and reuse the CGTase in the production of cyclodextrins from milled corn starch, an ultrafiltration membrane bioreactor system was employed. In a batch operation with ultrafiltration, the conversion yield was increased 57% compared with that without ultrafiltration. Operating conditions for the continuous production of cyclodextrins in the membrane bioreactor were optimized by taking into consideration the filtration rate and the conversion yield as follows: initial starch concentration, 7% (w/v); starch feeding rate, 240 mg/h; CGTase loading, 350 units/initial gram starch. When cyclodextrins were continuously produced in the membrane bioreactor under optimized conditions, 340 units of CGTase was require to produce 1 g of cyclodextrins for 48 h, while in the case of conventional batch operation, 1 g of cyclodextrins was produced for 24 h by 1410 units of CGTase. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 104-110 
    ISSN: 0006-3592
    Keywords: baculovirus ; insect cell culture ; cell death ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The death process of baculovirus-infected insect cells was divided into two phases: a constant viability (or delay) phase characterized by a delay time (td) and a first-order death phase characterized by a half-life (t1/2). These two parameters were used in conjunction with the n-target theory to classify the kinetics of cell death under various conditions, including different multiplicity of infection (MOI), host cell lines, virus types, incubation volumes, cell density and extracellular L(+)-lactate and ammonium concentrations. Two groups of kinetic effects were found: one characterized by a constant number of hypothetical targets and the other by decreased numbers of hypothetical targets. The first group includes effects such as MOI, virus types, and host cell lines. The second includes the effects of environmental perturbations, such as incubation volume, cell density, and extracellular concentrations of L(+)-lactate and ammonium. Although the underlying mechanisms of these effects are as yet unknown, the death kinetics of infected cells significantly affects the recombinant protein production. In general, foreign protein production does not correlate with the cell life after infection © 1993 John Wiley & Sons, Inc.
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  • 27
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    Biotechnology and Bioengineering 41 (1993), S. 118-128 
    ISSN: 0006-3592
    Keywords: intracellular pH ; 9-aminoacridine ; bioreactor ; on-line measurement ; yeast ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method has been developed to continuously measure the intracellular pH (pHi) of cells cultivated in a bioreactor in an on-line fashion over extend time periods. The methods is attractive in its simplicity and involves the use of a fluorescent pHi indicator 9-aminoacridine (9A A) which is a week base. An expression has been derived to calculate changes in pHi from measured 9AA-fluorescence changes. The indicator 9AA was found t be nontoxic to yeast cells at concentrations used to measure pHi (7 μM). The fluorescence of nicotinamide adenine dinucleotide (NADH) molecules did not interfere significantly with the measurement of 9AA-fluorescence. The pHi change in yeast cell following the addition of a proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) measured by 9AA compared favorably with that measured by the well-established pHi, indicator (which is however unsuitable for on-line applications in a bioreactor) bis-carboxyethyl carboxy fluorescein (BCECF). The pHi of yeast under substrate starved conditions was 6.4 units. The responses of pHi of yeast cells to induced metabolic transitions were studied. Under aerobic condition, pHi increased by 0.12 unit following a 100-ppm glucose pulse addition and by 0.25 unit following a 300-ppm ethanol pulse addition. Under anaerobic condition, pHi increased by 0.1 unit following a 500-ppm glucose pulse addition. Comparison of pHi with other indicators of cellular metabolic state suggests that pHi is a cellular metabolic state indicator. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 148-155 
    ISSN: 0006-3592
    Keywords: plasmid stability ; Streptomyces lividans ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Plasmid plJ303 stability in Streptomyces lividans cultures has been studied by measuring plasmid copy number under various growth conditions. An increase in mean plasmid copy number was normally seen during early rapid growth in both shaken culture and stirred vessel fermentations at 28°C. Maximum copy numbers were consistently attained in early stationary phase followed by a decline (of variable amount) upon further incubation. The imposition of environmental stress (high growth temperature, i.e., 37°C, and low dissolved oxygen tension, i.e., 〈5% air saturation) led to a plasmid copy number of zero and a 50% reduction, respectively. Interestingly, the relative proportions of plasmid topoisomers changed with time since progressively more supercoiled forms were observed throughout the stationary phase. Plasmid dimers were also observed in some cultures, and no evidence of structural plasmid instability was found. In general, this host-vector system seemed remarkably stable under normal growth conditions. However, copious organic acid production by the host was observed and was thought to be undesirable for good heterologous gene expression of a secreted protein. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
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  • 30
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    Biotechnology and Bioengineering 41 (1993), S. 253-262 
    ISSN: 0006-3592
    Keywords: scaleup ; plant cell suspension culture ; secondary metabolism ; gas composition ; shear forces ; ajmalicine production ; Catharanthus roseus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of kLa, aeration rate, CO2 production rate, and influent gas phase CO2 concentration on the liquid phase CO2 concentration are discussed. © 1993 John Wiley & Sons, Inc.
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  • 31
    ISSN: 0006-3592
    Keywords: Continuous Culture ; two-liquid-phase system ; recombinant E. coli-alk system ; bioconversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Escherichia coli is able to grow on sugars in the presence of a bulk n-alkane phase. When E. coli is equipped with the alk genes from Pseudomonas oleovorans, the resulting recombinant strain converts n-alkanes into the corresponding alkanoic acids. To study the effects of growth rate and exposure to a bulk apolar phase on the physiology and the productivity of E. coli, we have grown this microorganism in two-liquid-phase continuous cultures containing 5% (v/v) n-octane.In contrast to batch cultures of wild-tape E. coli grown in the presence of n-octane, cells remained viable during the entire continuous culture, which lasted 200 h. Bioconversion of n-octane to n-octanoic acid by a recombinant E. coli (alk+) in a two-liquid-phase continuous culture was made possible by optimizing both the recombinant host strain and the conditions of culturing the organism. Continuous production in such two-phase systems has been maintained for the least 125 h without any changes in the product concentration in the fermentation medium. The volumetric productivity was determined as a function of growth rate and showed a maximum at a dilution rate D = 0.32 h-1, reaching a continuous production rate of 0.5 g octanoate/L · h (4 tons/m3 · year). © 1993 John Wiley & Sons, Inc.
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  • 32
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    Biotechnology and Bioengineering 41 (1993), S. 280-286 
    ISSN: 0006-3592
    Keywords: adsorption ; ion exchange ; equilibrium ; QAE dextran ; bovine serum albumin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH ≥ 5.05 and by the Freundlich equation of pH 4.8, which is close to pl ≃ 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH ≥ 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm3, BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO42-,-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO42- + H2O4--from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl--and H2PO4--form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 287-295 
    ISSN: 0006-3592
    Keywords: carboxylic and amino acids ; supported ; emulsion ; hybrid liquid membranes ; facilitated transport ; uphill pumping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Liquid-liquid extraction and membrane separation are well-known separation method of extensive industrial application. Their incorporation into liquid membranes has the potential of several advantages, some of which are of particular interest for the recovery of carboxylic and amino acids: selectivities higher than those attainable by current separation methods, saving on energy costs for final concentration of separated products, high fluxes, compact installation, and low capital and operation costs. Stability of the liquid advantages, can be secured by utilizing extractant blocking polymeric membranes, Applicability, process consideration, and economic implications for recovery for carboxylic and amino acids by various extractant/membrane combinations are discussed. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 296-302 
    ISSN: 0006-3592
    Keywords: shear measurement ; cell culture reactors ; dissolved oxygen probe ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: When a dissolved oxygen (DO) probe is submerged in an air-saturated cell culture medium the thickness of the liquid film that exists outside the membrane of a DO probe changes with hydrodynamic shear. The response of the DO probe thus varies with the hydrodynamic shear environment near the DO probe in cell culture reactors. The thickness of the liquid film was estimated by using a three-layer model, which describes the flow of DO molecules through the liquid layer, the membrane, and the electrolyte, to the cathode of a DO probe. According to the three-layer model, the current output of the DO probe was a strong function of thickness of the liquid film outside the membrane of the DO probe. A correlation between shear rates on the surface of the probe and the DO saturation reading was obtained by using two concentric cylinders with a rotating inner cylinder. This correlation was then used to characterize the local hydrodynamic shear environment in a cell culture reactor. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 325-329 
    ISSN: 0006-3592
    Keywords: chemostat ; enzyme overproduction ; plasmid stability ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of plasmid-mediated metabolic burden of on the expression of the host genes and its consequences on the plasmid maintenance were studied in carbon-limited chemostat culture of Escherichia coli 1EA(pBR322) subject to selection for strains overproducing chromosomally coded ribitol dehydrogenase. The chemostat population became rapidly heterogeneous and the competition among evolved strains was found to be crucial for the kinetics of the plasmid loss from the culture. The selective disadvantages in growth rate associated with plasmid carriage in the parent-like and ribitol dehydrogenase-overproducing strains was estimated. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 316-324 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; acetic acid ; inhibition ; glycine ; methionine ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Among amino acids screened for their potential to relieve wild and recombinant Escherichia coli from the negative effects of acetic acid, glycine, and methionine showed a sparing effect. In the presence of 2 g/L of acetic acid, addition of 0.5 g/L of glycine or methionine resulted in either a complete recovery or a further enhancement in the specific growth rate, while the enhancement was significant but not fully complete in the presence of 4 g/L of acetic acid. The addition of 0.5 g/L of methionine alleviated the negative effect of acetic acid on recombinant E. Coli growth to produce more β-lactamase, which was encoded by plasmid pUC18. In continuous fermentation the methionine effect on recombinant. E. coli metabolism depended on dilution rate; at high dilution rates, above 0.4 h-1, the methionine addition enhanced β-lactamase production and reduced acetic acid formation, while at low dilution rates, below 0.3 h -1, the effect was reversed. In def-batch fermentation with wild-type E. Coli, cell growth rate and cell yield from glucose were enhanced with methionine addition, while the acetic acid concentration reached over 4 g/L. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 341-346 
    ISSN: 0006-3592
    Keywords: gas antisolvent process ; protein powders ; supercritical fluids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Gas antisolvent (GAS) expansion of dimethylsulfoxide (DMSO) and N,N-dimethylformamide (DMFA) solutions with supercritical carbon dioxide was used to produce biologically active powders of insulin. Powders with 90% of the particles smaller than 4 μm and 10% smaller than 1 μm were obtained under all conditions tested when the process was operated continuously, with small liquid droplets sprayed into a flowing supercritical continuum. Slow pressurization of the stagnant protein solution resulted in larger particles. In vivo tests on rats revealed no differences between the biological activity of processed and unprocessed insulin, GAS processing of organic solution appears to be a reliable and effective method for the production of dry, biologically active microparticulate powders of peptides and proteins. © 1993 John Wiley & Sons, Inc.
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  • 38
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    Biotechnology and Bioengineering 41 (1993), S. 347-353 
    ISSN: 0006-3592
    Keywords: reactor startup ; anaerobic reactor ; acetic acid ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The startup of anaerobic fluidized bed reactors, which use Manville R-633 beads as the growth support media, acetate enriched bacterial culture as the inoculum, and acetic acid as the sole substrate, is studied. Tow startup strategies are evaluated: one based on maximum and stable substrate utilization and another based on maximum substrate loading controlled by reactor pH. The startup process is characterized using a number of operational parameters.The reactors again excellent total organic carbon (TOC) removal (i.e., 〉 97% at a feed concentration of 5000 mg TOC/L) and stable methane production (i.e., 0.90 L CH4/g TOC, where TOCr is TOC removed) at a early stage of the startup process, regardless of the strategies applied. The loading can be increased rapidly without the danger of being overloaded. Significant losses of growth support media and biomass caused by gas effervescence at higher loadings limits the maximum loading that can be safely applied during startup process.A high reactor immobilized biomass inventory is achievable using the porous growth support media (e.g., Manville 633 beads). A rapid increase in loading creates a substrate rich environment that yields more viable reactor biomass. Both substrate utilization rate (batch and continuous) and immobilized biomass inventory stabilize concomitantly at the late stage of the startup process, indicating the attainment of steady-state conditions in reactors. Therefore, they are better parameters that TOC removal and methane production for characterizing the entire startup process of aerobic fluidized bed reactor.The strategy based on maximum substrate loading controlled by reactor pH significantly shortens the startup time. In this case, the reactor attains steady-state conditions approximately 140 days after startup. On the other hand, a startup time of 200 days is required when the strategy based maximum substrate utilization is adopted. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 370-379 
    ISSN: 0006-3592
    Keywords: immobilized cell ; kinetics ; Monod growth Kinetics ; substrate utilization kinetics ; physicochemical effects in immobilized cell culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A methodology is described for measurement if immobilized and suspended cell growth and substrate utilization kinetics parameters. Substrate utilization and growth kinetics were compared between immobilized and suspended cells for toluene degrading Pseudomonas strains K3-2 and 2,4-dichlorophenoxyacetic acid (2,4-D) degrading strain DBO131(pR0101), respectively. Kinetic parameters were estimated using nonlinear parameter estimation methods and compared between the immobilized and suspended Pseudomonas cells to determine the effect of immobilization on cellular growth and substrate utilization. Factors influencing the experimental design included calculated oxygen flux rates, primary carbon substrate flux rates, and shear stresses on the immobilize cell. Statistical interpretation of the cellular reaction rate parameters indicates that only the growth kinetics of the toluene system were significantly altered upon immobilization. Substrate utilization kinetics remained unchanged upon immobilization. The substrate growth associated half-saturation constant (Kg) for the toluene system increased by 30-fold and the maximum specific growth rate (μmax) decreased by 2-fold upon immobilization. Implication of these results for experimental determination of cellular kinetic parameters and for immobilization cell bioreactors design are discussed. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 390-393 
    ISSN: 0006-3592
    Keywords: organic solvents ; enzyme catalysis ; immiscibility with water ; hydrophobicity of solvents ; dipole moment dielectric constant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The question of whether the solvent's water-immiscibility is relevant to enzymatic activity was addressed by assaying four different hydrolases (three lipases and one protease) in nine anhydrous solvents of similar hydrophobicities of which four were infinitely miscible with water and five were not. For no enzyme was a jump in activity observed upon a transition from water-miscible to water-immiscible solvent. The relevance of solvent apolarity to enzymatic efficiency was also examined. To this end, three groups of isomeric anhydrous solvents were selected where within each group of isomeric anhydrous solvents were selected where within each group one solvent was apolar (i.e., lacked a permanent dipole moment). For none of the four enzymes studied was activity significantly higher in apolar solvents than in their polar counterparts. Thus we conclude that often-cited solvent's immiscibility with water and apolarity by themselves are irrelevant to enzymatic activity. © 1993 John Wiley & Sons, Inc.
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  • 41
    ISSN: 0006-3592
    Keywords: affinity precipitation ; β-glucosidase ; cellobiose production ; cellulase ; chitosan ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Removal of β-glucosidase (BG) from cellulase is essential to the enzymatic production of cellobiose from cellulose because of the high reactivity of BG with cellobiose to form glucose. Chitosan is a reversibly soluble-insoluble polymer depending on pH, and it has an affinity with the other components, endo-β-1,4-glucanase and cellobiohydrolase, or cellulase. The affinity precipitation technique using chitosan is an effective way to fractionate cellulase for the above purpose. Hydrolysis experiments of cellulose with the residual fractionated enzyme gave higher cellobiose contents in the soluble sugar products. © 1993 John Wiley & Sons, Inc.
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  • 42
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    Biotechnology and Bioengineering 41 (1993), S. 422-428 
    ISSN: 0006-3592
    Keywords: entropy of growth ; Escherichia coli K-12 ; entropy of anabolism ; entropy change ; entropy of formation ; entropy of formation of cells ; cellular entropy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The ΔSf′ of one unit carbon formula weight of Escherichia coli K-12 cells, when grown on succinic acid, was calculated to be -80.13 J/deg. This value could then be used to calculate the entropy change accompanying the anabolism and metabolism of succinic acid to be 30.82 J/deg and 32.40 J/mol deg, respectively. The entropy of one unit carbon formula weight of dried E. Coli K-12 cells is calculated to be 94.40 J/deg, which when divided by the mass of these cells becomes 3.90 J/g deg. The corresponding entropy of succinic acid is 2.77 J/g deg, making it apparent that the entropy per unit mass of the cells is greater than that of the substrate. It might be thought that because the cells appear to be so much more complex than the substrate, the cells should have a lesser entropy per unit mass than the substrate. That this does not appear to be true leads to the conclusion that the macromolecular organization (informational content?) of the cells contributes only in a very minor way to the total physical entropy of cells. © 1993 John Wiley & Sons, Inc.
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  • 43
    ISSN: 0006-3592
    Keywords: shear ; air-lift loop reactor ; growth rate ; cell size ; hybridoma cell ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To study the effects of the growth rate of the hybridoma cell Mn12 on productivity, cell cycle, cell size, and shear sensitivity, six continuous cultures were run at dilution rate of 0.011, 0.021, 0.023, 0.030, 0.042, and 0.058 h-1. This particular hybridoma cell appeared to be unstable in continuous culture with respect to specific productivity, as a sudden drop occurred after about 30 generations in continuous culture, accompanied by the appearance of two populations with respect to the cytoplasmic lgG content. The specific productivity increased with increasing growth rate. The shear sensitivity of the cell, as measured in a small air-lift loop reactor, increased with increasing growth rate. The mean relative cell size, as determined with a flow cytometer, increased with increasing growth rates. Furthermore, the fraction of cells in the S phase increased, and the fraction of cells in the G1/G0 phase decreased with increasing growth rates. © 1993 John Wiley & Sons, Inc.
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  • 44
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    Biotechnology and Bioengineering 41 (1993), S. 451-458 
    ISSN: 0006-3592
    Keywords: immobilized glucose isomerase ; substrate protection ; reactor analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of substrate protection on enzyme deactivation was studied in a differential bed and a packed bed reactor using a commercial immobilized glucose isomerase (Swetase, Nagase Co.). Experimental data obtained from differential bed reactor were analyzed based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrate was considered. The deactivation constant of the enzyme-substrate complex was found to be about half of that of the free enzyme. The mathematical analysis describing the performance of a packed bed reactor under the considerations of the effects of substrate protection, diffusion resistance, and enzyme deactivation was studied. The system equations for the packed bed reactor were solved using an orthogonal collocation method. The presence of substrate protection and the diffusion effect within the enzyme particles resulted in an axial variation of effectiveness factor, ηD, along the length of the packed bed. The axial distribution profile of ηD was found to be dependent on the operation temperature, Based on the effect of substrate protection, a better substrate feed policy could be theoretically found for promoting productivity in long-term operation. © 1993 John Wiley & Sons, Inc.
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  • 45
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    Biotechnology and Bioengineering 41 (1993), S. 868-877 
    ISSN: 0006-3592
    Keywords: surface proteins ; hydrodynamic injury ; HL60 cells ; Pluronic F68 ; flow cytometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Flow cytometry was used to examine the effect of hydrodynamic forces in surface aerated stirred tank bioreactors on the quantity of CD13 and CD33 surface proteins of Hl60 (human promyelocytic leukemia) cells. A step increase in agitation of the 2-L bioreactors from 80 to 400 rpm reduced the apparent growth rate and the average CD13 and CD33 content per HL60 cell. The effects on the two surface proteins were observed within 30-60 min following the increase in the agitation and preceded observed effects on cell growth by at least 10 h. Upon reduction of the agitation rate back to 80 rpm, the CD13 and CD33 content recovered (in ca. 10 h) for CD13 and ca. 29h for (CD33) to the levels of the control culture whose agitation rate was maintained at 80rpm. The CD13 and CD33 cell content was reduced even at agitation rates (270 rpm) that did not affect cell proliferation. Pluronic F68 (a commonly used shear protectant) had a protective effect on the CD33 content per cell of cultures subjected to hydrodynamic injury but no effect on the CD13 cell content. Possible bioprocessing and physiological implications of these findings are discussed © 1993 Wiley & Sons, Inc.
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  • 46
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    Biotechnology and Bioengineering 41 (1993), S. 489-492 
    ISSN: 0006-3592
    Keywords: reverse micelles ; back-extraction ; silica ; proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to use reverse micellar solutions successfully for the separation of proteins, good methods are needed to recover the biomolecules into an aqueous environment after solubilization into organic micellar media. Usually the recovery is accomplished by equilibrating the protein-loaded reverse micellar solution with a water phase containing an appropriate salt (back-transfer). In this article we describe an alternative “back extraction” procedure which is based on the addition of silica to the protein-containing reverse micellar solution. In this way, the water is stripped from the reverse micellar solution. [i.e., bis(2-ethylhexyl) sodium sulfosuccinate (AOT)/isooctane/water] and the proteins adsorb to the silica particles. The adsorption process is shown to be practically quantitative. The subsequent recovery of the proteins form the silica into an aqueous solution turns out to be most efficient at alkaline pH (pH 8); 60-80 of the total protein (α-chymotrypsin or trypsin) could be recovered. The specific enzyme activity at the end of the whole cycle can be as high as 80-100%. The procedure is applied also for the back extraction from micellar solutions in which, instead of AOT, a biocompatible surfactant such as a synthetic short-chain lecithin was used. It is shown that the recovery of a α-chymotrypsin and trypsin is also achievable under these conditions in quite good yield and under good maintenance of the enzyme's catalytic activity. © 1993 John Wiley & Sons, Inc.
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  • 47
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    Biotechnology and Bioengineering 41 (1993), S. 887-893 
    ISSN: 0006-3592
    Keywords: bilirubin oxidase ; enzymatic activity ; liquid/solid two-phase system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The activity of bilirubin oxidase toward bilirubin was studied in a liquid/solid two-phase low-water organic system using a simple spectrophotometric assay to follow the reaction. The enzyme was lyophilized from aqueous solution before being suspended in the organic solvent reaction medium. The activity was significantly influenced by the properties of the aqueous medium from which the enzyme was lyophilized, specifically its pH, and the quantity and nature of the buffering species. Analyses of these effect showed that the role of buffering species in such systems went beyond their effect in fixing the protonation state of the enzyme. The activity was also influenced by the quantity of water added to the organic solvent reaction medium. The reaction was shown to follow Michaelis-Menten Kinetics, and Km and kcat were determined. The liquid/solid two-phase system studied was extensively compared to a previously studied water-in-oil microemulsion system © 1993 Wiley & Sons, Inc.
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  • 48
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    Biotechnology and Bioengineering 41 (1993), S. 970-978 
    ISSN: 0006-3592
    Keywords: Methanothermus fervidus ; culture of extreme thermophiles ; methanogenic archaea ; high temperature fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Basic issues in the culture of the extremely thermophilic archaeon, Methanothermus fervidus, have been investigated, including culture medium formulation, substrate yield and product yield coefficient, growth rate and stoichiometry, and H2 uptake kinetics. The pH optimum for growth of this organism was estimated at 6.9. Growth medium buffered with PIPES instead of bicarbonate supported both increased growth rate and maximum biomass concentration. Substitution of titanium(III) citrate for the reducing agent sodium sulfide improved culture performance as well. However, independent adjustment of iron and nickel concentrations from 11 to 111 μM, respectively, and carbon dioxide partial pressure from 5 to 20 psia did not impact the culture of M. fervidus significantly. An elemental balance approach was utilized to aid in design of a defined medium to support growth to a target maximum biomass concentration of at least 1.0 g dry wt/L. The growth of this organism was limited by H2 availability in this reformulated culture medium. The maximum growth rate and biomass concentration achieved in anaerobic vials with the defined medium was 0.16 h-1 and 0.74 g dry wt/L, respectively. This maximum biomass concentration was a 72% improvement over that obtained with a literature-based defined medium. The Monod parameter, Ks, with H2 as limiting substrate, was estimated at 1.1 ± 0.4 psia (55 ± 20 μM in the broth), based on a H2 consumption study. Representative values for the substrate yield, YX/CO2, and product yield coefficient, YCH4/X, were determined experimentally to be 1.78 ± 0.04 g dry wt/mol CO2, and 0.52 ± 0.01 mol CH4/g dry wt, respectively. A bench-scale fermentation system suitable for the culture of extremely thermophilic anaerobes was designed and constructed and proved effective for the culture of M. fervidus. © 1993 Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 74-80 
    ISSN: 0006-3592
    Keywords: two-stage chemostats ; T7 promoter ; recombinant Escherichia coli ; bioreactor model ; cell residence time ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A two-stage culture strategy was studied for continuous high-level production of a foreign protein in the chemically inducible T7 expression system. The first stage is dedicated to the maintenance of plasmid-bearing cells and the second stage to the target protein synthesis by induction of cells coming from the first stage. On entering the second stage, recombinant cells undergo a gradual induction of the target gene expression. These plasmid-bearing cells experience dynamic changes in intracellular compositions and specific growth rates with their individual residence times. Therefore, the overall cultural characteristics in the production stage are really averages of the contributions from the various cells with different residence times. The behavior of the two-stage culture is described by a model, which accounts for dynamic variations of cell growth and protein synthesis rates with cell residence times. Model simulations were compared with experimental results at a variety of operating conditions such as inducer concentration and dilution rate. This model is useful for understanding the behavior of two-stage continuous cultures. © 1993 John Wiley & Sons, Inc.
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  • 50
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    Biotechnology and Bioengineering 42 (1993), S. 59-73 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; amino acids ; nucleotides ; biosynthesis ; linear optimization ; metabolic fluxes ; metabolic engineering ; stoichiometry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Microbial metabolism provides at mechanism for the conversion of substrates into useful biochemicals. Utilization of microbes in industrial processes requires a modification of their natural metabolism in order to increase the efficiency of the desired conversion. Redirection of metabolic fluxes forms the basis of the newly defined field of metabolic engineering. In this study we use a flux balance based approach to study the biosynthesis of the 20 amino acids and 4 nucleotides as biochemical products. These amino acids and nucleotides are primary products of biosynthesis as well as important industrial products and precursors for the production of other biochemicals. The biosynthetic reactions of the bacterium Escherichia coli have been formulated into a metabolic network, and growth has been defined as a balanced drain on the metabolite pools corresponding to the cellular composition. Theoretical limits on the conversion of glucose, glycerol, and acetate substrates to biomass as well as the biochemical products have been computed. The substrate that results in the maximal carbon conversion to a particular product is identified. Criteria have been developed to identify metabolic constraints in the optimal solutions. The constraints of stoichiometry, energy, and redox have been determined in the conversions of glucose, glycerol, and acetate substrates into the biochemicals. Flux distributions corresponding to the maximal production of the biochemicals are presented. The goals of metabolic engineering are the optimal redirection of fluxes from generating biomass toward producing the desired biochemical. Optimal biomass generation is shown to decrease in a piecewise linear manner with increasing product formation. In some cases, synergy is observed between biochemical production and growth, leading to an increased overall carbon conversion. Balanced growth and product formation are important in a bioprocess, particularly for nonsecreted products. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 81-86 
    ISSN: 0006-3592
    Keywords: human immunodeficiency virus ; gp41 ; recombinant fusion protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gp41 polypeptide of human immunodeficiency virus (HIV) contains an immunosuppressive domain, an epitope which elicits specific cytolytic T cell responses to HIV, and a complement Clq interactive domain. In addition, a synthetic peptide called CS3, derived from gp41 (amino acids 576-593 of gp160) and contiguous with the major immunodominant domain, binds to cellular proteins and may be important in HIV entry/fusion. In order to further investigate the role of the CS3 region of gp41 in cellular binding and to investigate other properties of gp41, sufficient quantities of this polypeptide must be readily available. We have therefore cloned the region of the HIV genome between nucleotides 7891 and 8188 (corresponding to amino acids 541-639 of gp160) into a series of procaryotic expression vectors. The resulting clones express a recombinant polypeptide of gp41 (r41). Two of these recombinants, pMAL-cRl/r41 and pGEMEX-2/r41, expressed the highest and most consistent levels of r41 as judged by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. With the pMAL-cRl/r41 construct, r41 was expressed as a fusion to the maltose-binding protein (MBP) and, following purification by affinity chromatography, was cleaved from MBP by factor Xa protease digestion. MBP/r41 may be useful for studies of a reported gp41 cellular binding domain and may facilitate studies involving other functions ascribed to this region of gp41. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 1112-1120 
    ISSN: 0006-3592
    Keywords: luminol chemiluminescence ; peroxidase ; hydrogen peroxide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship. © 1993 Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 1121-1128 
    ISSN: 0006-3592
    Keywords: flux control coefficient ; metabolic control analysis ; enzyme kinetics ; glycolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Determination of the control coefficients allows the identification of rate-controlling steps in a reaction system. However, the measurement of the flux control coefficients in a biochemical system is not a trivial task, except for some special cases. We have developed a theoretical basis for the direct determination of these coefficients from dynamic responses. In order to show the validity of this methodology experimentally, the dynamic approach is applied to an in vitro reconstituted partial glycolytic pathway to determine the flux control coefficients of hexokinase and phosphofructokinase. It is shown that the dynamic approach gives consistent results, which agree well with values obtained by the direct enzyme titration method. The detailed procedure and potential applications to other systems, such as immobilized enzyme or cell reactors, are discussed. © 1993 Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 55
    ISSN: 0006-3592
    Keywords: unsteady state ; kinetic parameters ; Pichia stipitis ; D-xylose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A quick technique for determination of kinetic parameters of fermentation processes is proposed and applied to the transformation of D-xylose into ethanol by Pichia stipitis. The commonly used method to evaluate these parameters is based on achieving several steady states. In the proposed procedure, μm and Ks can be determined from only one steady state, by provoking a disturbance over it, after allowing the system to return to the original conditions. The main difference between the steady and unsteady state methods is the required fermentation time; while the former method lasted 350 h, the latter required a period 25 times lower. Kinetic and stoichiometric parameters were determined with both methods under anoxic and limited oxygen concentration conditions. Results from the two methods were compared, giving only 2% and 4.5% differences in the values of Ks and μm and a little over 4% for μm were the deviations under the latter ones. © 1993 Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1-10 
    ISSN: 0006-3592
    Keywords: mycelial kinetics ; symmetric branching tree ; microscopic parameters ; macroscopic parameters ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model, linking microscopic to macroscopic parameters of the kinetics of mycelial growth is presented. The model consists of two parts: (a) a microscopic description, based on the assumption that growth of a mycelium can be represented approximately by the growth of a symmetric binary tree, where the branching level (microscopic state variable) is logarithmically related to the number of tips and segments; and (b) a macroscopic description which makes use of the microscopic description in order to define the parameters related to the evolution of biomass (macroscopic state variable) as a function of time. The latter uses a distribution of arrested tips in a population of mycelia, in order to estimate the fraction of non-growing biomass in terms of a power law function with coefficient, n, of the biomass concentration. The microscopic description explains the fact that the germ tube specific growth rate of Aspergillus nidulans measured in a growth chamber, is about the double the specific growth rate of this organism, when measured in shake flasks. It predicts that the length of the hyphal growth unit of the mycelium of Geotrichum candidum would be approximately the double the germ tube length measured at the time just before the first branching event. It also allows the derivation of useful expressions for predicting macroscopic parameters, such as the maximal specific growth rate, the initial amount of biomass, and the amount of biomass before the branching process starts. Those estimates are done in terms of microscopic quantities, i.e., the amount of germinated spores, the diameters of the spores and hyphae, the average rate of tip extension, and the average internodal segment length. Estimation of coefficient n by fitting the macroscopic description to a growth curve of A. niger gives an indication on the degree of skewness of the distribution of arrested mycelia. Estimated macroscopic parameters are in relative good agreement with measured average segment length. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 178-184 
    ISSN: 0006-3592
    Keywords: recombinant β-galactosidase fusion protein ; chelating peptide ; immobilized metal affinity chromatography ; immobilized enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The strong interaction of hexa-histidine fusion proteins with metal chelate adsorbents was utilized to immobilize β-galactosidase with a hexa-histidine peptide at the N-terminus to the Ni2+-nitrilotriacetic acid adsorbent. The fusion protein was cloned and expressed in Escherichia coli. The purified soluble fusion protein showed the same specific activity as the purified β-galactosidase and retained 64 percent of its β-galactosidase activity when bound to the adsorbent. To demonstrate the potential of the immobilized β-galactosidase in organic chemistry, allyl-β-D-galactosidase was synthesized from lactose and allyl alcohol on a gram scale. The same enzyme preparation was reused in three subsequent batches to prepare the model compound with high yield. © 1993 John Wiley & Sons, Inc.
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  • 58
    ISSN: 0006-3592
    Keywords: adhesion ; protein adhesion ; S-layers ; hydrophobic interactions ; protein hydration ; van der Waals forces ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The adhesion forces between various surfaces were measured using the “surface forces apparatus” technique. This technique allows for the thickness of surface layers and the adhesion force between them to be directly measured in controlled vapor or liquid environments. Three types of biological surfaces were prepared by depositing various lipid-protein monolayers (with thicknesses ranging from 1 to 4 nm) on the inert, molecularly smooth mica surface: (i) hydrophobic lipid monolayers; (ii) amphiphilic polyelectrolyte surfaces of adsorbed polylysine; and (iii) deposited bacterial S-layer proteins. The adhesion, swelling, and wetting properties of these surfaces was measured as a function of relative humidity and time. Initial adhesion is due mainly to the van der Waals forces arising from nonpolar (hydrophobic) contacts. Following adhesive contact, significant molecular rearrangements can occur which alter their hydrophobic-hydrophilic balance and increase their adhesion with time. Increased adhesion is generally enhanced by (i) increased relative humidity (or degree of hydration); (ii) increased contact time; and (iii) increased rates of separation. The results are likely to be applicable to the adhesion of many other biosurfaces, and show that the hydrophobicity of a lipid or protein surface is not an intrinsic property of that surface but depends on its environment (e.g., on whether it is in aqueous solution or exposed to the atmosphere), and on the relative humidity of the atmosphere. It also depends on whether the surface is in adhesive contact with another surface and - when considering dynamic (nonequilibrium) conditions - on the time and previous history of its interaction with that surface. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 351-356 
    ISSN: 0006-3592
    Keywords: microencapsulation ; selection ; secretion ; yeast ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed a microencapsulation selection method which allows the rapid and quantitative screening of 〉106 yeast cells for enhanced secretion of Aspergillus awamori glucoamylase. The method provides a 400-fold single-pass enrichment for high-secreting mutants, and can be straightforwardly adapted for application to growth-based selection schemes with other microorganisms and enzymes. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 449-454 
    ISSN: 0006-3592
    Keywords: artificial seed ; sodium alginate ; chitosan ; complex coacervation ; encapsulation ; embryoid viability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Survival of secondary embryoids of winter oilseed rape (Brassica napus ssp. oleifera cv. Primor) has been used as an assay for the development of artificial seeds involving complex coacervation of alginate (polyanion) with chitosan (polycation). Germination frequency of 100% was achieved for encapsulated embryoids when alginate formed the inner matrix and chitosan the outer layer. When the matrix makeup was reversed, there was no germination of embryoids. The artificial seeds produced were hardened in dilute alkaline solutions of NaOH and Ca(OH)2. An optimum setting time could be selected based on a quantitative measurement of resistance of hardened capsules to compression and the germination frequency of the encapsulated embryoids. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 440-448 
    ISSN: 0006-3592
    Keywords: anomeric specificity ; adaptation ; sugar transport ; mannose-phosphotransferase system ; Lactococcus cremoris ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Glucose uptake in Lactococcus lactis subsp. cremoris FD1 occurs via the mannose phosphotransferase system (Man-PTS), which is quite unspecific and allows transport of many different sugars and sugar analogues. It was previously shown (Benthin, S., Nielsen, J., Villadsen, J. Biotechnol. Bioeng. 40:137-146, 1992) that the kinetics of in vivo glucose uptake in a glucose-limited chemostat culture is best described by assuming that the glucose transport system has two anomer-specific sites with a relative uptake rate of 36% through the α-site. In the present study, the existence of anomer-specific sites on Man-PTS is shown by experiments where α-glucose, β-glucose, mannose, and 2-deoxyglucose are added to glucose-limited chemostat cultures. A quantitative description of the competitive uptake of the involved sugars at the two sites is given. In a mannose-limited chemostat culture, the relative glucose flux via the α-site is 50%, corresponding to a change toward the equilibrium composition of mannose (68%). Furthermore, when the feed to a mannose-limited chemostat culture is changed to glucose, the rate of change of relative glucose flux through the α-site corresponds to constitutive synthesis of Man-PTS with 36% α-site stoichiometry in new cells. When N-acetylglucosamine (73% α-anomer at equilibrium) is the limiting substrate, the relative glucose flux through the α-site is also 48% to 50%. With a feed of α-glucose generated enzymatically from nonmetabolizable sucrose the relative glucose flux through the α-site can be as high as 78%. Finally, growth in the presence of nonmetabolizable α-methylglucoside leads to formation of cells with a relative glucose flux through the α-site of 29% to 30%. The adaptation of the flux distribution between the α- and β-site is tentatively explained by the hypothesis that two integral membrane proteins of Man-PTS are involved in this process. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 465-468 
    ISSN: 0006-3592
    Keywords: glycidol ; enantioselective esterification ; lipase ; organic solvents ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35°C. The enzyme exhibited maximum activity at a water content of 13 ± 2% (w/w). The enantiomeric purity obtained was 83 ± 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. © 1993 John Wiley & Sons, Inc.
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  • 63
    ISSN: 0006-3592
    Keywords: penicillin G acylase ; Kluyvera citrophila ; immobilization-stabilization of penicillin G acylase ; stabilization of multimeric enzymes ; reactivation of enzyme derivatives ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed a strategy for immobilization-stabilization of penicillin G acylase (PGA) from Kluyvera citrophila by controlled multipoint covalent attachment to agarose-aldehyde gels. This enzyme is composed by two dissimilar subunits noncovalently bound. Thus, in this article we establish clear correlations between enzyme stabilization and the multipoint immobilization and/or between enzyme stabilization and the involvement of the two subunits in the attachment of them to the support. We have demonstrated that important thermal stabilizations of derivatives were only obtained through a very intense enzyme-support multipoint attachment involving the whole enzyme molecule. In this way, we have prepared derivatives preserving more than 90% of catalytic activity and being more than 1000-fold more stable than soluble and one-point attached enzyme. In addition, the involvement of the two subunits in the covalent attachment to the support has proved to be essential to develop interesting strategies for reactivation of inactivated enzyme molecules [e.g., by refolding of immobilized PGA after previous unfolding with urea and sodium dodecyl sulfate (SDS)]. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 469-479 
    ISSN: 0006-3592
    Keywords: cellulase ; cellulose ; adsorption ; kinetics ; mathematical model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two fractions of substrate in microcrystalline cellulose which differ in their adsorption capacities for the cellulases and their susceptibility to enzymatic attack have been identified. On the basis of a two-substrate hypothesis, mathematical models to describe enzyme adsorption and the kinetics of hydrolysis have been derived. A new nonequilibrium approach was chosen to predict cellulase-cellulose adsorption. A maximum binding capacity of 76 mg protein per gram substrate and a half-maximum saturation constant of 26 filter paper units (FPU) per gram substrate have been calculated, and a linear relationship of hydrolysis rate vs. adsorbed protein has been found. The fraction of substrate more easily hydrolyzed, as calculated from hydrolysis data, represents 19% of the total effective substrate concentration. This fraction is only slightly different from that of other celluloses and has been estimated to be 27% and 30% for NaOH- and H3PO4-swollen cellulose, respectively. The effective substrate concentration is equal to the maximum amount of the substrate which can be converted during exhaustive hydrolysis. This in turn is determined by the overall degradability of the substrate by the cellulases (85-90% for microcrystalline cellulose) and by the cellobiose concentration during hydrolysis. The kinetic model is based on a summation of two integrated first-order reactions with respect to the effective substrate concentration. Furthermore, it includes the principal factors influencing the reaction rates: the ratio of filter paper and β-glucosidase units per gram substrate and the initial substrate concentration. © 1993 John Wiley & Sons, Inc.
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  • 65
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    Biotechnology and Bioengineering 42 (1993), S. 480-486 
    ISSN: 0006-3592
    Keywords: CHO ; PI-PLC ; heterologous glipiated proteins ; controlled release ; GPI anchor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. © 1993 John Wiley & Sons, Inc.
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  • 66
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    Biotechnology and Bioengineering 42 (1993), S. 657-666 
    ISSN: 0006-3592
    Keywords: β-galactosidase ; lactose ; β-galactopyranoside ; synthesis ; organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Commercially available lactase (β-D-galactoside galactohydrolase, EC 3.2.1.23) enzymes produced from Kluyveromyces fragilis and Kluyveromyces lactis were accessed as catalysts for use in the production of β-galactopyranosides of various alcohols using lactose as galactosyl donor. The yield of galactoside was enhanced by using the highest practical concentrations of both lactose and alcohol acceptor. The concentrations and thus yield, were limited by the solubility of the substrates. The increase in galactoside yield with increasing lactose concentration appeared to be specific to the lactose substrate and not due to water activity alterations, because addition of maltose to a fixed concentration of lactose had no effect. During the course of the reaction, the yield of galactoside peaked after around 70% to 80% of the lactose was consumed, due to hydrolysis of the product by the enzyme. A wide variety of compounds with primary or secondary hydroxyl groups could act as acceptors, the essential requirement being at least some water solubility. Addition of organic cosolvents had little effect on galactoside yield except when it increased the water solubility of sparingly soluble alcohols. Some galactosides were synthesized on a gram scale to determine practical product recoveries and improve purification methods for large-scale synthesis. Initial purification by hydrophobic chromatography (for galactosides of hydrophobic alcohols) or strong anion-exchange chromatography (for galactosides of hydrophilic alcohols) separated galactosides, galactobiosides, and higher oligomers from reducing sugars. A facile separation of the galactoside and galactobioside could then be effected by flash chromatography on silica gel. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 759-764 
    ISSN: 0006-3592
    Keywords: Lipolysis ; modeling ; lipases ; reversed micelles ; fatty acid inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Triglyceride hydrolysis using Chromobacterium viscosum lipase B in a reversed micellar media was investigated in a batch-type reactor. The dynamic evolution of the product concentration was analyzed using several mechanistic models, both from the literature and developed in this work. A kinetic model with nonlinear product inhibition was found to be the most adequate for the description of batch hydrolysis data over an extensive range of time and substrate concentration. The obtained rate equation described the time course of not only the reactions performed in this work, at different water contents (W0 = 7, 24) and pH values, but also the experimental results obtained in the literature with a Candida rugosa lipase. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 765-771 
    ISSN: 0006-3592
    Keywords: lipolysis ; lipases ; reversed micelles ; modeling ; second-order model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Olive oil hydrolysis using Chromobacterium viscosum lipase B in a reversed micellar media was investigated in a membrane reactor. The dynamic evolution of the product concentration both in the concentrate and permeate stream was analyzed using a mechanistic model previously developed by us and further modified in this work. A kinetic law with a second-order dependence in the substrate concentration and nonlinear product inhibition was found to be the most adequate for the description of the hydrolysis data over an extensive range of time and substrate concentration. These findings are discussed in terms of the specific interactions occurring in the membrane reactor. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 785-787 
    ISSN: 0006-3592
    Keywords: biosorption ; biosorbent ; Penicillium ; biomass ; lead ; wastewater treatment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The removal of lead ions from aqueous solutions by adsorption on nonliving Penicillium chrysogenum biomass was studied. Biosorption of the Pb+2 ion was strongly affected by pH. Within a pH range of 4 to 5, the saturated sorption uptake of Pb+2 was 116 mg/g dry biomass, higher than that of activated charcoal and some other microorganisms. At pH 4.5, P. chrysogenum biomass exhibited selectivity for Pb+2 over other metal ions such as Cd+2, Cu+2, Zn+2, and As+3 Sorption preference for metals decreased in the following order: Pb 〉 Cd 〉 Cu 〉 Zn 〉 As. The sorption uptake of Pb+2 remained unchanged in the presence of Cu+2 and As+3, it decreased in the presence of Zn+2, and increased in the presence of Cd+2. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 788-791 
    ISSN: 0006-3592
    Keywords: lipase ; organic solvents ; sugar acylation ; polymeric materials ; solubilization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: By complexing glucose and other mono- and disaccharides with phenylboronic acid, they were solubilized in many organic solvents. Pseudomonas sp. lipoprotein lipase readily acylated such solubilized sugars in these solvents (no reaction was observed without phenylboronic acid, i.e., when sugars are insoluble in the reaction medium). Solubilized D-glucose was enzymatically acylated with vinyl acrylate on a preparative scale, and the resultant 6-O-acryloylglucose was chemically polymerized to yield a sugar-based polyacrylate with a molecular weight of 14,000 Da. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 793-799 
    ISSN: 0006-3592
    Keywords: L. erythrorhizon ; shikonin ; carbon and nitrogen feeding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Stationary phase cell suspension cultures of Agrobacterium tumefaciens transformed Lithospermum erythrorhizon respond to additions of sucrose-rich (C-rich) medium with a 2-3-fold increase in the accumulation of shikonin derivatives and a 3-3.5-fold increase in the accumulation of soluble phenolics while showing a modest (10-30%) increase in cell concentration. Conversely, the addition of nitrate-rich (N-rich) medium resulted in 25-35% increase in biomass concentration but only 2-9% increase in shikonin production and ∼ 3% increase in the yield of soluble phenolics. Repeated additions of C-rich medium resulted in only a modest (less than 10%) improvement in shikonin production over the levels obtained after the first application. No obvious correlation could be discerned between intracellular ATP levels or protein synthesis patterns and the pattern of shikonin accumulation following the addition of C-rich medium, suggesting that the precursor diversion mechanism is not generally applicable in our cell line. It was found that alternating feeding of N-rich and C-rich media could be used as an effective strategy for enhancing the productivity of plant secondary metabolite. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 807-814 
    ISSN: 0006-3592
    Keywords: peroxidase-catalyzed polymerizations ; numerical modeling ; Monte-Carlo simulations ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Numerical and Monte Carlo simulations of horseradish peroxidase-catalyzed phenolic polymerizations have been performed. Kinetic constants for the simulations were fit to data from the oxidation and polymerization of bisphenol A. Simulations of peroxidase-catalyzed phenolic polymerization were run as a function of enzyme concentration and radical transfer and radical coupling rate constants. Predictions were performed with respect to conversion vs. time and number average molecular weight and polydispersity vs. conversion. It is shown that the enzymatic polymerization of phenols can be optimized with respect to high molecular weights by employing low enzyme concentrations and phenols with low radical coupling rate constants coupled with relatively high radical transfer rate constants. Such phenols may be identified by using linear free energy relationships that relate radical reactivity to electron donating/withdrawing potential of the phenolic substituent. © 1993 John Wiley & Sons, Inc.
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  • 73
    ISSN: 0006-3592
    Keywords: chemostat ; glucose limitation ; glycosylation ; CHO cells ; interferon-γ ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited chemostat culture was studied over a range of dilution rates (D = 0.008 to 0.20 h-1). The specific growth rate (μ) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h-1 (μmax = 0.025 h-1) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-γ (IFN-γ). The specific rate of glucose uptake increased linearly with μ, and the saturation constant for glucose (Ks) was calculated to be 59.6 μM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-γ production increased with μ in a manner indicative of a growth-related product. Despite changes in the IFN-γ production rate and cell physiology, the pattern of IFN-γ glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-γ. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1014-1018 
    ISSN: 0006-3592
    Keywords: cyanobacteria ; surfactant ; hydrogen ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Addition of Tween 85 to aqueous suspensions of Anabaena variabilis induced photosynthetic evolution of hydrogen over a time span of several weeks: As much as 148 nmol H2/h · mg dry weight was produced in the first week by a suspension containing 4.2 mg dry weight of cells and 77 mM Tween 85. The chemical structure of Tween 85 was a necessary prerequisite for inducing hydrogen production, as compounds such as Tween 20, 60, and 80 had a quite different effect. There was a coupling between photosynthetic oxygen evolution and hydrogen evolution: Hydrogen evolution started to be effective only when oxygen evolution subdued. The presence of heterocysts in A. variabilis was also required for the Tween-induced hydrogen production. Based on these observations, possible mechanisms for the photosynthetic effect of Tween 85 are advanced and discussed. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 273-279 
    ISSN: 0006-3592
    Keywords: glucoamylase expression ; Aspergillus nidulans, recombinant ; growth kinetics ; heterologous protein production kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In the work, a study of cell growth and the regulation of heterologous glucoamylase synthesis under the control of the positively regulated alcA promoter in a recombinant Aspergillus nidulans is presented. We found that similar growth rates were obtained for both the host and recombinant cells when either glucose or fructose was employed as sole carbon and energy source. Use of the potent inducer cyclopentanone in concentrations greater than 3 mM resulted n maximum glucoamylase concentration and maximum overall specific glucoamylase concentration over 80 h of batch cultivation. However, cyclopentanone concentrations in excess of 3 mM also showed an inhibitory effect on spore germination as well as fungal growth. In contrast, another inducer, threonine, had no negative effect on spore germination even when concentrations of up to 100 mM were used with either glucose or fructose as carbon source. Glucoamylase synthesis in the presence of glucose plus either inducer did not begin until glucose was totally depleted, suggesting strong catabolite repression. Similar results were obtained when fructose was employed, although low levels of glucoamylase were detected before fructose depletion, suggesting partial catabolite repression. The highest enzyme concentration (570 mg/L) and overall specific enzyme concentration (81 mg/g cell) were observed in batch culture when cyclopentanone was the inducer and fructose the primary carbon source. A maximum glucoamylase concentration of 1.1 g/L and an overall specific glucoamylase concentration of 167 mg/g cell were obtained in a bioreactor using cyclopentanone as the inducer and limited-fructose feeding strategy, which nearly doubles the glucoamylase productivity from batch cultures. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1124-1126 
    ISSN: 0006-3592
    Keywords: dissipation ; thermodynamic efficiency ; biomass yield ; Gibbs energy dissipation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In a recent publication, Heijnen and van Dijken (Biotechnol. Bioeng. 39: 833-858, 1992) reviewed the state of the art regarding the use of macroscopic methods in the correlation of biomass yields in growing microorganisms. In their article, reference is made to this author's work of some 10 years ago.Heijnen and van Dijken introduce the Gibbs' energy dissipation as a concept with favorable characteristics compared with various other approaches, including thermodynamic efficiency, as introduced by this author.In this communication, it will be shown that the “dissipation” and the “thermodynamic efficiency” description are completely equivalent and that there can be no preference for one of these in terms of rigor or characteristics. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1131-1135 
    ISSN: 0006-3592
    Keywords: hybridoma ; instability ; immobilization ; monoclonal antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Loss of monoclonal antibody (MAb) productivity in long-term, free-suspended cell culture is often attributed to the appearance of a nonproducing population of hybridoma cell (NP) in the culture which has a growth advantage over the producing population (P). However, when an NP appears in long-term culture of entrapped cells, it may not be able to take over the whole culture in a short period of time due to the limited growth of the entrapped cells. In order to examine the hypothesis that entrapped cells can have improved stability of MAb productivity due to limited cell growth, free-suspended cell culture and calcium alginate-entrapped cell culture with inocula consisting of a P and an NP were compared with regard to stability of MAb productivity in a repeated fed-batch culture. In free-suspended cell culture, the NP appeared to take over the whole culture within three batches, and thereby MAb production completely disappeared. In entrapped cell culture, an NP appeared to outgrow the P rapidly only during an exponential growth phase, resulting in a significant decrease in specific MAb productivity, qMAb, from 11.58 μg/106 cell/day to 2.76 μg/106 cell/day. However, when the cell growth was limited in entrapped cell culture, the NP no longer outgrew the P rapidly, as indicated by the stable value of qMAb. In addition, when the cells recovered from the alginate beads by citrate buffer treatment were subcultured in free-suspended cell culture, MAb production rapidly deteriorated and completely disappeared within two batches. Thus, the P present at a small fraction of viable cell concentration in the beginning of the free-suspended cell culture, which were previously entrapped in alginate beads, seemed to be outgrown rapidly by the NP. Taken together, the results obtained from these experiments support the hypothesis that the limited cell growth in entrapped cell culture, which keeps an NP from taking over the whole culture, is responsible, in part, for the improved stability of MAb productivity. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 581-592 
    ISSN: 0006-3592
    Keywords: Catharanthus roseus ; hairy roots ; vindoline ; indole alkaloids ; Agrobacterium rhizogenes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Hairy root cultures of Catharanthus roseus were established by infection of seedlings with Agrobacterium rhizogenes 15834. Approximately 150 transformants from four different. C. roseus cultivars were screened for desirable traits in growth and indole alkaloid production. Five hairy root clones grew well in liquid culture with doubling times similar to those reported for cell suspensions. Fast growing clones had similar morphologies, characterized by thin, straight, and regular branches with thin tips. The levels of key alkaloids, ajmalicine, serpentine, and catharanthine, in these five clones, also compared well with literature data from cell suspensions, yet HPLC and GC-MS data indicate the presence of vindoline in two clones at levels over three orders of magnitude greater than the minute amounts reported in cell culture. These results suggest that further optimization may result in hairy roots as a potential source of vindoline and catharanthine, the two monomers necessary to synthesize that antineoplastic drug, vinblastine. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
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    Biotechnology and Bioengineering 41 (1993), S. 599-602 
    ISSN: 0006-3592
    Keywords: optimal control ; iterative dynamic programming ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: By using penalty functions to handle state constraints, iterative dynamic programming can be used in a straightforward manner for the optimization of fedbatch fermentors. No computational difficulties were encountered and better results are obtained than previously reported in the literature for a fed-batch fermentor for biosynthesis of penicillin. © 1993 Johy Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1277-1286 
    ISSN: 0006-3592
    Keywords: chemotaxis ; growth rate ; migration ; bacteria ; transport coefficients ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In many natural environments, bacterial populations experience suboptimal growth due to the competition with other microorganisms for limited resources. The chemotactic response provides a mechanism by which bacterial populations can improve their situation by migrating toward more favorable growth conditions. For bacteria cultured under suboptimal growth conditions, evidence for an enhanced chemotactic response has been observed previously. In this article, for the first time, we have quantitatively characterized this behavior in terms of two macroscopic transport coefficients, the random motility and chemotactic sensitivity coefficients, measured in the stopped-flow diffusion chamber assay. Escherichia coli cultured over a range of growth rates in a chemostat exhibits a dramatic increase in the chemotactic sensitivity coefficient for D-fucose at low growth rates, while the random motility coefficient remains relatively constant by comparison. The change in the chemotactic sensitivity coefficient is accounted for by an independently measured increase in the number of galactose-binding proteins which mediate the chemotactic signal. This result is consistent with the relationship between macroscopic and microscopic parameters for chemotaxis, which was proposed in the mathematical model of Rivero and co-workers. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1287-1292 
    ISSN: 0006-3592
    Keywords: integrated optics ; grating coupler ; FIA ; on-line monitoring ; animal cell culture ; monoclonal antibody ; immunochemical sensor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A grating coupler was used for the on-line determination of monoclonal antibodies produced in perfused animal cell bioreactor. The device was connected with the culture vessel via a flow-injection analysis (FIA) system, which was controlled automatically. Specific antimouse lgG antibodies were immobilized on the surface of the sensor-chip. After injection of the sample, the binding of mouse lgG was observed in real time. The regeneration of the binding sites of the immobilized antibodies using an acidic solution allowed the on-line detection of produced monoclonal antibodies in the range of 10 to 150 μg/mL. In contrast to other techniques coupled to bioprocesses, the developed method represents a regenerable direct immunosensor. Results were compared with standard ELISA techniques (off-line) and a competitive immunochemical assay using the grating coupler (off-line). © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1301-1310 
    ISSN: 0006-3592
    Keywords: Optimization ; multigradient search ; xylitol conversion ; Candida guilliermondii ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new method (named a “jumping spider”) is introduced for the optimization of slow biotechnological processes. The more traditional sequential experimentation (i.e., gradient search, simplex, etc.) is not well suited for slow dynamic processes, e.g., plant cell culture and differentiation. Therefore, a more simultaneous approach is proposed. A large number of initial experiments are performed, on the basis of which several of the initial experiments are selected as starting points. A search is then performed simultaneously from several gradient directions and the optimum is estimated by a quadratic approximation. In simulations, the spider generally climbs up the slopes quickly and the final estimator yields good maximum point estimates even on a complex topography. The spider may even approach more than one local maximum point simultaneously. As a model application, the average xylitol conversion rate of Candida guilliermondii was optimized in relation to cultivation volume (oxygen availability) and the concentration of nitrogen and phosphorus in the medium. A threefold increase in xylitol production was obtained with three experimental steps. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 42 (1993), S. 1360-1366 
    ISSN: 0006-3592
    Keywords: oxygen toxicity ; anaerobic biofilm ; facultative and methanogenic bacteria ; wastewater treatment ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This research assessed the effect of oxygen exposure on the methanogenic activity of anaerobic granular sludges. The toxicity of oxygen to acetoclastic methanogens in five different anaerobic granular sludges was determined in serum flasks with effective gas-to-liquid volumes of 4.65 to 1. The amount of oxygen that caused 50% inhibition of the methanogenic activity after 3 days of exposure ranged from 7% to 41% oxygen in the head space. These results indicate that methanogens located in granular sludge have a high tolerance for oxygen. The most important factor contributing to the tolerance was the oxygen consumption by facultative bacteria metabolizing biodegradable substrates. Uptake of oxygen by these bacteria creates anaerobic microenvironments where the methanogenic bacteria are protected. The results also indicate that methanogens in sludge consortia still have some tolerance to oxygen, even in the absence of facultative substrate for oxygen respiration. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 162-164 
    ISSN: 0006-3592
    Keywords: power law ; xanthan ; CMC, shear rate ; viscosity ; Penicillium chrysogenum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Samples from fed-batch fermentations of Penicillium chrysogenum on complex medium are rheologically characterized. The behavior is well described by a power law model for which the parameters are estimates. Furthermore, two types of model media are characterized and compared with the real fermentation samples. Xanthan solutions are found to mimic the rheological properties of the filamentous fungi much better than carboxymethyl cellulose (CMC) solutions. © 1993 John Wiley & Sons, Inc.
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  • 86
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    Biotechnology and Bioengineering 41 (1993) 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 87
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    Biotechnology and Bioengineering 41 (1993), S. 684-684 
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
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  • 88
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    Biotechnology and Bioengineering 41 (1993), S. 685-692 
    ISSN: 0006-3592
    Keywords: hybridoma cells ; bubble oxygenation ; silicone tubing oxygenation ; perfusion culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A perfusion culture system was developed to investigate the oxygenation of high-density hybridoma cell cultures. The culture system was composed of a stirred-tank bioreactor and an external microfiltration hollow fiber cartridge for medium perfusion. Cell growth and antibody production were examined with large bubble (≈5 mm in diameter), micron-sized bubble (≈ 80 μm in diameter), and silicone tubing oxygenation techniques. Comparable cell growth and monoclonal antibody (MAb) production were found for both the micron-sized and large oxygenation methods, provided that large bubbles were enriched with pure oxygen. Relatively low cell growth and MAb production were attained with the bubble-free silicone tubing oxygenation. It is concluded that direct bubble oxygenation can be applied successfully in high-density animal cell cultures, provided that the culture medium is supplemented with Pluronic F-68. The accumulation of ammonia in the culture medium rather than oxygen limitation was found to be one of the possible problems that eventually inhibited cell growth. This and the fouling of the filtration cartridge during long-term cultivation were found to be more problematic than simple bubble oxygenation of high-density cell culture. The micron-sized bubble oxygenation method is highly recommended for high-density animal cell cultures, provided that Pluronic F-68 is supplemented into the culture medium. © 1993 John Wiley & Sons, Inc.
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  • 89
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    Biotechnology and Bioengineering 41 (1993), S. 693-699 
    ISSN: 0006-3592
    Keywords: phenol ; biodegradation ; biofilter ; Pseudomonas putida ; deodorization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The purpose of this study is to investigate the feasibility of biologically removing phenol from waste gases by means of a biofilter using a Pseudomonas putida strain. Two series of both batch and continuous tests have been performed in order to ascertain the microbial degradation of phenol. For the preliminary batch tests, carried out in order to test the effective feasibility of the process and to investigate their kinetic behavior, two different microbial cultures belonging to the Pseudomonas genus have been employed, a heterogeneous culture and a pure strain of P. putida. The results of these comparative investigation showed that the pure culture is more efficient than the mixed one, even when the latter has undergone three successive acclimatization tests. The continuous experiments have been conducted during a period of about 1 year in a laboratory-scale column, packed with a mixture of peat and glass beads, and utilizing the pure culture of P. putida as microflora and varying the inlet phenol concentration from 50 up to 2000 mg m-3. The results obtained show that high degrees of conversion can be obtained (0.93/0.996) operating at a residence time of 54 s. © 1993 John Wiley & Sons, Inc.
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  • 90
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    Biotechnology and Bioengineering 41 (1993), S. 700-706 
    ISSN: 0006-3592
    Keywords: anaerobic digestion ; cheese whey ; UASB reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The anaerobic digestion of cheese whey was studied in a UASB reactor. The profiles of the reactor, i.e., the distributions of the substrate concentration and pH under different operating conditions were developed. From the concentrations of substrates measured at various levels above the bottom of the reactor, two reaction stages, namely acidogenesis and methanogenesis, were distinguished. The instability caused by high influent concentration was interpreted as the accumulation of VFAs in the acidogenic stage beyond the assimilative capacity of the methanogenic stage. A range of stable operating conditions was predicted from the results of the profile measurements. The optimal influent concentration was found to be between 25 and 30 g COD/L at an HRT of 5 days for system stability. Other options fro stability control were discussed. © 1993 John Wiley & Sons, Inc.
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  • 91
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    Biotechnology and Bioengineering 41 (1993), S. 204-210 
    ISSN: 0006-3592
    Keywords: immobilization ; interesterification ; cocoa butter equivalent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to investigate quantitatively the interesterification reaction, triolein and stearic acid were used as substrates and eight commercially available lipases were tested for their suitability for the reaction. Three fungal lipase preparations were found to be suitable. The hydrolytic activity of the commercial lipases was tested with olive oil, and it 2was noted that there was no correlation between their hydrolytic and interesterification activities. Among the lipases tested, Mucor miehei lipase was chosen for further study because of it high protein content and its relatively high hydrolytic and interesterification activities, both of which are required for effective interesterification. The effect of water activity of the interesterification reaction was investigated. interesterification activity was shown to be maximum at the water activity of 0.25. As the water activity of the lipase increased, hydrolysis of triglyceride was accelerated. At zero water activity, high conversion was achieved, although interesterification activity was relatively lower than that at the water activity of 0.25. A new and simple immobilization method was developed in order to render hydrophobicity to the lipase and hence to improve the interesterification activity of the lipase. The lipase was immobilized covalently with glutaraldehyde or with six alkyl chains as spacers onto Florisil (magnesium silicate, a inorganic matrix). Interesterification activity of the immobilized lipase with the hydrophobic spacers were increased against that of re lipase. The increase of activity was up to 8-fold that of the original activity of free lipase when the spacer was 7-aminoheptanoic acids. Relatively high stability of the immobilized lipase was shown in a continuous packed bed column reactor with a half-life of 97 days. © 1993 John Wiley & Sons, Inc.
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  • 92
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    Biotechnology and Bioengineering 41 (1993), S. 745-754 
    ISSN: 0006-3592
    Keywords: viscoelsticity ; cell culture ; oscillatory dynamic shear ; steady shear ; shear sensitivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Data on viscous (η′) and elastic (η″) components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62′D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 μm as the mean diameter of cells) showed the yield stress at 550 dyne/cm2 that was 1.8 times higher than the value of HeLa cell suspension (22 μm mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm2 for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 ∼ 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, \documentclass{article}\pagestyle{empty}\begin{document}$ \eta = \eta _0 \exp [K\dot \gamma ^{ - \beta } \phi /(1 - K''\sigma \phi _c /D)] $\end{document} was derived based on the cell concentration, the cell morphology, and the steady shear rate. The β, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s-1, for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field. © 1993 John Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 755-760 
    ISSN: 0006-3592
    Keywords: biopolymers ; metal binding ; polymer-metal binding ; calcium alginate beads ; copper binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A linear absorption model (LAM) is used to describe the process of metal binding to spherically shaped biopolymers particles. The LAM was solved using a numerical algorithm which calculates diffusivities of metal ion in biopolymer gels. It assumes attainment of rapid metal-biopolymer binding equilibrium accompanied by rate limiting diffusion of the metal ions through the gel. The model was tested using batch experiments in which copper (Cu2+) binding with calcium alginate beads was investigated. Biopolymer density in the beads was varied between 2% and 5%. The diffusion coefficient of Cu2+ calculated from the LAM ranged from 1.19 × 10-9 to 1.48 × 10-9 m2 s-1 (average 1.31 ± 0.21 × 10-9 m2 s-1), independent of biopolymer density. The LAM has theoretical advantages over the shrinking core model (shell progressive model). The latter calculated an unreasonable exponential increase in the diffusion coefficient as density of alginate polymer in the bead increased. © 1993 John Wiley & Sons, Inc.
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  • 94
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    Biotechnology and Bioengineering 41 (1993), S. 761-770 
    ISSN: 0006-3592
    Keywords: ultrafiltration membrane bioreactor ; reversed micelles ; lipase ; product separation ; lipolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The enzymatic hydrolysis of olive oil using Chromobacterium viscosum lipase B encapsulated in reversed micelles of dioctyl sodium sulfosuccinate (AOT) in isooctane was investigated in an ultrafiltration ceramic membrane reactor of tubular type, operating in a batch mode. Water concentration was found to be a critical parameter in the enzyme kinetics and hydrolysis yield of the reaction. The size of micelles, recirculation rate, and substrate concentration were found to be the major factors affecting the separation process. A correlation that enables the prediction of final conversion degrees in this bioreactor from the initial reaction conditions was established. © 1993 Wiley & Sons, Inc.
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  • 95
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    Biotechnology and Bioengineering 41 (1993), S. 771-780 
    ISSN: 0006-3592
    Keywords: Catharanthus roseus ; glucose limitation ; growth kinetics ; phosphate limitation ; plant cell suspension culture ; structured growth model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The growth of plant cell suspension cultures of Catharanthus roseus in batch fermentors was studied at different initial phosphate levels of the medium. On the basis of the observations and existing knowledge with respect to phosphate metabolism in cultured C. roseus cells, a structured mathematical model was developed for the description of the kinetics of growth and intracellular accumulation of glucose and phosphate, as a function of glucose and phosphate supply. It was shown that the model offers not only good description of the growth of the cells in batch culture at different initial phosphate levels, but also provided a satisfactory description of the growth in glucose limited chemostats. © 1993 Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 781-790 
    ISSN: 0006-3592
    Keywords: Escherichia coli ; recombinant ; fed batch ; high cell density ; trypsin ; fermention ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fed-batch techniques were employed to obtain high cell density cultures (92-100 g DCW/L) of Escherichia coli strain X90 producing a recombinant serine protease, rat anionic trypsin, secreted to the periplasm. The specific growth rate was controlled to minimize growth-inhibiting acetate formation by utilizing an exponential feeding profile determined from mass balance equation. The volumetric yield of recombinant rat anionic trypsin was 56 mg/L, and the final cell density was 92 g DCW/L when the culture was induced in the late logarithmic phase. However, when the culture was induced in the early logarithmic phase, the volumetric yield was 13 mg/L and the final cell density was 14 g DCW/L. Thus, the induction timing is shown to have a significant effect on the final cell density as well as the overall volumetric yield of the recombinant protease. © 1993 Wiley & Sons, Inc.
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    Biotechnology and Bioengineering 41 (1993), S. 833-836 
    ISSN: 0006-3592
    Keywords: phospholipase D ; adsorptive immobilization ; calcium ; stabilization ; immobilization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Immobilization of phospholipase D from cabbage was studied with the aim of stabilizing the enzyme for its use in synthesis of phospholipids. It was shown that phospholipase D can be immobilized by adsorption to polymeric carriers containing long chain anchor groups such as octadecyl, octyl, or other alkyl residues. Starting from the crude enzyme, phospholipase D activity is preferentially bound (up to 100%) in competition with contaminating proteins. A prerequisite of high binding rates is the presence of calcium ions, which play a mediating role in the adsorption process. The maximum activity of the carrier-enzyme complexes depends upon the calcium concentration in the immobilization process and the carrier material (≥10mM CaCl2 with octadecyl-Si40, ≥40 mM CaCl2 with octyl-sepharose and butyl-fractogel). Immobilization of phospholipase D to octyl-sepharose was shown to result in a distinctly increased storage stability and an enlarged pH-optimum range for the catalytic activity. Operational stability of different phospholipase D-carrier complexes was compared. © 1993 Wiley & Sons, Inc.
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  • 98
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    Biotechnology and Bioengineering 41 (1993), S. 837-845 
    ISSN: 0006-3592
    Keywords: broad host range expression plasmids ; expression system for Pseudomonas ; T7 promoter ; T7 polymerase ; T7 terminator ; lacl and laclq regulated expression in Pseudomonas ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An IPTF-regulated broad host range expression system was constructed using compatible broad host range plasmids, the T7 RNA polymerase, and T7 promoter sequences. The system is implemented by the coexistence of two plasmids. The first contains the T7 RNA polymerase gene under the control of lacl or laclq genes and lacUV5 promoter. The second encodes the T7 promoter upstream of a multicloning site. IncP1 or IncP4 T7 promoter plasmids, and IncP1, IncP4 or IncW T7 RNA polymerase plasmids were constructed. The expression from the IncP1 promoter plasmids in the presence of the IncP4 polymerase plasmids was tested by in vivo lacZ fusions and vivo labeling of proteins. In this combination, the use of lacq improves the regulation levels in Escherichia coli, whereas, in Pseudomonas phaseolicola, a 28.5-fold regulation was obtained with lacl, Although the level of lacZ expression from the T7 promoter in P. phaseolicola is low compared with E. coli, it is similar to levels obtained with the pm promoter in Pseudomonas putida when the differences in the copy number of the expression vectors are taken into consideration © 1993 Wiley & Sons, Inc.
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  • 99
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    Biotechnology and Bioengineering 41 (1993), S. 846-853 
    ISSN: 0006-3592
    Keywords: enzymatic hydrolysis ; cellulose ; β-glucosidase ; SSF ; ethanol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biochemical conversion of cellulosic biomass to ethanol, a promising alternative fuel, can be carried out efficiently and economically using the simultaneous saccharification and fermentation (SSF) process. The SSF integrates the enzymatic hydrolysis of cellulose to glucose, catalyzed by the synergistic action of cellulase and β-glucosidase, with the fermentative synthesis of ethanol. Because the enzymatic step determines the ethanol. Because the enzymatic step determines the availability of glucose to the ethanologenic fermentation, the kinetic of cellulose hydrolysis by cellulase and β-glucosidase and the susceptibility of the two enzymes to inhibition by hydrolysis and fermentation products are of significant importance to the SSF performance and were investigated under realistic SSF conditions. A previously developed SSF mathematical model was used to conceptualize the depolymerization of cellulose. The model was regressed to the collected data to determine the values of the enzyme parameters and was found to satisfactorily predict the kinetics of cellulose hydrolysis. Cellobiose and glucose were identified as the strongest inhibitors of cellulase and β-glucosidase, respectively. Experimental and modeling results are presented in light of the impact of enzymatic hydrolysis on fuel ethanol production. © 1993 Wiley & Sons, Inc.
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  • 100
    ISSN: 0006-3592
    Keywords: α-amylase production ; recombinant Saccharomyces cerevisiae ; PGK promote ; SUC2 promoter ; GAL7 promoter ; on-line glucose analyzer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cloned gene expression in recombinant Saccharomyces cerevisiae 20B-12 containing three different plasmids was compared in batch and fed-batch cultures. The plasmids pNA3, pNA7, and pNA9 contain the α-amylase gene under the control of SUC2, PGK, and GAL7 Promoters, respectively. The synthesis of α-amylase was therefore induced by low glucose concentration for the SUC2 and PGK promoters, and by galactose for GAL7 promoter. The specific cell growth rates were similar among cells harboring the three different plasmids; they decreased from 0.35 to 0.38 h-1 during the cell growth phase to 0.03 to 0.06h-1 during the production phase. The secretory α-amylase activity of cells harboring plasmid pNA7 was 129 U/mL in fed-batch culture, which was 1.4 and 2 times as high as the activities of cells harboring plasmids pNA3 and pNA9, respectively. The secretion ratios (amount of extracellular α-amylase activity/amounts of total α-amylase activity) of cells harboring plasmids pNA3, pNA7, and pNA9 were 91.4%, 94.5%, and 95.3%, respectively. © 1993 Wiley & Sons, Inc.
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