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  • Biochemistry and Biotechnology  (879)
  • 1995-1999  (879)
  • 1940-1944
  • 1997  (879)
  • 1943
  • 1
    Electronic Resource
    Electronic Resource
    Influence of the nature of modifier in the enzymatic activity of chemical modified semipurified lipase from Candida rugosa (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 252-260 
    Details
    ISSN: 0006-3592
    Keywords: lipase ; chemical modification ; stability ; esterification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Semipurified lipase of Candida rugosa (CRSL) was subjected to chemical modification, and the activities of the modified lipase, in hydrolysis and esterification reactions, were examined. The esterification reactions were carried out in the absence and presence of isooctane. When the enzyme was modified with polyethylene glycol (PEG), two methodologies were studied. The activation of PEG with p-NO2-phenylchloroformate gives better biocatalysts than those obtained with cyanuric chloride-PEG. The chemical modification with PEG increases the stability of pure lipases in isooctane at 50°C (extreme conditions). The chemically modified enzymes are useful for biotransformations in organic solvents. In addition the nitration of tyrosines with tetranitromethane was also studied. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 252-260, 1997.
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  • 2
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    Effect of hypoosmotic stress on hybridoma cell growth and antibody production (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 565-570 
    Details
    ISSN: 0006-3592
    Keywords: hybridoma ; hypoosmotic stress ; specific antibody productivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To investigate the response of hybridoma cells to hypoosmotic stress, S3H5/γ2bA2 and DB9G8 hybridomas were cultivated in the hypoosmolar medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% serum] resulting from sodium chloride subtraction. Both hybridomas showed similar responses to hypoosmotic stress in regard to cell growth and antibody production. The cell growth and antibody production at 276 mOsm/kg were comparable to those at 329 mOsm/kg (standard DMEM). Both cells grew well at 219 mOsm/kg, though their growth and antibody production were slightly decreased. When the osmolality was further decreased to 168 mOsm/kg, the cell growth did not occur. When subjected to hyperosmotic stress, both cells displayed significantly enhanced specific antibody productivity (qAb). However, the cells subjected to hypoosmotic stress did not display enhanced qAb. Taken together, both hyperosmotic and hypoosmotic stresses depressed the growth of S3H5/γ2bA2 and DB9G8 hybridomas. However, their response to hypoosmotic stress in regard to qAb was different from that to hyperosmotic stress. © 1997 John Wiley & Sons, Inc. Biotechnol Biong 55: 565-570, 1997.
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  • 3
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    Production of recombinant bacterial endoglucanase as a co-product with ethanol during fermentation using derivatives of Escherichia coli KO11 (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 547-555 
    Details
    ISSN: 0006-3592
    Keywords: ethanol ; cellulose ; hemicellulose ; endoglucanase ; cellulase ; lignocellulose ; biomass ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This study demonstrates a new approach to reduce the amount of fungal cellulase required for the conversion of cellulose into ethanol. Escherichia coli KO11, a biocatalyst developed for the fermentation of hemicellulose syrups, was used to produce recombinant endoglucanase as a co-product with ethanol. Seven different bacterial genes were expressed from plasmids in KO11. All produced cell-associated endoglucanase activity. KO11(pLOI1620) containing Erwinia chrysanthemi celZ (EGZ) produced the highest activity, 3,200 IU endoglucanase/L fermentation broth (assayed at pH 5.2 and 35°C). Recombinant EGZ was solubilized from harvested cells by treatment with dilute sodium dodecyl sulfate (12.5 mg/ml, 10 min, 50°C) and tested in fermentation experiments with commercial fungal cellulase (5 filter paper units/g cellulose) and purified cellulose (100 g/L). Using Klebsiella oxytoca P2 as the biocatalyst, fermentations supplemented with EGZ as a detergent-lysate of KO11(pLOI1620) produced 14%-24% more ethanol than control fermentations supplemented with a detergent-lysate of KO11(pUC18). These results demonstrate that recombinant bacterial endoglucanase can function with fungal cellulase to increase ethanol yield during the simultaneous saccharification and fermentation of cellulose. © 1997 Wiley & Sons, Inc. Biotechnol Bioeng 55: 547-555, 1997.
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  • 4
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    Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5′ cassette system (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 577-580 
    Details
    ISSN: 0006-3592
    Keywords: mRNA stability ; hairpins ; gene expression control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An expression system has been developed for the introduction of DNA cassettes into the region between the transcription and translation start sites of a gene of interest. This cassette system was used to engineer mRNA stability through the introduction of hairpins at the 5′ end. A synthetic DNA cassette was designed so that the resulting mRNA hairpin would be positioned one nucleotide from the 5′ mRNA end. The hairpin-containing mRNA exhibited a half-life 3 times that of the mRNA with no hairpin, resulting in increases in both mRNA and protein levels. These results indicate that it is possible to engineer mRNA stability as an additional means of controlling gene expression. © 1997 John Wiley & Sons Inc. Biotechnol Bioeng 55: 557-580, 1997
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  • 5
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    Application of membrane-based preferential transport to whole broth processing (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 581-591 
    Details
    ISSN: 0006-3592
    Keywords: adsorptive membranes ; oscillatory flow ; integrated processes ; in situ product recovery ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Preferential transport in adsorptive membranes can be used to selectively remove biochemicals directly from fermentation broths. During preferential transport, an adsorbing solute is selectively transported across the membrane while nonadsorbing solutes and cells are retained by the membrane. This technique was used to separate lysozyme directly from a feed containing lysozyme, myoglobin, and yeast cells. We found that because the oscillatory flows used in preferential transport involve strokes that are close to symmetric, they are very efficient in alleviating cake formation due to cell deposition on the membrane surface. Theoretical results suggest that, by optimizing process variables, preferential transport can lead to a continuous concentrated stream of the adsorbing protein. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 581-591, 1997.
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  • 6
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    In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae: II. Mathematical model (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 592-608 
    Details
    ISSN: 0006-3592
    Keywords: Saccharomyces cerevisiae ; metabolic modeling ; sensitivity analysis ; glycolysis ; compartmentation ; transient response ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model of glycolysis in Saccharomyces cerevisiae is presented. The model is based on rate equations for the individual reactions and aims to predict changes in the levels of intra- and extracellular metabolites after a glucose pulse, as described in part I of this study. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. The model structure and experimental observations are related to the aerobic growth of the yeast. The model is based on material balance equations of the key metabolites in the extracellular environment, the cytoplasm and the mitochondria, and includes mechanistically based, experimentally matched rate equations for the individual enzymes. The model includes removal of metabolites from glycolysis and TCC for biosynthesis, and also compartmentation and translocation of adenine nucleotides. The model was verified by in vivo diagnosis of intracellular enzymes, which includes the decomposition of the network of reactions to reduce the number of parameters to be estimated simultaneously. Additionally, sensitivity analysis guarantees that only those parameters are estimated that contribute to systems trajectory with reasonable sensitivity. The model predictions and experimental observations agree reasonably well for most of the metabolites, except for pyruvate and adenine nucleotides. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 592-608, 1997.
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  • 7
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    A novel membrane reactor design for controlled studies of interacting populations (simulation of the interaction between microorganism and plant suspension cultures) (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 609-615 
    Details
    ISSN: 0006-3592
    Keywords: interacting populations ; membrane reactor ; induced metabolic changes ; elicitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The design of a reactor in which two interacting cell populations (microorganisms and plants) could grow under controlled conditions was considered. In this reactor, the cell populations are separated by a membrane which permits semi-in vivo study of induced interaction-specific changes in metabolism. In this paper, the interaction of suspension culture of Nicotiana tabacum (tobacco) and the Oomycete, Phytophthora nicotiana was simulated. The results of the computer simulation show the induced metabolic changes as a consequence of the biological interaction. The paper introduces a novel approach in the strategy for the study of interacting population in suspension cultures. This type of system has potential applications in studies of the regulation of secondary metabolism and for the production of high values pharmaceuticals. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 609-615, 1997.
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  • 8
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    Application of fluid mechanic and kinetic models to characterize mammalian cell detachment in a radial-flow chamber (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 616-629 
    Details
    ISSN: 0006-3592
    Keywords: cell adhesion ; radial-flow chamber ; hydrodynamic shear ; detachment kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The strength of adhesion and dynamics of detachment of murine 3T3 fibroblasts from self-assembled monolayers were measured in a radial-flow chamber (RFC) by applying models for fluid mechanics, adhesion strength probability distributions, and detachment kinetics. Four models for predicting fluid mechanics in a RFC were compared to evaluate the accuracy of each model and the significance of inlet effects. Analysis of these models indicated an outer region at large radial positions consistent with creeping flow, an intermediate region influenced by inertial dampening, and an inner region dominated by entrance effects from the axially-oriented inlet. In accompanying experiments patterns of the fraction of cells resisting detachment were constructed for individual surfaces as a function of the applied shear stress and evaluated by comparison with integrals of both a normal and a log-normal distribution function. The two functions were equally appropriate, yielding similar estimates of the mean strength of adhesion. Further, varying the Reynolds number in the inlet, Red, between 630 and 1480 (corresponding to volumetric flow rates between 0.9 and 2.1 mL/s) did not affect the mean strength of adhesion. For these same experiments, analysis of the dynamics of detachment revealed three temporal phases: 1) rapid detachment of cells at the onset of flow, consistent with a first-order homogeneous kinetic model; 2) time-dependent rate of detachment during the first 30 sec. of exposure to hydrodynamic shear, consistent with the first-order heterogeneous kinetic model proposed by Dickinson and Cooper (1995); and 3) negligible detachment, indicative of pseudo-steady state after 60 sec. of flow. Our results provide rigorous guidelines for the measurement of adhesive interactions between mammalian cells and prospective biomaterial surfaces using a RFC. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 616-629, 1997.
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  • 9
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    Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 693-700 
    Details
    ISSN: 0006-3592
    Keywords: glucose ; lactate ; real-time determination ; hematopoietic cell culture ; colony-forming cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Glucose and lactate metabolic rates were evaluated for cultures of cord blood (CB) mononuclear cell (MNC), peripheral blood (PB) MNC, and PB CD34+ cell cultures carried out in spinner flasks and in T-flasks in both serum-containing and serum-free media. Specific glucose uptake rates (qgluc, in micromoles per cell per hour) and lactate generation rates (qlac) correlated with the percentage of colony-forming cells (CFC) present in the culture for a broad range of culture conditions. Specifically, the time of maximum CFC percentage in each culture coincided with the time of maximum qgluc and qlac in cultures with different seeding densities and cytokine combinations. A two-population model (Qlac = α[CFC] + β([TC] - [CFC]), where [TC] is total cell concentration; Qlac is volumetric lactate production rate in micromoles per milliliter per hour; α is qlac for an average CFC; and β is qlac for an average non-CFC) was developed to describe lactate production. The model described lactate production well for cultures carried out in both T-flasks and spinner flasks and inoculated with either PB or CB MNC or PB CD34+ cells. The values for α and β that were derived from the model varied with both the inoculum density and the cytokine combination. However, preliminary results indicate that cultures carried out under the same conditions from different samples with similar initial CD34+ cell content have similar values for β and β. These findings suggest that it should be possible to use lactate production data to predict the harvest time that corresponds to the maximum number of CFC in culture. The ability to harvest ex vivo hematopoietic cultures for transplantation when CFC are at a maximum has the potential to speed the rate at which immunocompromised patients recover. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 693-700, 1997.
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  • 10
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    A model for light distribution and average solar irradiance inside outdoor tubular photobioreactors for the microalgal mass culture (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 701-714 
    Details
    ISSN: 0006-3592
    Keywords: tubular photobioreactors ; light distribution ; average solar irradiance ; light attenuation ; microalgae mass culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A mathematical model to estimate the solar irradiance profile and average light intensity inside a tubular photobioreactor under outdoor conditions is proposed, requiring only geographic, geometric, and solar position parameters. First, the length of the path into the culture traveled by any direct or disperse ray of light was calculated as the function of three variables: day of year, solar hour, and geographic latitude. Then, the phenomenon of light attenuation by biomass was studied considering Lambert-Beer's law (only considering absorption) and the monodimensional model of Cornet et al. (1900) (considering absorption and scattering phenomena). Due to the existence of differential wavelength absorption, none of the literature models are useful for explaining light attenuation by the biomass. Therefore, an empirical hyperbolic expression is proposed. The equations to calculate light path length were substituted in the proposed hyperbolic expression, reproducing light intensity data obtained in the center of the loop tubes. The proposed model was also likely to estimate the irradiance accurately at any point inside the culture. Calculation of the local intensity was thus extended to the full culture volume in order to obtain the average irradiance, showing how the higher biomass productivities in a Phaeodactylum tricornutum UTEX 640 outdoor chemostat culture could be maintained by delaying light limitation. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 701-714, 1997.
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  • 11
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    Effect of agitation intensities on fungal morphology of submerged fermentation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 715-726 
    Details
    ISSN: 0006-3592
    Keywords: fungal morphology ; pellets ; hyphae ; hair of pellets ; agitation intensity ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Both parallel fermentations with Aspergillus awamori (CBS 115.52) and a literature study on several fungi have been carried out to determine a relation between fungal morphology and agitation intensity. The studied parameters include hyphal length, pellet size, surface structure or so-called hairy length of pellets, and dry mass per-wet-pellet volume at different specific energy dissipation rates. The literature data from different strains, different fermenters, and different cultivation conditions can be summarized to say that the main mean hyphal length is proportional to the specific energy dissipation rate according to a power function with an exponent of -0.25 ± 0.08. Fermentations with identical inocula showed that pellet size was also a function of the specific energy dissipation rate and proportional to the specific energy dissipation rate to an exponent of -0.16 ± 0.03. Based on the experimental observations, we propose the following mechanism of pellet damage during submerged cultivation in stirred fermenters. Interaction between mechanical forces and pellets results in the hyphal chip-off from the pellet outer zone instead of the breakup of pellets. By this mechanism, the extension of the hyphae or hair from pellets is restricted so that the size of pellets is related to the specific energy dissipation rate. Hyphae chipped off from pellets contribute free filamentous mycelia and reseed their growth. So the fraction of filamentous mycelial mass in the total biomass is related to the specific energy dissipation rate as well.To describe the surface morphology of pellets, the hyphal length in the outer zone of pellets or the so-called hairy length was measured in this study. A theoretical relation of the hairy length with the specific energy dissipation rate was derived. This relation matched the measured data well. It was found that the porosity of pellets showed an inverse relationship with the specific energy dissipation rate and that the dry biomass per-wet-pellet volume increased with the specific energy dissipation rates. This means that the tensile strength of pellets increased with the increase of specific energy dissipation rate. The assumption of a constant tensile strength, which is often used in literature, is then not valid for the derivation of the relation between pellet size and specific energy dissipation rate. The fraction of free filamentous mycelia in the total biomass appeared to be a function of the specific energy dissipation in stirred bioreactors. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 715-726, 1997.
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  • 12
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    On-line green fluorescent protein sensor with LED excitation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 921-926 
    Details
    ISSN: 0006-3592
    Keywords: green fluorescent protein ; sensor ; on-line monitoring ; quantitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:921-926, 1997.
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  • 13
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    Comparison of Trichoplusia ni BTI-Tn-5B1-4 (high five™) and Spodoptera frugiperda Sf-9 insect cell line metabolism in suspension cultures (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 909-920 
    Details
    ISSN: 0006-3592
    Keywords: baculovirus ; insect cells ; metabolism ; Sf-9; high five™ ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Nutrient utilization and byproduct accumulation were monitored in Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Five™) cell lines during growth and following viral infection in suspension cultures in order to develop a better understanding of cell metabolism and to acquire information relevant to large scale fed-batch bioreactors. The utilization of glucose, dissolved oxygen, and amino acids were monitored in Sf-9 cell cultures grown in Sf-900 II serum-free medium (SFM) and in High Five™ cell cultures grown in both Sf-900 II and Express Five SFM. Using the optimal medium for each cell line, i.e., Sf-900 II SFM for Sf-9 cells and Express Five SFM for High Five™ cells, the cell growth rate, maximum cell density, specific glucose and glutamine utilization rates, and specific alanine production rate were comparable during cell growth. In addition, the expression level of recombinant human tissue plasminogen activator was comparable in the two cell lines on a per cell basis. It was found, however, that lactate and ammonia accumulated in High Five™ cell cultures, but not in Sf-9 cell cultures. In addition, High Five™ cells utilized asparagine more rapidly than glutamine, whereas Sf-9 cells consumed only minimal asparagine, and the oxygen utilization rate was significantly higher in High Five™ cell cultures. It was also found that the medium had a significant effect on High Five™ cell metabolism, e.g., the specific glucose utilization rate and the specific lactate and alanine production rates were significantly higher in Sf-900 II SFM than in Express Five SFM. In addition, the maximum cell density and specific asparagine utilization rate were significantly higher in Express Five SFM. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:909-920, 1997.
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  • 14
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    An activity coefficient model for proteins (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 940-940 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: No abstract.
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  • 15
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    Kinetic studies of fusarium solani pisi cutinase used in a gas/solid system: Transesterification and hydrolysis reactions (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 1-8 
    Details
    ISSN: 0006-3592
    Keywords: transesterification ; hydrolysis ; water activity ; cutinase ; gas ; bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fusarium solani cutinase supported onto Chromosorb P was used to catalyze transesterification (alcoholysis) and hydrolysis on short volatile alcohols and esters in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrates and removed reaction products simultaneously. A kinetic study was performed under differential operating conditions in order to get initial reaction rates. The effect of the hydration state of the biocatalyst on the kinetics was studied for 3 conditions of hydration (aw = 0.2, aw = 0.4 and aw = 0.6), the alcoholysis of propionic acid methyl ester with n-propanol, and for 5 hydration levels (from aw = 0.2 to aw = 0.6) for the hydrolysis of propionic acid methyl, ethyl or propyl esters. F. solani cutinase was found to have an unusual kinetic behavior. A sigmoid relationship between the rate of transesterification and the activity of methyl propionate was observed, suggesting some form of cooperative activation of the enzyme by one of its substrate. For the hydrolysis of short volatile propionic acid alkyl esters, threshold effects on the reaction rate, highly depending on the water activity and the substrate polarity, are reported. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 1-8, 1997.
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  • 16
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    Kinetics and equilibria of condensation reactions between monosaccharide pairs catalyzed by Aspergillus niger glucoamylase (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 9-22 
    Details
    ISSN: 0006-3592
    Keywords: condensation reactions ; disaccharides ; equilibria ; glucoamylase ; kinetics ; monosaccharides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Arabinose, fructose, galactose, myo-inositol, lyxose, mannose, ribose, and xylose were incubated individually and with glucose in the presence of Aspergillus niger glucoamylase at pH 4.5 and 45°C. Glucoamylase condenses galactose, glucose, and mannose individually into disaccharides. It also produces mixed disaccharides when each of the eight carbohydrates is incubated with glucose. Many products were identified by gas chromatography of the derivatized reaction mixtures followed by mass spectroscopy of the individual chromatographic peaks. Galacto-, gluco-, or mannopyranosyl rings appear to be present at the nonreducing ends of all the disaccharides produced. Molecules linked through primary hydroxyl groups have the highest equilibrium constants of all products formed, since these bonds are thermodynamically favored. However, glucoamylase is capable of forming bonds with many available hydroxyl groups, as previously demonstrated when it was incubated with glucose alone. Formation rates of different bonds linking different residues vary widely. These results demonstrate that glucoamylase has a wide selectivity toward residues it will condense into disaccharides and toward bonds it will form between them. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 9-22, 1997.
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  • 17
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    Interaction of trout hemoglobin with H2O2: a chemiluminescence study (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 79-85 
    Details
    ISSN: 0884-3996
    Keywords: trout hemoglobin ; H2O2 chemiluminescence ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H2O2 using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H2O2 produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H2O2. © 1997 John Wiley & Sons, Ltd.
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    Effects of the composition of bacteriological growth media on a chemiluminometric assay of β-galactosidase in Escherichia coli (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 165-175 
    Details
    ISSN: 0884-3996
    Keywords: chemiluminescence ; β-galactosidase ; luminescent background ; quenching ; bacteriological growth media ; 1,2-dioxetanes ; coliforms ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The effects of the composition of bacteriological growth media on the light output in a chemiluminometric assay of β-galactosidase in Escherichia coli using 1,2-dioxetane substrates has been studied. In this assay a basic conflict exists between conditions that promote optimal bacterial growth and those conducive to maximal chemiluminescence. Common medium ingredients such as yeast or beef extract, protein hydrolysates and lactose suppress light emission and/or lead to high backgrounds. Quenching of light emission is probably partly due to light absorption by medium ingredients such as oxgall, and partly to interference with the reaction triggering the chemiluminescent process. Elevated backgrounds are caused by the presence of high concentrations of protein hydrolysates, which interact with the alkali in the accelerator solution. Only two purposely developed media, i.e. ILM and Colicult™ are shown to reconcile the requirements of growth support with that of optimal luminescent properties. © 1997 John Wiley & Sons, Ltd.
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    Effect of aliphatic aldehydes on the lipid peroxidation and chemiluminescence of biological systems under oxidative stress (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 141-148 
    Details
    ISSN: 0884-3996
    Keywords: lipid peroxidation ; aldehydes ; chemiluminescence ; oxidative stress ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The effect of several aliphatic aldehydes on lipid peroxidation was evaluated by measuring the oxygen uptake rate, thiobarbituric acid-reactive products formation and the emitted visible chemiluminescence intensity. Measurements were carried out in brain homogenates and erythrocyte plasma membrane and liver microsomal fractions. In all systems studied, aldehydes (25 mmol/L) (e.g. acetaldehyde, 2,2-dimethylpropanal), increased the intensity of the luminescence associated with the oxidation process. In contrast, aldehyde incorporation decreased TBARS production and the rate of oxygen uptake. The increased luminescence intensity is explained in terms of secondary reactions of aldehyde derived free radicals. These results clearly indicate that extreme care must be exercized in the intepretation of chemiluminescence data in the presence of aldehydes. © 1997 John Wiley & Sons, Ltd.
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    A two-intermediate model for imidazole-promoted peroxyoxalate chemiluminescence (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 241-248 
    Details
    ISSN: 0884-3996
    Keywords: peroxyoxalate chemiluminescence ; imidazole-catalysed mechanism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A mechanism is proposed for imidazole-catalysed peroxyoxalate chemiluminescence. The reaction model includes a sequential formation of 1-aroxalylimidazole and 1,1′-oxalyldiimidazole as light-producing reaction intermediates. The suggestion is supported by the kinetic data obtained for the reaction of imidazole with bis(4-nitrophenyl) oxalate and on the recently reported ability of 1,1′-oxalyldiimidazole to function as an efficient chemiluminescence reagent. The relative contributions of different catalytic pathways and hydrolytic side-reactions are discussed © 1997 John Wiley & Sons, Ltd.
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    Chemiluminescent immunoassay (CLIA) for salmon growth hormone (GH) (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 271-275 
    Details
    ISSN: 0884-3996
    Keywords: chemiluminescent immunoassay ; acridinium ester ; fish ; salmon ; growth hormone ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of growth hormone (GH) in salmonid species. The CLIA for salmon GH was performed using the sandwich method with anti-GH IgG as the first antibody and chemiluminescent acridinium ester-labelled specific anti-GH F(ab′)2 as the second antibody. The measurable range of salmon GH in the CLIA was 39-1250 pg/mL using a short assay (1 day) protocol and 3.9-125 pg/mL in a longer (2-day) assay. The dilution curve in the CLIA of serum from masu salmon (Oncorhynchus masou) was parallel to the standard curve of recombinant chum salmon (Oncorhynchus keta) GH. Seasonal changes of serum GH levels were measured in 1 year-old masu salmon cultivated in a pond from March to November. Their serum GH levels increased during smoltification from March to April, achieved a maximum level of 21 ng/mL in August, and then declined gradually to 11 ng/mL in October. © 1997 John Wiley & Sons, Ltd.
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    Enhanced chemiluminescence reaction applied to the study of horseradish peroxidase stability in the course of p-iodophenol oxidation (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 299-308 
    Details
    ISSN: 0884-3996
    Keywords: enhanced chemiluminescent reaction ; horseradish peroxidase ; enzyme inactivation ; p-iodophenol ; free radical ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The enhanced chemiluminescence reaction (ECL) was applied to the study of horseradish peroxidase (HRP) inactivation during the oxidation of p-iodophenol. Enzyme inactivation was shown to be the main reason for light decay in the course of the reaction. No individual effect of luminol and p-iodophenol as enhancer on HRP activity towards 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) was detected, enzymatic activity loss was detected only in the course of the ECL reaction. HRP activity towards ABTS (a colorimetric substrate) fell in a similar manner to the decay in light emission. The reactive radical species formed during enhancer oxidation were suggested as the main inactivating agents. The similarity of changes in light intensity and enzymatic activity allows one to apply the ECL reaction for testing potential stabilizers of HRP. The loss of enzyme activity can be partially explained by non-specific interaction of radical species with protein globule. The addition of bovine serum albumin provided almost complete protection of peroxidase from inactivation. This confirms the non-specific inactivation with highly reactive endogenous intermediates through the modification of a protein globule. © 1997 John Wiley & Sons, Ltd.
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    Superoxide anion reacts with enzyme intermediate in the bacterial luciferase reaction competitive with intramolecular electron transfer (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 15-20 
    Details
    ISSN: 0884-3996
    Keywords: bioluminescence ; bacteria ; luciferase ; superoxide anion ; dimethyl sulfoxide ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Addition of KO2 in dimethyl sulfoxide (DMSO) to the in vitro bacterial luciferase reaction subsequent to its initiation resulted in a biphasic decay of light emission. The first and more rapid phase is attributed to quenching by DMSO. With DMSO alone the continuing decay is kinetically the same as in a control reaction. With KO2 added the second decay phase is more rapid and dependent on the KO2 concentration. The enhanced decay is attributed to superoxide anion generated from KO2 reacting without light emission with an enzyme peroxy intermediate, breaking down of the peroxide bond through intermolecular electron transfer from the superoxide anion, in competiton with an intramolecular electron transfer from the N(5) position of the flavin ring, which normally leads to the production of the excited luciferase-dihydroflavin-4a-hydroxide. © 1997 John Wiley & Sons, Ltd.
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    Current awareness: abstracts from the symposium  -  luminescence assays for industry, 22-23 September 1997, University of Cambridge, UKOrganised by Cambridge Research & Technology Transfer Ltd. (1997)
    New York : Wiley-Blackwell
    Journal of Bioluminescence and Chemiluminescence 12 (1997), S. 93-112 
    Details
    ISSN: 0884-3996
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: No Abstract
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    Selective displacement chromatography of proteins (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 119-129 
    Details
    ISSN: 0006-3592
    Keywords: selective displacement chromatography ; protein purification ; on-line monitoring ; ion-exchange chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In contrast to high molecular weight polyelectrolyte displacers, the efficacy of low molecular weight displacers are dependent on both mobile phase salt and displacer concentration. This sensitivity to the operating conditions opens up the possibility of carrying out selective displacement where the product(s) of interest can be selectively displaced while the low affinity impurities can be desorbed in the induced salt gradient ahead of the displacement train, and the high affinity impurities either retained or desorbed in the displacer zone. This type of displacement combines the operational advantages of step gradient and the high resolution inherent in a true displacement process, in a single operation. Theoretical expressions are presented for establishing selective displacement operating conditions (initial salt concentration, displacer concentration) based on the Steric Mass Action parameters of the displacer and the linear Steric Mass Action parameters of the feed proteins. Experimental results are presented to elucidate the concept of selective displacement in both cation and anion exchange systems. A mixture of α-lactalbumin and β-lactoglobulin A and B has been used for anion-exchange systems; a four-protein mixture consisting of ribonuclease B, bovine and horse heart cytochrome c, and lysozyme has been employed in cation exchange systems. This article also demonstrates that on-line monitoring can be readily employed for the selective displacement process, thus facilitating the scale-up and control of the process. This work sets the stage for the development of robust large scale high resolution separations using selective displacement chromatography. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 119-129, 1997.
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    Modeling assembly, aggregation, and chaperoning of immunoglobulin G production in insect cells (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 106-116 
    Details
    ISSN: 0006-3592
    Keywords: BiP ; immunoglobulin ; aggregation ; mathematical model ; chaperones ; baculovirus-insect cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A model for immunoglobulin G (IgG) production in the baculovirus-insect cell system was developed that incorporates polypeptide synthesis, oligomer assembly, protein aggregation, and protein secretion. In addition, the capacity of a chaperone to protect heavy and light chain polypeptides from protein aggregation was considered by including in vitro chaperone-peptide binding and dissociation kinetic constants from the literature. Model predictions were then compared to experiments in which the chaperone immunoglobulin heavy chain binding protein, BiP, was coexpressed by coinfecting insect cells with BiP-containing baculovirus. The model predicted a nearly twofold increase in intracellular and secreted IgG that was similar to the behavior observed experimentally after approximately 3 days of coexpressing heterologous IgG and BiP. However, immunoglobulin aggregation was still significant in both the model simulation and experiments, so the model was then used to predict the effect of strategies for improving IgG production even further. Increasing expression of the chaperone BiP by 10-fold over current experimental levels provided a 2.5-fold increase in secreted IgG production over IgG assembly without BiP. Alternatively, the expression of BiP earlier in the baculovirus infection cycle achieved a twofold increase in protein secretion without requiring excessive BiP production. The potential effect of cochaperones on BiP activity was considered by varying the BiP binding and release constants. The utilization of lower binding and release kinetic constants led to a severalfold increase in IgG secretion because the polypeptides were protected from aggregation for greater periods. An optimized strategy for chaperone action would include the rapid peptide binding of a BiP-ATP conformation along with the slow peptide release of a BiP-ligand conformation. However, even with an optimized chaperoning system, limitations in the secretion kinetics can result in the accumulation of intracellular IgG. Thus, the entire secretory pathway must be considered when enhanced secretion of heterologous proteins is desired. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 106-116, 1997.
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    Elevated Fis expression enhances recombinant protein production in Escherichia coli (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 138-144 
    Details
    ISSN: 0006-3592
    Keywords: rrn promoter ; rRNA synthesis ; restricted growth ; ribosome ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A genetic strategy to enhance recombinant protein production is discussed. A small DNA bending protein, Fis, which has been shown to activate rRNA synthesis upon a nutrient upshift, was overexpressed in E. coli strain W3110 carrying vector pUCR1. Overexpression of Fis during exponential growth was shown to activate rrn promoters to different extents. A 5-fold improvement in chloramphenicol acetyltransferase (CAT) production in cultures with elevated Fis level was observed in shake-flask cultivations. A similar improvement in the culture performance was also observed during fed-batch fermentation; the specific CAT activity increased by more than 50% during the fed-batch phase for cultures with elevated Fis expression. In contrast, no increase in specific CAT activity was detected for cultures carrying pUCR2, expressing a frame-shift Fis mutant. Expression of Fis from a complementary vector, pKFIS, restored CAT production from W3110:pUCR2 to approximately the same level as cultures carrying pUCR1, indicating that the enhancement in CAT production was indeed Fis-dependent. The framework presented here suggests that differential activation in recombinant protein production may be achieved with differential Fis overexpression. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 138-144, 1997.
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    Integration of charged membrane into perstraction system for separation of amino acid derivatives (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 162-167 
    Details
    ISSN: 0006-3592
    Keywords: perstraction ; amino acid derivative ; partition coefficient ; charged membrane ; electrostatic rejection ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The integration of a charged membrane into a perstraction system for high selective separation is reported. A mixture of N-(benzyloxycarbonyl)-L-aspartic acid (ZA), L-phenylalanine methyl ester (PM), and N-(benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (ZAPM) was used as the model solution. The aqueous phase containing ZA, PM, and ZAPM was adjusted to pH 6 and was contacted with tert-amyl alcohol through a charged membrane. Seven different ion-exchange membranes and two different microfiltration membranes were tested for the separation system. Only ZAPM could permeate into the organic phase through SELEMION AMV and ASV. The separations between ZA and ZAPM and between PM and ZAPM were performed by biphasic extraction and electrostatic rejection, respectively. The permeabilities of ZAPM were higher than those of PM for all experiments using the ion-exchange membranes, although the molecular weight of ZAPM is larger than that of PM. The membrane that had a smaller pore size showed higher ZAPM selectivity. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 162-167, 1997.
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    Properties of free and immobilized lipase from Pseudomonas cepacia (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 181-189 
    Details
    ISSN: 0006-3592
    Keywords: lipase ; immobilization ; polypropylene support ; Pseudomonas cepacia ; kinetic parameters ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The purified lipase from Pseudomonas cepacia (PS, Amano) was immobilized on a commercially available microporous polypropylene support. The enzyme was rapidly and completely adsorbed on the support. Special attention was devoted to the demonstration of the lack of diffusional limitations, either internal or external, when a soluble substrate (p-nitrophenylacetate, pNPA) was used. The activity yield was high (100%) with pNPA and very low (0.4%) with p-nitrophenylpalmitate (pNPP). These values clearly showed that the immobilized enzyme was fully active as soon as activity was assayed on a soluble substrate rather than an insoluble one. With the latter one, the low activity was due mainly to a slow rate of substrate diffusion inside the porous support. The same diffusional phenomenon could explain the complete change of fatty acid specificity of the immobilized lipase. After immobilization, the lipase was mainly specific for short chain fatty acid esters, whereas the free enzyme was mainly specific for long chain esters. The activity-versus-temperature profiles were not greatly affected by immobilization with maximal reaction rates in the range 45° to 50°C for both enzyme preparations. However, immobilization increased enzyme stability mainly by decreasing the sensitivity to temperature of the inactivation reaction. Half-lives at 80°C were 11 and 4 min for the immobilized and free enzymes, respectively. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 181-189, 1997.
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    Quantitative assessment of uncertainty in the optimization of metabolic pathways (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 145-161 
    Details
    ISSN: 0006-3592
    Keywords: metabolic pathways ; parametric uncertainty ; chance-constrained programming ; nonlinear optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The S-System formalism provides a popular, versatile and mathematically tractable representation of metabolic pathways. At steady-state, after a logarithmic transformation, the S-System representation reduces into a system of linear equations. Thus, the maximization of a particular metabolite concentration or a flux subject to physiological constraints can be expressed as a linear programming (LP) problem which can be solved explicitly and exactly for the optimum enzyme activities. So far, the quantitative effect of parametric/experimental uncertainty on the S-model predictions has been largely ignored. In this work, for the first time, the systematic quantitative description of modeling/experimental uncertainty is attempted by utilizing probability density distributions to model the uncertainty in assigning a unique value to system parameters. This probabilistic description of uncertainty renders both objective and physiological constraints stochastic, demanding a probabilistic description for the optimization of metabolic pathways. Based on notions from chance-constrained programming and statistics, a novel approach is introduced for transforming the original stochastic formulation into a deterministic one which can be solved with existing optimization algorithms. The proposed framework is applied to two metabolic pathways characterized with experimental and modeling uncertainty in the kinetic orders. The computational results indicate the tractability of the method and the significant role that modeling and experimental uncertainty may play in the optimization of networks of metabolic reactions. While optimization results ignoring uncertainty sometimes violate physiological constraints and may fail to correctly assess objective targets, the proposed framework provides quantitative answers to questions regarding how likely it is to achieve a particular metabolic objective without exceeding a prespecified probability of violating the physiological constraints. Trade-off curves between metabolic objectives, probabilities of meeting these objectives, and chances of satisfying the physiological constraints, provide a concise and systematic way to guide enzyme activity alterations to meet an objective in the face of modeling and experimental uncertainty. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 145-161, 1997.
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    Response of the central metabolism of Corynebacterium glutamicum to different flux burdens (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 168-180 
    Details
    ISSN: 0006-3592
    Keywords: metabolic fluxes ; metabolite balance ; NMR spectroscopy ; amino acid production ; bidirectional fluxes ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1-13C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5-phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of 13C enrichments on exchange fluxes enabled the transketolase-catalyzed exchange rate (2 pentose 5-phosphate ↔ sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5-phosphate + erythrose 4-phosphate ↔ fructose 6-phosphate + glyceraldehyde 3-phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L-glutamate. Because we had already quantified the fluxes for L-lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause-and-effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L-lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168-180, 1997.
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    Modeling and optimization of a batch process for in vitro RNA production (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 210-220 
    Details
    ISSN: 0006-3592
    Keywords: ribonucleic acids ; transcription ; optimization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: RNA molecules are commonly produced in vitro by transcription, utilizing a DNA template, an RNA polymerase enzyme, and nucleoside triphosphate substrates (NTPs). In addition to the full-length RNA molecule coded for by the DNA template, significant amounts of shorter RNA molecules are produced. A simplified model of this complex transcription process is presented, with the shorter RNA molecules lumped into a single pool. The rate equations do not depend on the stoichiometry of the RNA molecule of interest, which facilitates application of the model to other RNA molecules. Optimal initial conditions for batch in vitro RNA transcription to produce a dodecamer RNA containing three different nucleotides have been predicted using the model. The predicted optimal values for equimolar NTPs are 10 to 15 mM initial concentration for each NTP and 50 to 60 mM for magnesium acetate, yielding a maximum final dodecamer concentration of 0.8 ± 0.1 mM at the 90% confidence interval. Experimental data agree well with the model results. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 210-220, 1997.
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    Enzymatic condensation of cholecystokinin CCK-8 (4-6) and CCK-8 (7-8) peptide fragments in organic media (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 456-463 
    Details
    ISSN: 0006-3592
    Keywords: enzymatic fragment condensation ; α-chymotrypsin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetically controlled condensation reaction of Z-Gly-Trp-Met-OR1 (R1: Et, Al, Cam) and H-Asp-(OR2)-Phe-NH2 (R2: H, But) catalyzed by α-chymotrypsin deposited onto polyamide in organic media was studied. The effect of the drying process of the enzyme-support preparation, substrate concentrations, reaction medium, acyl donor, and nucleophile structure on both enzymatic activity and pentapeptide yield was investigated. The immobilized preparation directly equilibrated at aw = 0.113, gave higher enzymatic activities than dried with vacuum first, and then equilibrated at aw = 0.113. The addition of triethylamine to the reaction medium increased dramatically the enzymatic activity. However, the pentapeptide yield was affected neither by the drying procedure nor by the addition of triethylamine. The donor ester Z-Gly-Trp-Met-OAl gave initial reaction rates 2.6 times higher than the conventional ethyl ester derivative but rendered similar yields. The best results were obtained using Z-Gly-Trp-Met-OCam as acyl-donor ester; 80% yield and initial reaction rates 4 times higher than the ethyl ester derivative. In all cases, acetonitrile containing Tris-HCl 50 mM pH 9 buffer (0.5% v/v) and triethylamine (0.5% v/v) was found to be the best reaction system. Under these conditions, it was possible to use the nucleophile H-Asp-Phe-NH2 with β-unprotected aspartic acid residue. In this case, 50% yield was obtained, but economic considerations could lead to select it as nucleophile. Finally, the fragment condensation reaction was carried out at gram scale, obtaining a 39% yield which included the reaction, removal of protecting groups and purification steps. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 456-463, 1997.
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    Production of recombinant proteins in transgenic plants: Practical considerations (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 473-484 
    Details
    ISSN: 0006-3592
    Keywords: transgenic plants ; recombinant protein ; gene expression ; downstream processing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This review is based on our recent experience in producing the first commercial recombinant proteins in transgenic plants. We bring forward the issues that have to be considered in the process of selecting and developing a winning transgenic plant production system. From the production point of view, transcription, posttranscription, translation, and posttranslation are important events that can affect the quality and quantity of the final product. Understanding the rules of gene expression is required to develop sound strategies for optimization of recombinant protein production in plants. The level of recombinant protein accumulation is critical, but other factors such as crop selection, handling and processing of transgenic plant material, and downstream processing are equally important when considering commercial production. In some instances, the cost of downstream processing alone may determine the economic viability of a particular plant system. Some of the potential advantages of a plant production system such as the high levels of accumulation of recombinant proteins, glycosylation, compartmentalization within the cell, and natural storage stability in certain organs are incentives for aggressively pursuing recombinant protein production in plants. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 473-484, 1997.
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    Ammonium toxicity in different cell lines (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 530-537 
    Details
    ISSN: 0006-3592
    Keywords: ammonium ; cell culture ; cell cycle ; cell death ; cell growth ; Jurkat cells, GH4 cells ; LLC-PK1 cells ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The toxic effect of ammonium upon a variety of cell lines of lymphoid (Jurkat), pituitary (GH4), and renal (LLC-PK1) origin was studied. Millimolar concentrations of the ion mildly affected the growth of GH4 cells and prevented the growth of LLC-PK1 cells. The ion did not lead to the death of LLC-PK1 cells but it produced morphologic changes in these cells. The effects of ammonium upon Jurkat cells were different because cells died after accumulating at S phase. Cell death was due to apoptosis and might be related to ammonium-induced calcium mobilization from intracellular stores. These results indicate that the toxic effects caused by ammonium accumulation are different depending upon the cell type. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 530-537, 1997.
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    The continuous isolation of trypsin by affinity adsorbent recycling (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 538-545 
    Details
    ISSN: 0006-3592
    Keywords: affinity ; separation ; purification ; continuous ; trypsin ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method for the continuous affinity separation of proteins is described in which the adsorbent, in the form of a polymer belt, is recycled through feedstock and eluent liquid flows. As the belt is nonporous, contact between the solute and the ligand is not diffusion-dependent. Consequently, rapid cycle rates are possible. Soybean trypsin inhibitor immobilized on nylon was used as an affinity ligand for the isolation of trypsin. During a 30-h continuous run, trypsin was isolated from a crude preparation of bovine pancreas with a recovery of 30% to 40%. Approximately 18 mg of trypsin was obtained from 500 mg of protein using a total of approximately 10 μg of ligand. Electrophoretic analysis of the eluent showed that chymotrypsin, which also binds to SBTI, was the only major contaminant of the product. It was demonstrated that the highest rates of protein purification were obtained using solid/liquid contact times well below that required to achieve saturation of the affinity adsorbent. Slower adsorbent recycle rates, which achieved higher protein binding per unit area of belt, resulted in lower protein purification per unit time. The rate of purification was also dependent on the concentration of target protein in the adsorption chamber at steady state. As high concentrations increased losses from the chamber outflow, this resulted in a compromise between throughput and recovery during the adsorption phase. Under the conditions investigated, recoveries of over 60% were obtained, and a maximum throughput of approximately 2.5 mg trypsin per hour was achieved. Preliminary studies have shown that this can be improved by compartmentalizing the adsorption chamber, which can reduce losses from the adsorption chamber to less than 5%. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 538-545, 1997.
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    Immobilization of invertase on sepharose-linked enzyme glycosyl recognizing polyclonal antibodies (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 605-609 
    Details
    ISSN: 0006-3592
    Keywords: affinity immobilization ; glycoenzymes ; thermal stability ; non-inhibitory antienzyme antibodies ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Polyclonal antibodies directed against the yeast invertase glycosyls were raised by immunizing rabbits with neoglycoprotein-I and neoglycoprotein-II. The neoglycoproteins were prepared by separately coupling the N-linked large and small molecular weight yeast invertase oligosaccharides respectively to bovine serum albumin with the help of glutaraldehyde. Antibodies specifically recognizing the invertase oligosaccharides were purified from the sera of rabbits immunized with either neoglycoprotein using an affinity column of sepharose 4B-linked yeast invertase. Specific immunoaffinity supports for the immobilization of invertase were constructed by coupling the affinity-purified antineoglycoprotein-I or antineoglycoprotein-II antibodies to cyanogen bromide activated sepharose-4B. Both the affinity adsorbants were effective in binding and improving the thermal stability of invertase. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 605-609, 1997.
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    Reduction of albumin adsorption onto silicon surfaces by Tween 20 (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 618-625 
    Details
    ISSN: 0006-3592
    Keywords: albumin ; silicon ; hydrophobicity ; adsorption ; Tween 20 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The ability of Tween 20 to reduce the adsorption of albumin on silicon surfaces of different hydrophobicity was investigated by ellipsometry. As expected, protein adsorption was found to depend on the degree of hydrophobicity of the surfaces and on the concentration of the surfactant. A reduction of 90% in albumin adsorption on hydrophobic methylated surfaces by 0.05% Tween 20 was achieved, whereas a reduction of only 15% on hydrophilic surfaces was observed. Experiments of time-dependent protein adsorption in both pure protein and protein-surfactant mixtures were conducted to ascertain the stability of physically adsorbed Tween 20 films on intermediate silicon surfaces. It was found that the adsorbed Tween 20 film was robust and there was no evidence of exchange of the Tween molecules with albumin for up to 240 min exposure. Adsorption minima were confirmed to correlate with minima in contact angle and critical micelle concentration (CMC). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 618-625, 1997.
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    Effect of substrate concentration and nitrate inhibition on product release and heavy metal removal by a Citrobacter sp. (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 821-830 
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    ISSN: 0006-3592
    Keywords: Citrobacter ; actinides ; nitrate ; biomineralization ; biocatalysis ; phosphatase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A Citrobacter sp. accumulates heavy metals as cell-bound metal phosphates, utilizing phosphate released by the enzymatic cleavage of a phosphomonoester substrate. The effect of increased substrate (glycerol 2-phosphate, G2P) concentration on phosphate release and heavy metal accumulation was evaluated using a stirred tank reactor (STR) and a plug flow reactor (PFR). A significant improvement in metal removal was achieved with increased substrate concentration using immobilized Citrobacter cells in the PFR, which was not observed using free cells in the STR. Nitrate is an inhibitor of the Citrobacter phosphatase. This inhibition was concentration dependent and reversible. The rate of product release was restored by increasing the concentration of substrate (G2P). The ratio of rates of phosphate release under two different conditions (different nitrate and G2P concentrations) can be described by a equation developed from Michaelis-Menten kinetics. The concentration of substrate required for restoration of maximum velocity, Vmax, in a batch and continuous-flow system can be predicted by substitution and calculation; this was confirmed by an experiment in model systems using cell suspensions and polyacrylamide gel immobilized cells in a flow-though column. For use in industrial situations it may be uneconomical or infeasible to supply additional substrate. Bioreactor activity was also restored by increasing the flow residence time, in accordance with a Michaelis-Menten-based model to describe removal of lanthanum from nitrate-supplemented flow in a PFR. © 1997 John Wiley & Sons, Inc. Biotechnol Biotechnol Bioeng 55:821-830, 1997.
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    Modeling isotopomer distributions in biochemical networks using isotopomer mapping matrices (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 831-840 
    Details
    ISSN: 0006-3592
    Keywords: isotopomer mapping matrix ; isotopomer modeling ; metabolic flux analysis ; 13C NMR ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Within the last decades NMR spectroscopy has undergone tremendous development and has become a powerful analytical tool for the investigation of intracellular flux distributions in biochemical networks using 13C-labeled substrates. Not only are the experiments much easier to conduct than experiments employing radioactive tracer elements, but NMR spectroscopy also provides additional information on the labeling pattern of the metabolites. Whereas the maximum amount of information obtainable with 14C-labeled substrates is the fractional enrichment in the individual carbon atom positions, NMR spectroscopy can also provide information on the degree of labeling at neighboring carbon atom positions by analyzing multiplet patterns in NMR spectra or using 2-dimensional NMR spectra. It is possible to quantify the mole fractions of molecules that show a specific labeling pattern, i.e., information of the isotopomer distribution in metabolite pools can be obtained. The isotopomer distribution is the maximum amount of information that in theory can be obtained from 13C-tracer studies. The wealth of information contained in NMR spectra frequently leads to overdetermined algebraic systems. Consequently, fluxes must be estimated by nonlinear least squares analysis, in which experimental labeling data is compared with simulated steady state isotopomer distributions. Hence, mathematical models are required to compute the steady state isotopomer distribution as a function of a given set of steady state fluxes. Because 2n possible labeling patterns exist in a molecule of n carbon atoms, and each pattern corresponds to a separate state in the isotopomer model, these models are inherently complex. Model complexity, so far, has restricted usage of isotopomer information to relatively small metabolic networks. A general methodology for the formulation of isotopomer models is described. The model complexity of isotopomer models is reduced to that of classical metabolic models by expressing the 2n isotopomer mass balances of a metabolite pool in a single matrix equation. Using this approach an isotopomer model has been implemented that describes label distribution in primary carbon metabolism, i.e., in a metabolic network including the Embden-Meyerhof-Parnas and pentose phosphate pathway, the tricarboxylic acid cycle, and selected anaplerotic reaction sequences. The model calculates the steady state label distribution in all metabolite pools as a function of the steady state fluxes and is applied to demonstrate the effect of selected anaplerotic fluxes on the labeling pattern of the pathway intermediates. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:831-840, 1997.
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    Very high-level production and export in Escherichia coli of a cellulose binding domain for use in a generic secretion-affinity fusion system (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 854-863 
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    ISSN: 0006-3592
    Keywords: cellulose binding protein ; high-density fermentations ; recombinant protein production ; affinity purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel expression vector pTugA, previously constructed in our laboratory, was modified to provide kanamycin resistance (pTugK) and used to direct the synthesis of polypeptides as fusions with the C- or N-terminus of a cellulose binding domain which serves as the affinity tag in a novel secretion-affinity fusion system. Fed-batch fermentation strategies were applied to production in recombinant E. coli TOPP5 of the cellulose binding domain (CBD) from the Cellulomonas fimi cellulase Cex. The pTugK expression vector, which codes for the Cex leader sequence that directs the recombinant protein to the periplasm of E. coli, was shown to remain stable at very high-cell densities. Recombinant cell densities in excess of 90 g (dry cell weight)/L were achieved using media and feed solutions optimized using a 2n factorial design. Optimization of inducer (isophenyl-thio-β-D-galactopyranoside) concentration and the time of induction led to soluble, fully active CBDCex production levels in excess of 8 g/L. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:854-863, 1997.
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    Lactic acid fermentation in a recycle batch reactor using immobilized Lactobacillus casei (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 841-853 
    Details
    ISSN: 0006-3592
    Keywords: immobilized cells ; lactic acid bacteria ; adhesive fermentation ; poly(ethyleneimine) ; recycle batch reactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lactic acid production by recycle batch fermentation using immobilized cells of Lactobacillus casei subsp. rhamnosus was studied. The culture medium was composed of whey treated with an endoprotease, and supplemented with 2.5 g/L of yeast extract and 0.18 mM Mn2+ ions. The fermentation set-up comprised of a column packed with polyethyleneimine-coated foam glass particles, Pora-bact A, and connected with recirculation to a stirred tank reactor vessel for pH control. The immobilization of L. casei was performed simply by circulating the culture medium inoculated with the organism over the beads. At this stage, a long lag period preceded the cell growth and lactic acid production. Subsequently, for recycle batch fermentations using the immobilized cells, the reducing sugar concentration of the medium was increased to 100 g/L by addition of glucose. The lactic acid production started immediately after onset of fermentation and the average reactor productivity during repeated cycles was about 4.3 to 4.6 g/L · h, with complete substrate utilization and more than 90% product yield. Sugar consumption and lactate yield were maintained at the same level with increase in medium volume up to at least 10 times that of the immobilized biocatalyst. The liberation of significant amounts of cells into the medium limited the number of fermentation cycles possible in a recycle batch mode. Use of lower yeast extract concentration reduced the amount of suspended biomass without significant change in productivity, thereby also increasing the number of fermentation cycles, and even maintained the D-lactate amount at low levels. The product was recovered from the clarified and decolorized broth by ion-exchange adsorption. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:841-853, 1997.
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    Metabolic and physiological studies of Corynebacterium glutamicum mutants (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 864-879 
    Details
    ISSN: 0006-3592
    Keywords: Corynebacterium glutamicum mutants ; transconjugation ; intracellular flux analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The physiology and central carbon metabolism of Corynebacterium glutamicum was investigated through the study of specific disruption mutants. Mutants deficient in phosphoenolpyruvate carboxylase (PPC) and/or pyruvate kinase (PK) activity were constructed by disrupting the corresponding gene(s) via transconjugation. Standard batch fermentations were carried out with these mutants and results were evaluated in the context of intracellular flux analysis. The following were determined. (a) There is a significant reduction in the glycolytic pathway flux in the pyruvate kinase deficient mutants during growth on glucose, also evidenced by secretion of dihydroxyacetone and glyceraldehyde. The resulting metabolic overflow is accommodated by the pentose phosphate pathway (PPP) acting as mechanism for dissimilating, in the form of CO2, large amounts of accumulated intermediates. (b) The high activity through the PPP causes an overproduction of reducing power in the form of NADPH. The overproduction of biosynthetic reducing power, as well as the shortage of NADPH produced via the tricarboxylic acid cycle (as evidenced by a reduced citrate synthase flux), are compensated by an increased activity of the transhydrogenase (THD) enzyme catalyzing the reaction NADPH + NAD+↔NADP+ + NADH. The presence of active THD was also confirmed directly by enzymatic assays. (c) Specific glucose uptake rates declined during the course of fermentation and this decline was more pronounced in the case of a double mutant strain deficient in both PPC and PK. Specific ATP consumption rates similarly declined during the course of the batch. However, they were approximately the same for all strains, indicating that energetic requirements for biosynthesis and maintenance are independent of the specific genetic background of a strain. The above results underline the importance of intracellular flux analysis, not only for producing a static set of intracellular flux estimates, but also for uncovering changes occurring in the course of a batch fermentation or as result of specific genetic modifications. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:864-879, 1997.
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    Adhesion and biofilm development on suspended carriers in airlift reactors: Hydrodynamic conditions versus surface characteristics (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 880-889 
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    ISSN: 0006-3592
    Keywords: biofilm ; airlift reactor ; adhesion ; detachment ; surface characteristics ; Pseudomonas putida ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Adhesion and biofilm formation by Pseudomonas putida was studied using suspended carriers in laboratory airlift reactors. Standard, roughened, hydrophobic, and positively charged glass beads, sand, and basalt grains were used as carriers. The results clearly show that in airlift reactors hydrodynamic conditions and particle collisions control biofilm formation. In the reactors, on surfaces subjected to different shear levels, biofilm formation differed considerably. This could be described by a simple growth and detachment model. Increased surface roughness promoted biofilm accumulation on suspended carriers. The physicochemical surface characteristics of the carrier surface proved to be less important due to the turbulent conditions in the airlift reactors. Adhesion of P. putida to glass beads was poor, and results of an adhesion test under quiescent conditions were not predictive for adhesion and subsequent biofilm formation under reactor conditions. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:880-889, 1997.
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    Production of recombinant human antithrombin III on 20-L bioreactor scale: Correlation of supernatant neuraminidase activity, desialylation, and decrease of biological activity of recombinant glycoprotein (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 441-448 
    Details
    ISSN: 0006-3592
    Keywords: Chinese hamster ovary (CHO) cells ; glycoprotein ; recombinant human antithrombin III (rhAT III) ; neuraminidase activity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Chinese hamster ovary (CHO) cells producing the recombinant glycoprotein human antithrombin III (rhAT III) were batch cultivated in a 20-L bioreactor for 13 days. Neuraminidase activity in cell-free supernatant was monitored during cultivation and free sialic acid was determined by HPLC. Neu5Acα(2→3)Gal-specific Maackia amurensis and Galβ(1→4)GlcNAc-specific Datura stramonium agglutinin were used for determination of sialylated and desialylated rhAT III, respectively. A commercial test kit was used for evaluation of functional rhAT III activity. Supernatant neuraminidase as well as lactate dehydrogenase activity increased significantly during batch growth. The enhanced number of dead cells correlated with increased neuraminidase activity, which seemed to be principally due to cell lysis, resulting in release of cytosolic neuraminidase. Loss of terminally α(2→3) linked sialic acids of the oligosaccharide portions of rhAT III, analyzed in lectin-based Western blot and lectin-adsorbent assays, correlated with a decrease of activity of rhAT III produced throughout long-term batch cultivation. Thus, structural oligosaccharide integrity as well as the functional activity of recombinant glycoprotein depend on the viability and mortality of the bioreactor culture, and batches with a high number of viable cells are required to guarantee production of glycoproteins with maximum biological activity. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 441-448, 1997.
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    DNA encapsulation by an air-agitated, liquid-liquid mixer (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 464-470 
    Details
    ISSN: 0006-3592
    Keywords: microencapsulation ; microcapsules ; microspheres ; alginate ; emulsification ; DNA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Smooth and spherical alginate microspheres and nylon-membrane bound microcapsules were formed in an air-agitated, liquid-liquid mixer by emulsification/internal gelation and interfacial polymerization respectively. The mean diameter of the alginate microspheres ranged from 100 to 800 μm, and was controlled by process modifications. Increase in emulsifier concentration, gas flowrate, and emulsification time resulted in smaller microsphere size as did a decrease in liquid height. Increase in the dispersed phase viscosity resulted in a longer emulsification time required for approaching a minimum microsphere size. Microspheres could be formed with the proportion of dispersed phase approaching 30%. The yield of alginate microspheres was 70%, with losses attributed to incomplete recovery during washing and filtration operations. The yield of DNA encapsulation within the fraction of recovered microspheres, was 94%. The small loss was thought to occur by surface release during the washing of the microspheres. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 464-470, 1997.
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    Comparative studies of Escherichia coli strains using different glucose uptake systems: Metabolism and energetics (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 56 (1997), S. 583-590 
    Details
    ISSN: 0006-3592
    Keywords: 31P NMR ; PTS mutant ; Escherichia coli ; metabolism ; energetics ; glucose uptake system ; galactose symport system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Modifying substrate uptake systems is a potentially powerful tool in metabolic engineering. This research investigates energetic and metabolic changes brought about by the genetic modification of the glucose uptake and phosphorylation system of Escherichia coli. The engineered strain PPA316, which lacks the E. coli phosphotransferase system (PTS) and uses instead the galactose-proton symport system for glucose uptake, exhibited significantly altered metabolic patterns relative to the parent strain PPA305 which retains PTS activity. Replacement of a PTS uptake system by the galactose-proton symport system is expected to lower the carbon flux to pyruvate in both aerobic and anaerobic cultivations. The extra energy cost in substrate uptake for the non-PTS strain PPA 316 had a greater effect on anaerobic specific growth rate, which was reduced by a factor of five relative to PPA 305, while PPA 316 reached a specific growth rate of 60% of that of the PTS strain under aerobic conditions. The maximal cell densities obtained with PPA 316 were approximately 8% higher than those of the PTS strain under aerobic conditions and 14% lower under anaerobic conditions. In vivo NMR results showed that the non-PTS strain possesses a dramatically different intracellular environment, as evidenced by lower levels of total sugar phosphate, NAD(H), nucleoside triphosphates and phosphoenolpyruvate, and higher levels of nucleoside diphosphates. The sugar phosphate compositions, as measured by extract NMR, were considerably different between these two strains. Data suggest that limitations in the rates of steps catalyzed by glucokinase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase, and pyruvate kinase may be responsible for the low overall rate of glucose metabolism in PPA316. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 583-590, 1997.
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    Copper binding and release by immobilized transferrin: A new approach to heavy metal removal and recovery (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 01-09 
    Details
    ISSN: 0006-3592
    Keywords: transferrin ; conalbumin ; metalloprotein ; affinity chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Recently these laboratories have demonstrated that it is possible to use proteins as efficient, selective agents for heavy metal removal and recovery. In this study, transferrin was chemically bound to an insoluble support. The ability of immobilized transferrin to produce clean water was demonstrated. Copper loading was independent of feed concentration. The loaded copper could be readily eluted and concentrated into the gram per liter range. The mechanism of copper release was studied. It was shown that release was dependent on pH and the chelating ability of the stripping agent. Metal release occurred slowly at pH 〈 7. However, at low pH in the presence of a chelator, metal removal occurred much more efficiently. The binding constant of copper to immobilized transferrin was determined as a function of pH. This information was used to model metal binding and release to the protein/support matrix. © 1997 John Wiley & Sons, Inc.
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    Fouling effects of yeast culture with antifoam agents on microfilters (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 10-16 
    Details
    ISSN: 0006-3592
    Keywords: microfiltration ; fouling ; yeast ; antifoam agents ; depressurization ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The fouling effects of yeast fermentation broths of Candida utilis in the presence of various commercial antifoam agents (PPG2000, B5600, and G832) up to 4.0 mL/L were studied, using Millipore polyvinylidene fluoride 0.22-μm hydrophilic membranes (GVWP), in a stirred-cell system at 50 kPa and 700 rpm. PPG2000, which has a low value of work of adhesion (Wa of 0.81 mN/m), gave a steady flux of broth of 29 L/(h m2) and was found to have no significant fouling effect on the microfiltration of broth. G832, which has a high Wa, (26.0 mN/m) reduced the flux of the broth to 17 L/(h m2); i.e., by 42% when only 1.0 mL/L was used. However, B5600, which has a Wa of 14.3 mN/m, was found to enhance the flux of broth to 54 L/(h m2); i.e., by 86%, due to the preferential adsorption of the B5600 components onto the hydrophobic cell contents released. These results were reinforced by the depressurization experiments performed with both hydrophilic (GVWP) and hydrophobic (GVHP) membranes, using both young and aged broths. B5600 was found to be the optimum antifoam agent in this study in terms of membrane performance and defoaming efficiency. © 1997 John Wiley & Sons, Inc.
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    Alginate oligosaccharides as enhancers of penicillin production in cultures of penicillium chrysogenum (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 17-20 
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    ISSN: 0006-3592
    Keywords: P. chrysogenum ; alginate oligosaccharides ; oligomannuronate ; oligoguluronate ; penicillin G ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Oligosaccharide fragments were prepared by partial acid hydrolysis of sodium alginate and consisted of oligomannuronate (OM) and oligoguluronate (OG) blocks. Effects of the OM and OG blocks on penicillin G production by P. chrysogenum were investigated. The oligosaccharides were found to cause significant increases in penicillin G yields. OM blocks at concentrations 10 to 100 μg/mL were used to further evaluate the effects of the oligosaccharides, and were found to enhance the production of penicillin G in shaken flask cultures of P. chrysogenum P2 (high penicillin producer) and NRRL 1951 (low penicillin producer) at the test concentrations. There was an approximately 50% maximum increase in penicillin G yield from biomass in P. chrysogenum P2 cultures and 150% in P. chrysogenum NRRL 1951 cultures, when compared to control cultures without the oligosaccharides. © 1997 John Wiley & Sons, Inc.
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    Effect of organic solvents on enantioselectivity of protease catalysis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 26-31 
    Details
    ISSN: 0006-3592
    Keywords: protease ; transesterification ; enantioselectivity ; organic solvent ; solvent effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The protease-catalyzed transesterifications between N-trifluoroacetyl-DL-phenylalanine 2,2,2-trifluoroethyl ester and 1-propanol were studied in a variety of anhydrous organic solvents at 30°C. The protease preparations lyophilized from phosphate buffer solutions (pH 8.0) were used as catalysts. The organic solvent affected both rate of reaction and enantioselectivity differently. Proteases such as Aspergillus oryzae protease, subtilisin Carlsberg, and subtilisin BPN′ always preferred the L-enantiomer in both hydrophilic and hydrophobic solvents, indicating no inversion of the L-specificity in hydrophobic solvents such as toluene. However, enantioselectivity was rather poor, with E (enantiomeric ratio) values not exceeding even one order of magnitude except for acetonitrile. There was a weak inverse correlation between E values of subtilisin Carlsberg and solvent hydrophobicity (logP). Acetonitrile was a preferable solvent in terms of both rate of reaction and enantioselectivity (E= 15 to 25) for processing L-amino acid derivatives in organic media. Organic solvents generally have potential advantages of processing D-amino acid derivatives. © 1997 John Wiley & Sons, Inc.
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    Alcohol fermentation of starch by a genetic recombinant yeast having glucoamylase activity (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 21-25 
    Details
    ISSN: 0006-3592
    Keywords: starch fermentation ; recombinant yeast ; ethanol production ; glucoamylase activity ; fed-batch culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Alcohol fermentation of starch was investigated using a direct starch fermenting yeast, Saccharomyces cerevisiae SR93, constructed by integrating a glucoamylase-producing gene (STA1) into the chromosome of Saccharomyces cerevisiae SH1089. The glucoamylase was constitutively produced by the recombinant yeast. The ethanol concentration produced by the recombinant yeast was 14.3 g/L which was about 1.5-fold higher than by the conventional mixed culture using an amylolytic microorganism and a fermenting microorganism. About 60% of the starch was converted into ethanol by the recombinant yeast, and the ethanol yield reached its maximum value of 0.48 at the initial starch concentration of 50 g/L. The fed-batch culture, which maintains the starch concentration in the range of 30 to 50 g/L, was used to produce a large amount of ethanol from starch. The amount of ethanol produced in the fed-batch culture increased about 20% compared to the batch culture. © 1997 John Wiley & Sons, Inc.
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    Stability of nicotinamide adenine dinucleotide immobilized to cyanogen bromide activated agarose (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 41-48 
    Details
    ISSN: 0006-3592
    Keywords: nicotinamide adenine dinucleotide ; isourea ; imidocarbonate ; agarose ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The stability of NAD(H) immobilized to a crosslinked agarose support (Sepharose®-4B) was examined in buffer solutions at a pH of 7.0 and 8.5. Specifically, this study investigated particle attrition and ligand leakage rates from a cyanogen bromide activated agarose support. Particle attrition did not occur under the experimental conditions. Ligand leakage rates were found to be first order in immobilized ligand concentration with two labile populations of ligand. The two-population model is consistent with the cyanogen bromide coupling chemistry, which results in both an isourea and imidocarbonate ligand linkage. The rate of ligand leakage was found to occur over a time scale of days, with first order rate constants ranging from 0.007 to 0.15 d-1, depending on solution pH. © 1997 John Wiley & Sons, Inc.
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    Design and study of peptide-ligand affinity chromatography adsorbents: Application to the case of trypsin purification from bovine pancreas (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 49-57 
    Details
    ISSN: 0006-3592
    Keywords: chromatography ; enzyme purification ; peptide immobilization ; peptide ligand ; trypsin purification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The purification of trypsin from bovine pancreas was employed in a case study concerning the design and optimization of peptide-ligand adsorbents for affinity chromatography. Four purpose-designed tripeptide-ligands were chemically synthesized (〉95% pure), exhibiting an Arg residue as their C-terminal (site P1) for trypsin bio-recognition, a Pro or Ala in site P2, and a Thr or Val in site P3. Each tripeptide-ligand was immobilized via its N-terminal amino group on Ultrogel A6R agarose gel, which was previously activated with low concentrations of cyanuric chloride (10.5 to 42.5 μmol/g gel). Well over 90% of the peptide used was immobilized. Three different concentrations were investigated for every immobilized tripeptide-ligand, 3.5, 7.0, and 14 μmol/g gel. The KD values of immobilized tripeptide-trypsin complexes were determined as well as the purifying performance and the trypsin-binding capacity of the affinity adsorbents. The KD values determined were in good agreement with the trypsin purification performance of the respective affinity adsorbents. The tripeptide sequence H-TPR-OH displayed the highest affinity for trypsin (KD 8.7 μM), whereas the sequence H-TAR-OH displayed the lowest (KD 38 μM). Dipeptide-ligands have failed to bind trypsin. When the ligand H-TPR-OH was immobilized via its N-terminal on agarose, at a concentration of 14 μmol/g gel, it produced the most effective affinity chromatography adsorbent. This adsorbent exhibited high trypsin-binding capacity (approximately 310,000 BAEE units/mL of adsorbent); furthermore, it purified trypsin from pancreatic crude extract to a specific activity of 15,200 BAEE units/mg (tenfold purification), and 82% yield. © 1997 John Wiley & Sons, Inc.
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    Performance of a sulfide-oxidizing expanded-bed reactor supplied with dissolved oxygen (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 32-40 
    Details
    ISSN: 0006-3592
    Keywords: expanded-bed reactor ; sulfur ; Thiobacilli ; immobilization ; biofilm ; sludge ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The performance of a new sulfide-oxidizing, expanded-bed bioreactor is described. To stimulate the formation of well-settleable sulfur sludge, which comprises active sulfide-oxidizing bacterial biomass and elemental sulfur, the aeration of the liquid phase and the oxidation of sulfide to elemental sulfur are spatially separated. The liquid phase is aerated in a vessel and subsequently recirculated to the sulfide-oxidizing bioreactor. In this manner, turbulencies due to aeration of the liquid phase in the bioreactor are avoided. It appeared that, under autotrophic conditions, almost all biomass present in the reactor will be immobilized within the sulfur sludge which consists mainly of elemental sulfur (92%) and biomass (2.5%). The particles formed have a diameter of up to 3 mm and can easily be grinded down. Within time, the sulfur sludge obtained excellent settling properties; e.g., after 50 days of operation, 90% of the sludge settles down at a velocity above 25 m h-1 while 10% of the sludge had a sedimentation velocity higher than 108 m h-1. Because the biomass is retained in the reactor, higher sulfide loading rates may be applied than to a conventional “free-cell” suspension. The maximum sulfide-loading rate reached was 14 g HS- L-1 d-1, whereas for a free-cell suspension a maximum loading rate of 6 g HS- L-1 d-1 was found. At higher loading rates, the upward velocities of the aerated suspension became too high so that sulfur sludge accumulated in the settling zone on top of the reactor. When the influent was supplemented with volatile fatty acids, heterotrophic sulfur and sulfate reducing bacteria, and possibly also (facultatively) heterotrophic Thiobacilli, accumulated within the sludge. This led to a serious deterioration of the system; i.e., the sulfur formed was increasingly reduced to sulfide, and also the formation rate of sulfur sludge declined. © 1997 John Wiley & Sons, Inc.
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    A Kalman filter algorithm and monitoring apparatus for at-line control of fractional protein precipitation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 58-70 
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    ISSN: 0006-3592
    Keywords: control ; monitoring ; fractional precipitation ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Downstream processing operations are often carried out blind in the process timescale since product monitoring on-line is not common. Knowledge of the location and concentration of the product and key contaminants is complementary to other process information for process development and, if available on-line in conjunction with a suitable model, control. This article sets out to demonstrate a model describing a two-cut fractional protein precipitation process and how this may be used for control of the process to maximize yield in the face of variable process stream conditions. Estimation of the model parameters is achieved by means of data-fitting by least squares and in comparison prediction by a Kalman filter algorithm. A description and error analysis of equipment for at-line monitoring of the soluble product in a pilot plant environment is presented which includes a micro-centrifuge necessary to clarify small volumes of sample prior to analysis. Finally, an account of the successful implementation of this equipment and the Kalman filter algorithm for control at bench scale is given where conditions in the process stream are deliberately disturbed to test the control operation. © 1997 John Wiley & Sons, Inc.
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    Effect of acetaldehyde on Saccharomyces cerevisiae and Zymomonas mobilis subjected to environmental shocks (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 71-78 
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    ISSN: 0006-3592
    Keywords: Zymomonas ; yeast ; acetaldehyde ; ethanol ; stress ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The lag phase of Saccharomyces cerevisiae subjected to a step increase in temperature or ethanol concentration was reduced by as much as 60% when acetaldehyde was added to the medium at concentrations less than 0.1 g/L. Maximum specific growth rates were also substantially increased. Even greater proportional reductions in lag time due to acetaldehyde addition were observed for ethanol-shocked cultures of Zymomonas mobilis. Acetaldehyde had no effect on S. cerevisiae cultures started from stationary phase inocula in the absence of environmental shock and its lag-reducing effects were greater in complex medium than in a defined synthetic medium. Acetaldehyde reacted strongly with the ingredients of complex culture media. It is proposed that the effect of added acetaldehyde may be to compensate for the inability of cells to maintain transmembrane acetaldehyde gradients following an environmental shock. © 1997 John Wiley & Sons, Inc.
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    Preparation of magnetically susceptible polyacrylamide/magnetite beads for use in magnetically stabilized fluidized bed chromatography (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 79-87 
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    ISSN: 0006-3592
    Keywords: polyacrylamide ; magnetic ; stabilized ; fluidized bed ; chromatography ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Spherical polyacrylamide/magnetite (PAM) composite beads, suitable for use in a magnetically stabilized fluidized bed (MSFB), were manufactured by a suspension polymerization method. Yield of beads depended on the type and concentration of buffer used during polymerization as well as the pH. More stabilizer was needed to prevent bead agglomeration as magnetite concentration increased. Bead diameter ranged from less than 60 to 600 μm, depending on reaction conditions, and the bead mean diameter and size distribution decreased with increasing impeller speed. The density and roundness factor of the beads were 1.19 ± 0.02 g cm-3 and 1.08 ± 0.03, respectively. The beads had high magnetization at a low applied magnetic field strength (60 mT at 75 kA m-1) and retained little residual magnetization (〈2 mT) after the field was removed. Incorporation of magnetite did not significantly affect the physical strength of the beads: the beads' average elastic modulus was 14 ± 4 kPa, similar to reported values for polyacrylamide gels (15.8 kPa). The beads were stable in a range of buffers from pH 1 to 10 and were resistant to microbial degradation. The fluidization and stabilization behavior of the beads was examined in a bench-scale MSFB. The minimum fluidization velocity (Umf) of the beads (0.035 mm s-1) allowed the MSFB to be operated at superficial velocities close to those used in HPLC systems. Against expectations, at high superficial velocities, the stabilized bed of the MSFB had a greater expansion than the unstabilized bed. The PAM beads could be derivatized and activated for soybean trypsin inhibitor immobilization by a standard carbodiimide method, and the affinity separation of trypsin from chymotrypsin was demonstrated. The PAM beads show excellent potential for use in MSFB chromatography. © 1997 John Wiley & Sons, Inc.
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    Abrasion of suspended biofilm pellets in airlift reactors: Importance of shape, structure, and particle concentrations (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 88-99 
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    ISSN: 0006-3592
    Keywords: biofilm structure ; detachment ; abrasion ; collisions ; airlift-reactor ; hydrodynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The detachment of biomass from suspended biofilm pellets in three-phase internal loop airlift reactors was investigated under nongrowth conditions and in the presence of bare carrier particles. In different sets of experiments, the concentrations of biofilm pellets and bare carrier particles were varied independently. Gas hold-up, bubble size, and general flow pattern were strongly influenced by changes in volume fractions of biofilm pellets and bare carrier particles. In spite of this, the rate of biomass detachment was found to be linear with both the concentration of biofilm pellets and the bare carrier concentration up to a solids hold-up of 30%. This implies that the detachment rate was dominated by collisions between biofilm pellets and bare carrier particles. These collisions caused an on-going abrasion of the biofilm pellets, leading to a reduction in pellet volume. Breakage of the biofilm pellets was negligible. The biofilm pellets were essentially ellipsoidal, which made three-dimensional size determination necessary. Calculating particle volumes from two-dimensional image analysis measurements and assuming a spherical shape led to serious errors. The abrasion rate was not equal on all sides of the biofilm pellets, resulting in an increasing flattening of the pellets. This flattening was oriented with the basalt carrier inside the biofilm and independent of the absolute abrasion rate. These observations suggest that the collisions causing abrasion are somehow oriented. The internal structure of the biofilms showed two layers, a cell-dense outer layer and an interior with a low biomass density. Taking this density gradient into account, the washout of detached biomass matched observed changes in volume of the biofilm pellets. No gradient in biofilm strength with biofilm depth was indicated. © 1997 John Wiley & Sons, Inc.
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    Nitrogen compounds in wine during its biological aging by two flor film yeasts: An approach to accelerated biological aging of dry sherry-type wines (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 159-167 
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    ISSN: 0006-3592
    Keywords: amino acid ; flor yeast ; L-proline ; urea ; wine aging ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Urea, ammonium, and free amino acid contents were quantified in biological aging of a young wine under two flor film forming yeast strains, Saccharomyces cerevisiae race capensis and S. cerevisiae race bayanus, and compared. Cell viability in the film was different for the two yeast strains. Thus, capensis maintained a much greater number of viable cells per surface area than bayanus and hence used greater amount of nitrogen compounds. The main source of nitrogen for the yeasts during the biological aging process was L-proline. The two yeast strains also differed in the amounts of assimilable nitrogen they utilized, in their preferences for amino acid consumption, and kinetics. To accelerate the aging process, the effect of controlled monthly aeration of the wine aged with capensis strain was investigated. The results revealed that short aeration did not appreciably increase the overall consumption of assimilable nitrogen, but consumption of some nitrogen compounds was accelerated (particularly L-proline, L-tryptophan, L-glutamic acid, ammonium ion, L-lysine, and L-arginine); the use of L-ornithine was inhibited; and GABA, L-methionine, and urea were depletes. Probably the aeration increases the aroma compounds, thereby producing wines with improved sensory properties. © 1997 John Wiley & Sons, Inc.
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    Influence of dissolved oxygen concentration on nitrite accumulation in a biofilm airlift suspension reactor (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 168-178 
    Details
    ISSN: 0006-3592
    Keywords: airlift reactor ; BAS reactor ; biofilm ; nitrification ; nitrite ; oxygen transfer ; residence time ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biofilm airlift suspension (BAS) reactor can treat wastewater at a high volumetric loading rate combined with a low sludge loading. Two BAS reactors were operated, with an ammonium load of 5 kg N/(m3 d), in order to study the influence of biomass and oxygen concentration on the nitrification process. After start-up the nitrifying biomass in the reactors gradually increased up to 30 g VSS/L. Due to this increased biomass concentration the gas-liquid mass transfer coefficient was negatively influenced. The resulting gradual decrease in dissolved oxygen concentration (over a 2-month period) was associated with a concomitantly nitrite build-up. Short term experiments showed a similar relation between dissolved oxygen concentration (DO) and nitrite accumulation. It was possible to obtain full ammonium conversion with approximately 50% nitrate and 50% nitrite in the effluent. The facts that (i) nitrite build up occurred only when DO dropped, (ii) the nitrite formation was stable over long periods, and (iii) fully depending on DO levels in short term experiments, led to the conclusion that it was not affected by microbial adaptations but associated with intrinsic characteristics of the microbial growth system. A simple biofilm model based on the often reported difference of oxygen affinity between ammonium and nitrite oxydizers was capable of adequately describing the phenomena.Measurements of biomass density and concentration are critical for the interpretation of the results, but highly sensitive to sampling procedures. Therefore we have developed an independent method, based on the residence time of Dextran Blue, to check the experimental methods. There was a good agreement between procedures.The relation between biomass concentration, oxygen mass transfer rate and nitrification in a BAS reactor is discussed. © 1997 John Wiley & Sons, Inc.
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    Production of micronic particles of biocompatible polymer using supercritical carbon dioxide (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 232-237 
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    ISSN: 0006-3592
    Keywords: supercritical fluid ; rapid expansion ; antisolvent ; biocompatible polymer ; drug delivery system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Three micronization techniques, based on the use of supercritical carbon dioxide, were investigated to produce microspheres of a natural biocompatible polysaccharide. Particles smaller than 20 μm were obtained by means of the rapid expansion of a supercritical solution method (RESS), both with and without cosolvents. The mean diameter of the particles was reduced to about 0.5 μm when a solution of the polymer in an organic solvent was expanded by using carbon dioxide as a supercritical antisolvent (SAS). The SAS process was operated both in a continuous and in a batch mode. The former leads to aggregated structures and fibers, and the latter to the formation of micronic spherical particles. It was found that the experimental temperature did not substantially affect the shape and dimension of the particles. A stronger dependence is shown with respect to the solute concentration in the starting solution. The proposed method is attractive as the basis of a new process for the preparation of drug delivery systems. © 1997 John Wiley & Sons, Inc.
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    Intrinsic kinetic parameters of substrate utilization by immobilized anaerobic sludge (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 220-225 
    Details
    ISSN: 0006-3592
    Keywords: anaerobic processes ; immobilized anaerobic sludge ; intrinsic kinetic parameters ; fixed-bed reactors ; horizontal-flow anaerobic immobilized sludge reactor ; polyurethane foam ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (robs) under a diversity of external mass transfer resistance conditions. The robs curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters rmax, the maximum specific substrate utilization rate, and Ks, the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30°C resulted in intrinsic rmax and Ks, respectively, of 0.330 mg chemical oxygen demand (COD) · mg-1 volatile suspended solids (VSS) · h-1 and 72 mg COD · L-1. In comparison with the values found in the literature, intrinsic rmax is significantly high and intrinsic Ks is relatively low. © 1997 John Wiley & Sons, Inc.
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    Determination of carbon dioxide evolution rate using on-line gas analysis during dynamic biodegradation experiments (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 243-252 
    Details
    ISSN: 0006-3592
    Keywords: carbon dioxide evolution rate ; mass transfer ; modeling ; biodegradation ; pH ; kinetics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Respirometry is a precious tool for determining the activity of microbial populations. The measurement of oxygen uptake rate is commonly used but cannot be applied in anoxic or anaerobic conditions or for insoluble substrate. Carbon dioxide production can be measured accurately by gas balance techniques, especially with an on-line infrared analyzer. Unfortunately, in dynamic systems, and hence in the case of short-term batch experiments, chemical and physical transfer limitations for carbon dioxide can be sufficient to make the observed carbon dioxide evolution rate (OCER) deduced from direct gas analysis very different from the biological carbon dioxide evolution rate (CER).To take these transfer phenomena into account and calculate the real CER, a mathematical model based on mass balance equations is proposed. In this work, the chemical equilibrium involving carbon dioxide and the measured pH evolution of the liquid medium are considered. The mass transfer from the liquid to the gas phase is described, and the response time of the analysis system is evaluated.Global mass transfer coefficients (KLa) for carbon dioxide and oxygen are determined and compared to one another, improving the choice of hydrodynamic hypotheses. The equations presented are found to give good predictions of the disturbance of gaseous responses during pH changes.Finally, the mathematical model developed associated with a laboratory-scale reactor, is used successfully to determine the CER in nonstationary conditions, during batch experiments performed with microorganisms coming from an activated sludge system. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 243-252, 1997.
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    Identification of linearity in the biofilm process and its operational utility (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 253-258 
    Details
    ISSN: 0006-3592
    Keywords: biofilm ; deep biofilm reactor (DBFR) ; kinetics ; linearity ; operational control ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Various reported field studies on the performance of biofilm reactors suggest that the linear control of the system is effective for maintaining the consistent treatment efficiency under changing environmental conditions. However, no theoretical basis is available in the literature to substantiate such a claim. In this article, inherent linearity of the biofilm process has been identified along with the conditions under which this linearity exists. Exploiting the linear state of the system, operational criteria for regulating the performance of the biofilm reactors are obtained. The utility and applicability of the developed criteria are numerically demonstrated. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 253-258, 1997.
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    Performance of a styrene-degrading biofilter containing the yeast Exophiala jeanselmei (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 259-266 
    Details
    ISSN: 0006-3592
    Keywords: waste gas ; styrene ; fungi ; biofilter performance ; biofilm ; Exophiala jeanselmei ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A general mathematical model developed for a description of pollutant degradation in a biofilm was used to evaluate the performance of a biofilter for the purification of styrene-containing gas. The biofilter contained perlite as an inert support on which a biofilm was present composed of a mixed microbial population containing the fungus Exophiala jeanselmei as a major styrene-degrading microorganism. Although styrene is a moderately hydrophobic compound, the biofilter was reaction limited at a styrene gas phase concentration of 0.1-2.4 g/m3. Limitation of biofilter performance by the mass transfer of styrene was only observed at styrene concentrations lower than 0.06 g/m3. A maximal styrene degradation rate of 62 g/(m3 · h) was maintaind for over 1 year. At a high styrene concentration, the maximal styrene degradation rate could be increased to 91 g/(m3 · h) by increasing the oxygen concentration in the gas from 20 to 40%. After 300 days of operation, the dry-weight biomass concentration of the filter bed was 41% (w/w), and an average biofilm thickness of 240-280 μm, but maximal up to 600 μm, was observed. Experimental results and model calculations indicated an effective biofilm thickness of about 80 μm. It is postulated that the thickness of the effective biofilm is determined by the oxygen availability in the biofilm. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 259-266, 1997.
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    Development of a new reversed micelle liquid emulsion membrane for protein extraction (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 267-273 
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    ISSN: 0006-3592
    Keywords: liquid emulsion membrane ; reversed micelles ; protein extraction ; α-chymotrypsin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new type of liquid emulsion membrane containing reversed micelles for protein extraction is introduced. A three-step extraction mechanism is proposed including solubilization, transportation, and release of the protein. The surfactants Span80 and sodium di(2-ethylhexyl)sulfosuccinate (AOT) are used to stabilize the membrane phase and to build up the reversed micelles, respectively. α-Chymotrypsin was used as the model protein. The condition in the internal phase inhibits the solubilization process of the already extracted protein back into reversed micelles. Concerning the solubilization, we studied the influence of the AOT concentration in the membrane phase and the ionic strength in the external phase. The extraction rate increases with higher AOT concentration and decreases with higher ionic strength. Using NaCl in the external phase led to better extraction results than using KCl. Maximum extraction results of 98% into the membrane phase and 65% into the internal phase were obtained. This condition retained 60% of the enzyme's activity. The concentration of KCl in the internal phase does not affect the solubilization rate but the release into the internal phase. By this way the ionic strength in the internal phase is used as the driving force for the protein release. The solubilization process is much faster than the diffusion and the releasing process, as found by variation of the extraction time. The influence of the operating conditions on the membrane swelling is also discussed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 267-273, 1997.
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    Monte Carlo simulation of the enzymatic lysis of yeast (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 290-295 
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    ISSN: 0006-3592
    Keywords: yeast lysis ; Monte Carlo simulation ; cell rupture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The overall reaction in the enzymatic lysis of yeast takes place in three major steps: (i) the two-layer wall is digested, (ii) the cell bursts under the osmotic pressure difference to release its intracellular material, and (iii) the intracellular material is digested by the enzymes still present in the solution. The first and third steps are continuous processes, adequately described by Michaelis-Menten kinetic models. The second step is a discrete event, statistical in nature. A model of engineering value should effectively bridge the gap between the two continuous processes (first and third steps). In this work, Monte Carlo simulations are used to identify a suitable function that captures the statistical nature of cell rupture and represents the rate of release of intracellular material. It is shown that the two-parameter beta distribution function serves this purpose most effectively. Comparisons with experimental results indicate that the cell rupture ratio is a widely distributed statistical function. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 290-295, 1997.
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    Microbial ecosystems in compost and granular activated carbon biofilters (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 296-303 
    Details
    ISSN: 0006-3592
    Keywords: biofilters ; microbial ecocystems ; compost ; granular activated carbon ; phospholipid fatty acid analysis ; POTW ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Compost and granular activated carbon biofilters operated at a wastewater treatment plant simultaneously removed low concentrations of hydrogen sulfide and volatile organic compounds. Through the use of phospholipid fatty acid analyses, the effects of declining pH caused by sulfide oxidation were established for microbial growth, microorganism stress, and microbial community structure. Microorganisms on both media demonstrated increases in microbial densities, varying degrees of environmental stress, and domination by gram-negative bacteria. However, the declining pH had little effect on compound removal, which was greater than 99% for the hydrogen sulfide and greater than 70% for the oxygenated and aromatic hydrocarbons. The microbial communities adjusted to difficult environmental conditions through acclimation of the species present or by growth of low-pH-tolerant species. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 296-303, 1997.
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    Processing and secretion of insulin-related peptides in an insulinoma cell line (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 283-289 
    Details
    ISSN: 0006-3592
    Keywords: regulated secretion ; insulin processing ; insulin secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Certain classes of prohormones and other neuroendocrine or endocrine-derived secretory proteins are post-translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine-derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine-derived cell line βTC-3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T-flask experiments with confluent cultures of βTC-3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversed-phase high-performance liquid chromatography (HPLC). Under conditions of steady-state secretion in glucose-rich growth medium the cells turned over their (pro)insulin inventory (90 ± 5% mature insulin) at 2-3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 μM) and isobutylmethylxanthine (1 mM), ∼80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 ± 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady-state rate with a treatment which attenuated calcium influx (20 μM verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only ∼50% converted to mature insulin, but 85 ± 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long-term and predominantly mature stores. Thus, a cyclic controlled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady-state secretion. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 283-289, 1997.
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    Analysis of secretory dynamics and development of media for the controlled secretion of insulin-related peptides from βTC-3 insulinoma cells (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 274-282 
    Details
    ISSN: 0006-3592
    Keywords: insulinoma cells ; regulated secretion ; insulin secretion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Controlled secretion of proteins from endocrine-derived cell lines has been proposed as a means to produce some classes of post-translationally modified proteins in bioreactors. Under the right biological and environmental conditions it may be possible to improve the product purity or quality relative to that obtained through steady (constitutive) secretion. The pancreatic-islet-derived cell line, βTC-3, was selected as a model system to explore the secretory dynamics of insulin under various combinations of stimulatory or inhibitory environmental conditions. The βTC-3 cells exhibited a glucose-mediated stimulus-response pattern which was saturated above 1 mM glucose and with an apparent “Kg” of 0.1 mM glucose. However, the kinetics of insulin synthesis were closely coupled to those of secretion such that βTC-3 cells cycled between saturating and basal levels of glucose were never perturbed far from an intracellular synthesis-secretion equilibrium. When more powerful and selective agents were used to control secretion, the system performance improved markedly. A combination of 1 mM isobytylmethylxanthine (IBMX) and 1 μM carbachol (with saturating levels of glucose) could discharge 75% of stored insulin in 2 h. When this treatment was followed by incubation in media adjusted to attenuate the influx of calcium into the cells, intracellular pools were efficiently replenished within 24 h. Calcium attenuating treatments included hyperpolarization with reduced potassium (1 mM), calcium channel blockade with the dihydropyridine verapamil (1 μM), and the direct mass-action effect of reduced environmental calcium (0.5 mM versus 1.8 mM). Other inhibitory treatments were explored, but these tended to reduce both insulin synthesis and secretion. The best recharging treatment found was a combination of verapamil (1 μM) with reduced calcium level (0.5 mM).To demonstrate the feasibility of a controlled secretion process, βTC-3 T-flask cultures were grown to confluence, then cycled through two periods of discharging (2 h) and recharging (20 h) with the best combinations of secretagogues and calcium attenuators. The overall process was quite efficient: Only 15% of the overall insulin secretion took place during the recharging episodes, and this residual secretion represented only 10% of the net insulin synthesis during these episodes. Discharging was very effective in the first episode (80% recovery of stored insulin), but slightly less efficient in subsequent discharging episodes, possibly due to a desensitization effect of the calcium attenuating media. Nevertheless, the regulated secretory pathway of βTC-3 cells could be successfully harnessed to a controlled secretion process for the selective recovery of stored insulin. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 274-282, 1997.
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    A structured model for Thiobacillus ferrooxidans growth on ferrous iron (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 310-319 
    Details
    ISSN: 0006-3592
    Keywords: T. ferrooxidans ; iron oxidation ; energetics ; model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A structured model for Thiobacillus ferrooxidans growth dependence on ferrous and ferric iron, arsenic, oxygen, carbon dioxide, pH, and temperature is presented. A new kinetic mechanism for ferrous oxidation by T. ferrooxidans is introduced. Data from several earlier experimental studies of T. ferroaxidans growth are used for model development. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 310-319, 1997.
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    Production and characterization of a plant α-hydroxynitrile lyase in Escherichia coli (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 332-338 
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    ISSN: 0006-3592
    Keywords: α-hydroxynitrile lyase ; cassava ; cyanogenesis ; cyanohydrin ; Escherichia coli expression vector ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The coding sequence of the cyanogenic α-hydroxynitrile lyase gene of Manihot esculenta Crantz (cassava) was cloned in the plasmid vector pMal-c2 and expressed in Escherichia coli strain JM105. DNA sequencing showed that the recombinant plasmid contained the same sequence as the cDNA clone pHNL10. Peptide sequencing of the recombinant protein showed that the N-terminus was heterogeneous, with either four or six additional amino acid residues compared with the native protein. Circular dichroism spectra indicated similar secondary structure contents for both proteins. Enzyme assays showed that specific activity of native and recombinant proteins were 0.24 and 0.26 mmol CN-/mg/min, respectively; that both proteins had optimal activity at 40°C and pH 5.5; and that both proteins were inhibited by the serine protease inhibitor phenyl-methane sulfonyl flouride (PMSF). Isoelectric focusing of native and recombinant protein revealed multiple isoforms for both proteins; the recombinant protein had a more basic mean isoelectric point (pl) (5.1) than the native protein (4.5). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 332-338, 1997.
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    Numerical modeling and uncertainties in rate coefficients for methane utilization and TCE cometabolism by a methane-oxidizing mixed culture (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 320-331 
    Details
    ISSN: 0006-3592
    Keywords: numerical modeling ; uncertainty ; statistics ; cometabolism ; trichloroethylene ; methanotroph ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The rates of methane utilization and trichloroethylene (TCE) cometabolism by a methanotrophic mixed culture were characterized in batch and pseudo-steady-state studies. Procedures for determination of the rate coefficients and their uncertainties by fitting a numerical model to experimental data are described. The model consisted of a system of differential equations for the rates of Monod kinetics, cell growth on methane and inactivation due to TCE transformation product toxicity, gas/liquid mass transfer of methane and TCE, and the rate of passive losses of TCE. The maximum specific rate of methane utilization (kCH4) was determined by fitting the numerical model to batch experimental data, with the initial concentration of active methane-oxidizing cells (X0a) also used as a model fitting parameter. The best estimate of kCH4 was 2.2 g CH4/g cells-d with excess copper available, with a single-parameter 95% confidence interval of 2.0-2.4 mg/mg-d. The joint 95% confidence region for kCH4 and X0a is presented graphically. The half-velocity coefficient (KS,CH4) was 0.07 mg CH4/L with excess copper available and 0.47 mg CH4/L under copper limitation, with 95% confidence intervals of 0.02-0.11 and 0.35-0.59 mg/L, respectively. Unique values of the TCE rate coefficients kTCE and KS,TCE could not be determined because they were found to be highly correlated in the model fitting analysis. However, the ratio kTCE/KS,TCE and the TCE transformation capacity (TC) were well defined, with values of 0.35 L/mg-day and 0.21 g TCE/g active cells, respectively, for cells transforming TCE in the absence of methane or supplemental formate. The single-parameter 95% confidence intervals for kTCE/KS,TCE and TC were 0.27-0.43 L/mg-d and 0.18-0.24 g TCE/g active cells, respectively. The joint 95% confidence regions for kTCE/KS,TCE and TC are presented graphically. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 320-331, 1997.
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    Regulation of growth and adhesion of cultured cells by insulin conjugated with thermoresponsive polymers (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 339-344 
    Details
    ISSN: 0006-3592
    Keywords: cell culture ; tissue engineering ; thermoresponsive polymer ; cell adhesion ; insulin conjugate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We developed a new biomaterial for use in cell culture. The biomaterial enabled protein-free cell culture and the recovery of viable cells by lowering the temperature without the aid of supplements. Insulin was immobilized and a thermoresponsive polymer was grafted onto a substrate. We investigated the effect of insulin coupling on the lower critical solution temperature (LCST) of the thermoresponsive polymer, poly(N-isopropylacrylamide-co-acrylic acid), using polymers that were ungrafted, or coupled with insulin. The insulin conjugates were precipitated from an aqueous solution at high temperatures, but they were soluble at low temperatures. The LCST was not significantly affected by the insulin coupling. The thermoresponsive polymer was grafted to glow-discharged polystyrene film and covalently conjugated with insulin. The surface wettability of the conjugate film was high at low temperatures and low at high temperatures. The amounts of immobilized insulin required to stimulate cell growth were 1-10% of the amount of free insulin required to produce the same effect. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. About half of the viable cells was detached from the film only by lowering the temperature. The recovered cells proliferated normally on new culture dishes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 339-344, 1997.
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    Phosphate uptake kinetics by Acinetobacter isolates (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 304-309 
    Details
    ISSN: 0006-3592
    Keywords: biological phosphorus removal ; activated sludge ; phosphate uptake kinetics ; overplus phenomenon ; luxury uptake ; Acinetobacter ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Acinetobacter isolates from activated sludge treatment plants of forest industry were used as model organisms for polyphosphate accumulating bacteria to study excess phosphate uptake by the overplus phenomenon as well as luxury uptake of phosphate during growth. The initial, rapid phosphate uptake by the phosphorus-starved Acinetobacter isolates (the overplus phenomenon) followed the Michaelis-Menten model (maximum initial phosphate uptake rate 29 mg P g-1 dry mass (DM) h-1, half-saturation constant for excess phosphate uptake 17 mg P L-1). During the rapid uptake no growth was observed, but most cells contained polyphosphate granules. Also growth and luxury uptake of phosphate could be modeled with the Michaelis-Menten equation (maximum phosphate uptake rate 3.7-12 mg P g-1 DM h-1, half-saturation constant for growth 0.47-6.0 mg P L-1, maximum specific growth rate 0.15-0.55 h-1). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 304-309, 1997.
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    On the pH memory of lyophilized compounds containing protein functional groups (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 345-348 
    Details
    ISSN: 0006-3592
    Keywords: fourier transform infrared (FTIR) spectroscopy ; lyophilization ; pKa ; protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Lyophilized proteins exhibit “pH memory,” i.e., their behavior in the solid form corresponds to the pH of the aqueous solution from which they were freeze dried. Herein, we directly tested whether the ionization state is “remembered” by model organic compounds containing various protein functional groups (amino, carboxyl, and phenolic). The fraction of ionized species was quantitated from the infrared spectra of both the aqueous and lyophilized states. The pKa values in the aqueous and lyophilized forms for each compound were found to be quite similar, within 0.3 units from each other. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 345-348, 1997.
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    Substrate utilization and mass transfer in an autotrophic biofilm system: Experimental results and numerical simulation (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 363-371 
    Details
    ISSN: 0006-3592
    Keywords: biofilm ; autotrophic bacteria ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: An autotrophic biofilm has been investigated for over 10 months in a biofilm tube reactor. The objective of this investigation was the verification and improvement of a biofilm model. The use of a Clark-type oxygen microelectrode in situ allowed the determination of the substrate flux in the biofilm. Also, the population dynamics of the autotrophic bacteria could be evaluated by varying the substrate conditions. Simulation of the experimental results showed that the liquid phase of the biofilm decreased with biofilm depth. This could be described by a logistic function. The density of the inert volume fraction was found to be higher than that of the viable bacteria. This was verified in a nonsubstrate phase of 5 weeks. Growth and decay of the autotrophic bacteria could be described by the growth, endogenous respiration, and death processes. Mass transfer coefficients at the bulk/biofilm interface were evaluated. They were found to be one order of magnitude higher than those known from hydrodynamics in tubes without a biofilm. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 363-371, 1997.
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    Can conformational changes be responsible for solvent and excipient effects on the catalytic behavior of subtilisin Carlsberg in organic solvents? (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 351-362 
    Details
    ISSN: 0006-3592
    Keywords: Fourier-transform infrared (FTIR) ; excipients ; lyophilization ; organic solvents ; subtilisin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We developed an FTIR (Fourier transform infrared) methodology for quantitatively assessing the secondary structure of proteins suspended in nonaqueous media. This methodology was used to measure the percentages of α-helices and β-sheets of subtilisin Carlsberg, prepared under different conditions, placed in various organic solvents. The title question was addressed with respect to some instances of markedly influencing the subtilisin activity in organic solvents reported in the literature. It is concluded that the mechanism of subtilisin activation by KCl and N-Ac-L-Phe-NH2 present in the aqueous solution of the enzyme prior to lyophilization may be due to their preservation of the secondary structure, otherwise altered by the dehydration. Likewise, subtilisin inactivation in the protein-dissolving solvent DMSO (dimethyl sulfoxide) is likely caused by enzyme denaturation (the loss of both α-helices and β-sheets). On the other hand, some other ligands, as well as protein nondissolving organic solvents, while greatly affecting the subtilisin activity, have little effect on its secondary structure, thus ruling out the causal relationship between the two. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 351-362, 1997.
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    Enzymatic assay of cholesterol by reaction rate measurements (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 391-396 
    Details
    ISSN: 0006-3592
    Keywords: assay ; cholesterol ; cholesterol oxidase ; free and total cholesterol ; cholesterol esterase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A dynamic method for free and total cholesterol assay based on the oxidation of cholesterol by cholesterol oxidase, and conversion of cholesteryl oleate to cholesterol by cholesterol esterase is discussed in this article. The reaction conditions for total cholesterol assay were a temperature of 310 K and pH of 7.4. For conversion of cholesteryl oleate to cholesterol, the samples were incubated with 0.6 unit/mL of cholesterol esterase and 0.02 g/mL of taurocholate. The determination of initial reaction rates in the oxidation of free cholesterol, which is directly related to the cholesterol concentration, was found to be rapid, reliable, and inexpensive. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 391-396, 1997.
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    Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 372-378 
    Details
    ISSN: 0006-3592
    Keywords: glucose ; lactate ; on-line monitoring ; mammalian cell culture ; fermentation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: On-line monitoring and control of cell culture fermentation is important for optimal and consistent production of biologicals. In this work, glucose and lactate concentrations are monitored on-line using a commercially available analyzer (Model 2700, Yellow Springs Instruments, Yellow Springs, OH) during batch and perfusion hybridoma cell fermentation. Cell free samples from the reactor are obtained using a 0.45 μm hollow fiber filtering system placed in a circulation loop. The samples were analyzed at specified times and the data are collected on a computer. A process control strategy was developed to control the concentrations of glucose and lactate in a perfusion reactor where the feed rate is adjusted to maintain their concentrations at desired set points. Hybridoma cells (A10G10) were cultivated in a high density perfusion culture where cell density increased from 2 to 14 million cells/mL. During this period the control algorithm successfully adjusted the perfusion rate while maintaining constant glucose and lactate concentrations. Glucose consumption and lactate accumulation rates as well as net lactate yield on glucose were monitored continuously during perfusion culture. These metabolic rates were observed to be independent of cell concentration and were used for the estimation of viable cell density in the reactor. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 372-378, 1997.
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    Peptide condensation activity of a neutral protease from Vibrio sp. T1800 (Vimelysin) (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 387-390 
    Details
    ISSN: 0006-3592
    Keywords: enzymatic synthesis ; peptide synthesis ; Vibrio ; protease ; thermolysin ; organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Condensation of Cbz-Asp and PheOMe catalyzed by a neutral protease from Vibrio sp. T1800 (Vimelysin: VLN) was studied. VLN showed a relatively higher catalytic activity of condensation and an apparently larger yield after 3 h or 24 h, in comparison with thermolysin (TLN), especially at lower pH and temperatures.VLN showed higher solvent-tolerance than TLN. TLhe apparent highest yield (25%) was obtained in 30% DMSO by using VLN; under similar conditions, TLN gave only about a half of this value. The rate of the condensation reaction per mole of enzyme (v/[E]o) in DMSO 50% at 37°C and pH 6.5 was 0.16 s-1 for VLN and 0.047 s-1 for TLN. In 30% ethanol VLN showed more than three-fold peptide yield than TLN after 5 h reaction. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 387-390, 1997.
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    Improvement in recombinant protein production in ppGpp-deficient Escherichia coli (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 379-386 
    Details
    ISSN: 0006-3592
    Keywords: ppGpp ; recombinant protein synthesis ; translational machinery ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Maintaining a metabolically productive state for recombinant Escherichia coli remains a central problem for a wide variety of growth-dependent biosynthesis. This problem becomes particularly acute under conditions of minimal cell growth such as fed-batch fermentations. In this, we investigated the possibility of manipulating the protein synthesis machinery of E. coli whereby synthesis of foreign proteins might be decoupled from cell growth. In particular, the effects of eliminating intracellular ppGpp on the synthesis of foreign proteins were studied in both batch and fed-batch operations. A significant increase in CAT production was observed from the ppGpp-deficient strain during both exponential and fed-batch phases. The increase in CAT production during exponential growth was accompanied by a simultaneous increase in CAT mRNA levels. Interestingly, CAT production was increased five-fold, while the level of CAT-specific mRNA increased only three-fold. Thus, eliminating intracellular ppGpp appears to have increase the production of recombinant protein by increasing not only the pool sizes of CAT mRNA but also possible alternations in the post-transcriptional processes. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 379-386, 1997.
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    Cell-substratum adhesion strength as a determinant of hepatocyte aggregate morphology (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 415-426 
    Details
    ISSN: 0006-3592
    Keywords: hepatocytes ; cell adhesion ; spheroids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cultured hepatocytes typically form multicellular aggregates which are either monolayered or spheroidal in morphology. We propose that the aggregate morphology resulting from a particular cell-substratum interaction has a biophysical basis: when cell contractile forces are greater than cell-substratum adhesion forces, spheroidal aggregates form; when cell contractile forces are weaker than cell-substratum adhesion forces, cells remain essentially spread and form monolayered aggregates. We tested this hypothesis by systematically varying the morphology of hepatocellular aggregates formed on substrata coated with a series of different concentrations of Matrigel, and correlating aggregate morphology with the cell-substratum adhesion strength measured in a shear flow detachment assay. Aggregate morphology was binary - spheroidal aggregates formed at low Matrigel concentrations and monolayered aggregates formed at high Matrigel concentrations. Cell-substratum adhesion strength was similarly binary, with low adhesion strengths correlated with spheroidal aggregates and high adhesion strengths correlated with formation of monolayered aggregates. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 415-426, 1997.
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    Acetylation of pea isolate in a torus microreactor (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 409-414 
    Details
    ISSN: 0006-3592
    Keywords: acylation ; pea isolate ; plant protein ; torus bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Acetylation, which acts on the amino groups of proteins, allows to increase the solubility and the emulsifying properties of pea isolate. Acetylation by acetic anhydride was carried out in a torus microreactor in semibatch and continuous conditions. The mixing characteristics, obtained by a residence time distribution (RTD) method, are the same in batch and continuous processes. The maximum acetylation degree reached by the torus reactor is higher than with the stirred reactor. Torus reactors are more efficient than stirred ones as shown by a conversion efficiency, defined by the quantity of modified lysine groups by consumed acetic anhydride. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 409-414, 1997.
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    Bioaffinity separation of trypsin using trypsin inhibitor immobilized in reverse micelles composed of a nonionic surfactant (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 406-408 
    Details
    ISSN: 0006-3592
    Keywords: bioaffinity separation ; reverse micelles ; trypsin-trypsin inhibitor ; nonionic surfactant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Trypsin inhibitor was converted to hydrophobic states by covalently combining cholesteryl groups using an acylation reaction, and was immobilized in reverse micelles composed of a nonionic surfactant. Using this reverse micellar phase containing trypsin inhibitor as an affinity ligand, trypsin was selectively separated with high recoveries from a mixture of several kinds of contaminating proteins by forward and backward extraction. No loss of activity of the recovered trypsin was observed through these operations. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 406-408, 1997.
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    Control of heterotrophic layer formation on nitrifying biofilms in a biofilm airlift suspension reactor (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 397-405 
    Details
    ISSN: 0006-3592
    Keywords: airlift reactor ; biofilm ; biofilm detachment ; control biofilm formation ; heterotrophic layer ; hydraulic retention time ; nitrification ; oxygen diffusion limitation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A Biofilm Airlift Suspension (BAS) reactor was operated with nitrifying biofilm growth and heterotrophic suspended growth, simultaneously converting ammonium and acetate. Growth of heterotrophs in suspension decreases the diffusion limitation for the nitrifiers, and enlarges the nitrifying capacity of a biofilm reactor. Neither nitrifiers nor heterotrophs suffer from additional oxygen diffusion limitation when the heterotrophs grow in suspension. Control of the location of heterotrophic growth, either in suspension or in biofilms over the nitrifying biofilms, was possible by manipulation of the hydraulic retention time. A time delay for formation and disappearance of the heterotrophic biofilms of 10 to 15 days was observed. Surprisingly, it was found that in the presence of the heterotrophic layers the maximum specific activity on ammonia of the nitrifying biofilms increased. The reason for the increase in activity is unknown. The effect of heterotrophic biofilm formation on oxygen diffusion limitation for the nitrifiers is discussed. Some phenomena compensating the increased mass transfer resistance due to the growth of a heterotrophic layer are also presented. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 397-405, 1997.
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    A new approach to continuous counter-current protein chromatography: Direct purification of malate dehydrogenase from a Saccharomyces cerevisiae homogenate as a model system (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 427-441 
    Details
    ISSN: 0006-3592
    Keywords: malate dehydrogenase ; protein chromatography ; Saccharomyces cerevisiae ; direct extraction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A novel technique for protein chromatography has been developed, which can be used to extract proteins from particulate-containing solutions (such as fermentation broths or preparations of disrupted cells) on a continuous basis, and delivers clarified streams of purified product. Adsorbents deployed in this type of contactor are based on PVA-coated perfluorocarbons derivitized with affinity ligands such as triazine dyes. In this article, we describe the application of this equipment for the continuous purification of malate dehydrogenase from an unclarified homogenate of Saccharomyces cerevisiae, using a Procion Red HE-7B-derivitized adsorbent. Although operating conditions were not optimized to produce a product of maximized purification factor, concentration, and yield, we have shown that MDH can be purified continuously in 78% yield at a rate of 70 U/min, with a purification factor of approximately 10. This corresponds to specific productivity of approximately 0.35 U/min per milliliter of settled adsorbent, a higher specific productivity than was feasible with the same adsorbent using expanded bed adsorption (EBA). © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 427-441, 1997.
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    Kinetics and modeling of GM-CSF production by recombinant yeast in a three-phase fluidized bed bioreactor (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 470-477 
    Details
    ISSN: 0006-3592
    Keywords: fluidized bed bioreactor ; recombinant ; yeast ; kinetics ; modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Continuous production of a recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) by Saccharomyces cerevisiae strain XV2181 (a/a, Trp 1) containing plasmid pαADH2 and immobilized on porous glass beads in a fluidized bed bioreactor was studied. Kinetic models for plasmid stability, cell growth, and protein production in the three-phase fluidized bed bioreactor were developed and used to study the effects of solid loading or cell immobilization on plasmid stability and recombinant protein production. With increasing cell immobilization or solid loading in the bioreactor, plasmid stability and protein production improved significantly. The improvements could be attributed to the decreased θ value, which is the plasmid loss probability during cell division and is an indication of segregational instability of the recombinant cell, and the increased α value, which is the ratio of the specific growth rate of a plasmid-carrying cell to that of a plasmid-free cell and is indicative of competitive stability of the recombinant cell culture. θ decreased from 0.552 to 0.042 and α increased from 0.351 to 0.991 when solid loading in the bioreactor was increased from 5% (v/v) to 33%. The model simulation also showed that the specific growth rate of cells in the bioreactor was lower at higher solid loading. This indicated that there was significant mass transfer limitation, particularly for oxygen transfer, when the total cell density in the bioreactor was high at high solid loading. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 470-477, 1997.
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    Chemical treatment of Escherichia coli: 1. Extraction of intracellular protein from uninduced cells (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 453-458 
    Details
    ISSN: 0006-3592
    Keywords: chemical permeabilization ; cell disruption ; urea ; EDTA ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Extraction of intracellular protein from Escherichia coli is traditionally achieved by mechanical disruption. A chemical treatment that destroys the integrity of the bacterial cell wall and could provide an alternative technique is examined in this study. Treatment with a combination of the chelating agent ethylenediaminetet-raacetate (EDTA) (greater than 0.3 mM) and the chaotropic agent urea (6 M) is highly effective at releasing protein from uninduced E. coli. The 6 M urea in the presence of 3 mM EDTA can release cytoplasmic protein from both logarithmic-phase and stationary-phase E. coli cells at levels equivalent to mechanical disruption. The concentrations of the two chemical agents were the major variables affecting the maximum levels of protein release. Several minor variables and interactions were also identified. The kinetics of protein release is first order. For 2, 4, and 6 M urea with 3 mM EDTA, the time constant is approximately 2.5 min independent of urea concentration. Kinetics for 3 mM EDTA without urea is considerably slower, with a time constant of 12.3 min. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 453-458, 1997.
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    Fermentation database mining by pattern recognition (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 443-452 
    Details
    ISSN: 0006-3592
    Keywords: fermentation ; database mining ; pattern recognition ; dbminer© ; decision trees ; wavelets ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A large volume of data is routinely collected during the course of typical fermentation and other processes. Such data provide the required basis for process documentation and occasionally are also used for process analysis and improvement. The information density of these data is often low, and automatic condensing, analysis, and interpretation (“database mining”) are highly desirable. In this article we present a methodology whereby process variables are processed to create a database of derivative process quantities representative of the global patterns, intermediate trends, and local characteristics of the process. A powerful search algorithm subsequently attempts to extract the specific process variables and their particular attributes that uniquely characterize a class of process outcomes such as high- or low-yield fermentations.The basic components of our pattern recognition methodology are described along with applications to the analysis of two sets of data from industrial fermentations. Results indicate that truly discriminating variables do exist in typical fermentation data and they can be useful in identifying the causes or symptoms of different process outcomes. The methodology has been implemented in a user-friendly software, named db-miner, which facilitates the application of the methodology for efficient and speedy analysis of fermentation process data. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 443-452, 1997.
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    A kinetic model for biological oxidation of ferrous iron by Thiobacillus ferrooxidans (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 478-486 
    Details
    ISSN: 0006-3592
    Keywords: Thiobacillus ferrooxidans ; kinetic model ; biological oxidation ; ferrous iron ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The kinetics of bacterial oxidation of ferrous iron in the presence of Thiobacillus ferrooxidans cells were studied using an initial-rate method. Measurements of the redox potential of the solution during the oxidation of ferrous iron were used to assess the initial rate of the reaction. Effects on the rate of reaction were determined for ferrous iron concentration in the range 0.25 to 30 kg m-3, bacterial concentration in the range 3.25 × 107 to 4.47 × 108 cells mL-1, and temperature in the range 20 to 35°C. Using these experimental results and an approach based on Michaelis-Menten kinetics, a model for biological oxidation of ferrous iron was developed. The model, which incorporates terms for the effect of temperature and substrate and cell inhibition, was successfully used to simulate the full range of experimental data obtained. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 478-486, 1997.
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    Uptake and release of inert fluorescence particles by mixed population biofilms (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 459-469 
    Details
    ISSN: 0006-3592
    Keywords: population dynamics ; biofilms ; fluorescent microbeads ; confocal scanning laser microscope (CSLM) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Inert fluorescent microparticles were used as tracers to investigate the dynamics of spatial distribution of particulate components in mixed population biofilms. The tracer bead spatial distributions in the biofilm were experimentally measured by sectioning the biofilms with a microslicer. The experimental results were compared with model simulations using the biofilm model (BIOSIM) to evaluate the assumption that advective transport (displacement) of particulates balances with cell growth in the model. The tracer beads could traverse throughout a biofilm 360 μm thick within less than 23 minutes, which cannot be explained solely by their attachment to the surface followed by molecular diffusion. Advective transport of the tracer beads via “voids and pores” could be responsible for such rapid bead penetration. Observation by confocal scanning laser microscopy (CSLM) clearly showed that the biofilm consisted of a thick loose surface layer, varying in thickness, and a semicontiguous base layer separated by water channels. About 80% of attached tracer beads remained in the biofilm for over 20 days. The trapped tracer beads were gradually transferred from the depth of the biofilm to the surface. The observed bead release rate was much slower than the model predictions. This is probably because the cell density increased predominantly near the substratum, resulting in an unbalance of advective transport of the tracer beads and cell growth. The pores, voids, and cell-free spaces in the biofilm were first filled with growing biomass, thereafter, displacement of the beads took place once the cell density reached certain levels. The model assumptions of the temporal and spatial constant cell density and the continuum concept (flat biomass) are clearly oversimplified and should be revised. It was concluded that the dynamics of the inert microbeads in the biofilm was strongly influenced by not only microbial growth, but also by the biofilm structure and growth pattern. Therefore, one dimensional modeling is not adequate for the accurate description of the transport of particulates in a biofilm. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 459-469, 1997.
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    Optimization by experimental design of polyacrylamide gel composition as support for enzyme immobilization by entrapment (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 497-506 
    Details
    ISSN: 0006-3592
    Keywords: immobilized enzymes ; polyacrylamide gels ; experimental design ; response surface methodology ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have developed a methodology based on experimental design, to optimize a polyacrylamide gel as the support for enzyme immobilization, taking advantage of all the properties which this type of gel has. Monomer and crosslinking agent proportions are responsible for both the porous structure and pore size of the gel. A correct selection of those variables and suitable synthesis conditions leads to an increase in the activity retained by the gel. The path of steepest ascent method was used to obtain the relative maximum activity. The maximum retained activity was chosen with a central composite design in terms of the gel composition. The retained activity in the network, loss activity in the wash water, and loss activity due to steric impediment or blockage was modeled in terms of the variables responsible for the gel structure. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 497-506, 1997.
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    Mathematical model for characterization of bacterial migration through sand cores (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 487-496 
    Details
    ISSN: 0006-3592
    Keywords: bacterial chemotaxis ; Escherichia coli ; random motility ; mathematical model ; sand core ; porous media ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The migration of chemotactic bacteria in liquid media has previously been characterized in terms of two fundamental transport coefficients - the random motility coefficient and the chemotactic sensitivity coefficient. For modeling migration in porous media, we have shown that these coefficients which appear in macroscopic balance equations can be replaced by effective values that reflect the impact of the porous media on the swimming behavior of individual bacteria. Explicit relationships between values of the coefficients in porous and liquid media were derived. This type of quantitative analysis of bacterial migration is necessary for predicting bacterial population distributions in subsurface environments for applications such as in situ bioremediation in which bacteria respond chemotactically to the pollutants that they degrade.We analyzed bacterial penetration times through sand columns from two different experimental studies reported in the literature within the context of our mathematical model to evaluate the effective transport coefficients. Our results indicated that the presence of the porous medium reduced the random motility of the bacterial population by a factor comparable to the theoretical prediction. We were unable to determine the effect of the porous medium on the chemotactic sensitivity coefficient because no chemotactic response was observed in the experimental studies. However, the mathematical model was instrumental in developing a plausible explanation for why no chemotactic response was observed. The chemical gradients may have been too shallow over most of the sand core to elicit a measurable response. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 487-496, 1997.
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    Study of stability of recombinant plasmids during the continuous culture of Bacillus stearothermophilus NUB3621 in nonselective medium (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 507-514 
    Details
    ISSN: 0006-3592
    Keywords: Bacillus stearothermophilus ; continuous culture ; plasmid stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The optimal culture conditions for Bacillus stearothermophilus NUB3621 (BGSC 9A5) in chemostat were studied. The results obtained showed that the optimal culture conditions in terms of biomass concentration and maximum growth rate were 65°C, pH 6.8 to 7.2. Dissolved oxygen became growth limiting at pO2 levels below 10%. Furthermore, this strain was transformed with three new hybrid vectors (pPAM2, pPCH2, or pPLY2) constructed by cloning in pRP9, a plasmid based on the thermophilic replicon, pBC1, and three heterologous genes: the α-amylase gene from Bacillus licheniformis, the cholesterol oxidase gene from Streptomyces sp., and the lipase gene from Pseudomonas fluorescens. The influence of several fermentative conditions on segregational and structural stability of the recombinant B. stearothermophilus NUB3621 transformants was studied.The parameters of plasmid loss, that is, rate of plasmid loss (R) and specific growth rate difference (δμ), were calculated. B. stearothermophilus NUB3621 carrying pRP9 showed great segregational stability in all the assayed conditions, exceeding more than 300 generations without significant plasmid loss, whereas NUB3621 carrying pPAM2, pPCH2, or pPLY2 exhibited relatively low plasmid stability. The segregational instability of the recombinant constructs increased by increasing the fermentation temperature, decreased by increasing the dilution rate, and was not affected by the level of dissolved oxygen. On the other hand, plasmid maintenance decreased in minimal medium if compared with the results obtained in complex medium. Restriction analyses carried out on cultures of NUB3621 carrying pRP9, pPAM2, pPCH2, or pPLY2, grown for 200 generations on nonselective media, revealed that all the clones tested contained the parental plasmids. These results indicate that the heterologous inserts did not affect the structural stability of the recombinant plasmids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 507-514, 1997.
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    Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a prospective consortium and its most effective isolate Serratia marcescens (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 515-522 
    Details
    ISSN: 0006-3592
    Keywords: RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine ; Serratia marcescens ; biotransformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The biotransformation of hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) has been observed in liquid culture by a consortium of bacteria found in horse manure. Five types of bacteria were found to predominate in the consortium and were isolated. The most effective of these isolates at transforming RDX was Serratia marcescens. The biotransformation of RDX by all of these bacteria was found to occur only in the anoxic stationary phase. The process of bacterial growth and RDX biotransformation was quantified for the purpose of developing a predictive type model. Cell growth was assumed to follow Monod kinetics. All of the aerobic and anoxic growth parameters were determined: μmax, Ks, and Yx/s. RDX was found to competitively inhibit cell growth in both atmospheres. Degradation of RDX by Serratia marcescens was found to proceed through the stepwise reduction of the three nitro groups to nitroso groups. Each of these reductions was found to be first order in both component and cell concentrations. The degradation rate constant for the first step in this reduction process by the consortium was 0.022 L/g cells · h compared to 0.033 L/g cells · h for the most efficient isolate. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 515-522, 1997.
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    Capture of microbial cells on brush-type polymeric materials bearing different functional groups (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 523-528 
    Details
    ISSN: 0006-3592
    Keywords: radiation-induced graft polymerization ; microbial cell capture ; tertiary amino group ; coexisting functional group ; capturing rate constant ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A brush-type microbial-cell-capturing polymeric material was prepared by radiation-induced grafting of an epoxy-group-containing monomer, glycidyl-methacrylate (GMA), onto a polyethylene-based fiber. The epoxy ring (EO) of GMA was opened with different degrees of introduction of diethylamine (DEA). The residual epoxy group was hydrophilized by ethanolamine (EA). The prepared DEA membranes with coexisting EO or EA groups were tested for their ability to capture Staphylococcus aureus and Escherichia coli cells. The DEA membrane (2.7 mol/kg of product of DEA group density) with coexisting EO groups (DEA-EO membrane) exhibited good S. aureus-cell-capturing ability with a capturing rate constant of 1.82 × 10-6 m/s, whereas the DEA membrane with coexisting EA groups (DEA-EA membrane) retarded capturing abilities for both S. aureus and E. coli cells. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 523-528, 1997.
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    Toluene degradation kinetics for planktonic and biofilm-grown cells of Pseudomonas putida 54G (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 535-546 
    Details
    ISSN: 0006-3592
    Keywords: toluene ; biofilm kinetics ; planktonic cell kinetics ; specific activity ; injury ; Pseudomonas putida 54G ; vapor phase bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Toluene degradation kinetics by biofilm and planktonic cells of Pseudomonas putida 54G were compared in this study. Batch degradation of 14C toluene was used to evaluate kinetic parameters for planktonic cells. The kinetic parameters determined for toluene degradation were: specific growth rate, μmax = 10.08 ± 1.2/day; half-saturation constant, KS = 3.98 ± 1.28 mg/L; substrate inhibition constant, KI = 42.78 ± 3.87 mg/L. Biofilm cells, grown on ceramic rings in vapor phase bioreactors, were removed and suspended in batch cultures to calculate 14C toluene degradation rates. Specific activities measured for planktonic and biofilm cells were similar based on toluene degrading cells and total biomass. Long-term toluene exposure reduced specific activities that were based on total biomass for both biofilm and planktonic cells. These results suggest that long-term toluene exposure caused a large portion of the biomass to become inactive, even though the biofilm was not substrate limited. Conversely, specific activities based on numbers of toluene-culturable cells were comparable for both biofilm and planktonically grown cultures. Planktonic cell kinetics are often used in bioreactor models to model substrate degradation and growth of bacteria in biofilms, a procedure we found to be appropriate for this organism. For superior bioreactor design, however, changes in cellular activity that occur during biofilm development should be investigated under conditions relevant to reactor operation before predictive models for bioreactor systems are developed. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 535-546, 1997.
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    Effects of rheological change by addition of carboxymethylcellulose in culture media of an air-lift fermentor on poly-D-3-hydroxybutyric acid productivity in autotrophic culture of hydrogen-oxidizing bacterium, Alcaligenes eutrophus (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 529-533 
    Details
    ISSN: 0006-3592
    Keywords: polyhydroxyalkanoate (PHA) ; Alcaligenes eutrophus ; autotrophic culture ; air-lift fermentor ; carboxy-methylcellulose (CMC) ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effects of rheological change by addition of sodium carboxymethylcellulose (CMC) to culture medium in an air-lift-type fermentor on autotrophic production of poly-(D-3-hydroxybutyric acid) [P(3HB)] by two-stage culture of Alcaligenes eutrophus is investigated. Addition of 0.05% CMC increased P(3HB) production rate during the P(3HB) accumulation phase to twice that of the control culture. It was thought that addition of a small amount of CMC was beneficial for production of P(3HB) employing the air-lift fermentor under safe autotrophic culture conditions in wich oxygen concentration was maintained below 6.9% (v/v). the volumetric mass transfer coefficient (KLa) observed in the presence of CMC is shown to correlated with the P(3HB) production rate obtained. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 529-533, 1997.
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