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  • Biochemistry and Biotechnology  (164)
  • 2020-2024
  • 1995-1999  (164)
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  • 1
    Electronic Resource
    Electronic Resource
    Conservative chemical modification of proteins to study the effects of a single protein property on partitioning in aqueous two-phase systems (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 290-299 
    Details
    ISSN: 0006-3592
    Keywords: proteins, modified ; partitioning in aqueous system ; thaumatin ; β-lactoglobulin ; BSA ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Relatively conservative modifications of three proteins were carried out to alter their surface properties. The protein properties modified were hydrophobicity and charge. This was done by acylation of amino groups with anhydrides. For the hydrophobic modification experiments, two proteins (β-lactoglobulin and bovine serum albumin [BSA]) and four anhydrides (hexanoic, butyric, succinic, acetic) were used. For the modification of surface charge the protein thaumatin was selected and various proportions of the free amino groups were blocked with acetic anhydride to give a series of proteins with differing isoelectric points. Detailed characterization and purification of selected modified proteins was carried out including molecular weight measurements and conformational analysis. The criteria used for selecting the modified proteins for subsequent investigation of their partitioning in aqueous two-phase systems (ATPS) is described. With a judicious choice of starting material it was found that limited chemical modifications to proteins could effectively alter surface hydrophobicity or charge almost independently, with little effect on other molecular properties. It appears, however, that the method for chemical modification and the reaction conditions must also be carefully controlled. © 1996 John Wiley & Sons, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface charge (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 309-315 
    Details
    ISSN: 0006-3592
    Keywords: surface charge ; proteins, modified ; partitioning in aqueous system ; thaumatin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A series of charge-modified thaumatins with different values of surface charge were partitioned in aqueous two-phase systems (ATPS) to study the effect of surface charge as a single property on partitioning. Electrophoretic mobility of the proteins in titration curves was used as a measure of surface charge. Four modified proteins derived from thaumatin with the following values of isoelectric point: 8.70, 8.15, 5.60, and 4.50 were used for partitioning. The resolution of the systems in terms of protein surface charge was calculated. Partitioning of modified thaumatins in PEG 4000/dextran systems with phosphate buffer, Tris buffer, NaCl, KCl, and sulfate salts was carried out. Among the sulfate salts tested, the addition of 50 mM Li2SO4 to the system buffered with phosphate gave the highest value of resolution for differences in surface protein charge (RSPC). It shows a decrease in the value of K (partition coefficient) with an increase in the protein's charge. The addition of 100 mM KCl to the system promoted the opposite effect on the RSPC value. Charge-modified proteins were partitioned in PEG/salt systems to investigate the ability of these systems for resolving differences in surface charge. The PEG/citrate system seemed to have almost no ability for resolving proteins on the basis of surface charge differences; PEG/phosphate systems had some capability for resolving differently charged proteins. The more negative proteins tended to have higher values of K than the more positively charged fractions. The use of charge-modified proteins allowed the investigation of the effect of protein surface charge on partitioning in aqueous two-phase systems independently from other protein parameters as they were prepared from a common parent protein thaumatin. This technique provides an interesting novel tool to investigate the effect of protein surface charge on partitioning in ATPS taking protein charge as an independent parameter. © 1996 John Wiley & Sons, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface hydrophobicity (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 49 (1996), S. 300-308 
    Details
    ISSN: 0006-3592
    Keywords: hydrophobicity ; proteins, modified ; partitioning in aqueous system ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Two different series of hydrophobically modified proteins were partitioned in a number of aqueous two-phase systems (ATPS) to investigate the effect of hydrophobicity as a single property on partitioning. The modified proteins were derived from β-lactoglobulin and bovine serum albumin (BSA). Measurement of the surface hydrophobicity of the proteins is important; hydrophobic interaction chromatography (HIC) was used for this purpose. The resolution of the systems (R) in terms of protein surface hydrophobicity and the intrinsic hydrophobicity (log P0) of the systems was established. The effect of the addition of NaCl to PEG/phosphate and PEG/dextran systems was analyzed in terms of the hydrophobicity difference between the phases and their ability to promote hydrophobic interactions between the protein surface and the PEG molecules. The values for R and log P0 differed somewhat depending on which group of modified proteins was used for partitioning. The addition of NaCl to PEG/phosphate systems promoted an increase in the values of R, showing an important effect on the resolution of the systems for protein surface hydrophobicity (twice as high when compared with systems without NaCl). For PEG/dextran systems, the addition of 9% NaCl (w/w) promoted an improvement in the resolution toward surface hydrophobicity with an increase of 60% on the value of R. © 1996 John Wiley & Sons, Inc.
    Additional Material: 6 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    Thermodynamic and kinetic parameters of lipase-catalyzed ester hydrolysis in biphasic systems with varying organic solvents (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 179-189 
    Details
    ISSN: 0006-3592
    Keywords: biphasic system ; activity coefficient ; organic solvents ; solvation ; hydrolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Kinetics of lipase-catalyzed hydrolysis of esters were modeled using reactant activities for aqueous-organic, biphasic systems. By using thermodynamic activities of the substrates in ordinary rate equations, the kinetic parameters were corrected for the contribution of substrate-solvent interactions and a uniform quantification of the substrates for lipase attached to the interface can be achieved. The kinetic parameters, on the basis of their thermodynamic activities, should be constant in different systems, provided that the solvents do not interfere with the binding of the substrates to the enzyme nor affect the catalytic mechanism. Experimental and computational methods on how to obtain the thermodynamic activities of the substrates are presented. Initial rates were determined for Pseudomonas cepacia lipase (PcL)-catalyzed hydrolysis of decyl chloroacetate in dynamic emulsions with various solvents. The thermodynamic equilibrium and corrected kinetic constants for this reaction appeared to be similar in various systems. The kinetics of PcL in an isooctane-aqueous biphasic system could be adequately described with the rate equation for a ping-pong mechanism. The observed inhibitory effect of decanol appeared to be a consequence of this mechanism, allowing the backreaction of the decanol with the chloroacetyl-enzyme complex. The kinetic performance of PcL in systems with toluene, dibutyl ether, and methyl isobutyl ketone could be less well described. The possible causes for this and for the remaining differences in corrected kinetic parameters are discussed. © 1995 John Wiley & Sons, Inc.
    Additional Material: 5 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260480303
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  • 5
    Electronic Resource
    Electronic Resource
    Analysis of cell-to-bubble attachment in sparged bioreactors in the presence of cell-protecting additives (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 407-419 
    Details
    ISSN: 0006-3592
    Keywords: cell-to-bubble attachment ; cell damamge ; cell protecting additives ; interfacial properties ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: To investigate the mechanisms of cell protection provided by medium additives against animal cell injury in sparged bioreactors, we have analyzed the effect of various additives on the cell-to-bubble attachment process using CHO cells in suspension. Cell-to-bubble attachment was examined using three experimental techniques: (1) cell-bubble induction time analysis (cell-to-bubble attachment times); (2) forming thin liquid films and observing the movement and location of cells in the thin films; and (3) foam flotation experiments. The induction times we measured for the various additives are as follows: no additive (50 to 500 ms), polyvinyl pyrrolidone (PVP: 20 to 500 ms), polyethylene glycol (PEG: 200 to 1000 ms), 3% serum (500 to 1000 ms), polyvinyl alcohol (PVA: 2 to 10 s), Pluronic F68 (5 to 20 s), and Methocel (20 to 60 s). In the thin film formation experiments, cells in medium with either F68, PVA, or Methocel quickly flowed out of draining thin liquid films and entered the plateau border. When using media with no additive or with serum, the flow of cells out of the thin liquid film and film drainage were slower than for media containing Pluronic F68. PVA, or Methocel. With PVP and PEG, the thin film drainage was much slower and cells remained trapped in the film. For the foam flotation experiments, a separation factor (ratio of cell concentration in the foam catch to that in the bubble column) was determined for the various additives. In the order of increasing separation factors (i.e., increasing cell attachment to bubbles), the additives are as follows: Methocel, PVA, Pluronic F68, 3% serum, serum-free medium with no additives, PEG, and PVP. Based on the results of these three different cell-to-bubble attachment experiments, we have classified the cell-protecting additives into three groups: (1) Pluronic F68, PVA, and Methocel (reduced cell-to-bubble attachment); (2) PEG and PVP (high or increased cell-to-bubble attachment); and (3) FBS (reduced cell attachment butslower drainage films compared with F68, PVA, and Methocel with some cell entrapment in those films). These phenomena are discussed in relation to the interfacial properties of the media reported in a companion Study (this issue). © 1995 John Wiley & Sons Inc.
    Additional Material: 8 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260470402
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  • 6
    Electronic Resource
    Electronic Resource
    Interfacial properties of cell culture media with cell-protecting additives (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 420-430 
    Details
    ISSN: 0006-3592
    Keywords: cell damamge ; shear protectant ; surface tension ; rheological interfacial properties ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In an effort to identify key rheological properties that contribute to cell protection against shear damage, we have measured surface shear and dilatationai viscosities, dynamic surface tension, foaminess, and foam stability for media containing cell-protecting additives. In a companion article,18 we found that cell-to-bubble attachment was decreased in media containing Methocel, Pluronic F68, or polyvinyl alcohol (PVA). In medium containing polyethylene glycol (PEG) or potyvinyl-pyrrolidone (PVP), attachment was increased. PEG, PVP, serum (FBS), and serum albumin (BSA) increased the surface viscosity of the air/medium surface (thus, producing a more rigid interface), whereas F68 and PVA lowered it greatly. Foaming experiments showed that Methocel, PEG, PVA, and F68 decreased the foam half-life while FBS, BSA, and PVP were foam stabilizers. Interestingly, the foam stability of CHO cell suspensions decreased significantly for cell concentrations higher than ca. 2 × 106 cells/mL. Nonviable CHO cells reduced foam stability further. Dynamic surface tension values of the media tested were found significantly differentfrom their static surface tension values. The interfacial properties measured and the results presented in the companion study suggest that the additives that lower dynamic surface tension the most (Methocel, F68, and PVA) correlate well with reduced cell-to-bubble attachment, and thus, cell protection. Reduced dynamic surface tension with these additives implies faster surfactant adsorption, mobile interfaces, lower surface viscosity, and foam destabilization. Because PEG and PVP resulted in increased cell-to-bubble attachment and had different interfacial properties, a different mechanism (compared with Methocel, PVP, and F68) is apparently responsible for their protective effect. Finally, cell protection offered by FBS and BSA is attributed to the foam stabilization properties provided by these additives. © 1995 John Wiley & Sons Inc.
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    URL: http://dx.doi.org/10.1002/bit.260470403
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  • 7
    Electronic Resource
    Electronic Resource
    In situ microscopy for on-line characterization of cell-populations in bioreactors, including cell-concentration measurements by depth from focus (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 47 (1995), S. 106-116 
    Details
    ISSN: 0006-3592
    Keywords: in situ microscopy ; on-line biomass determination ; cell concentration ; depth from focus ; image analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A new technique is presented which allows the use of a front-end sensor head for in situ and on-line characterization of cell concentration and cell size during fermentation. An epifluorescence microscope is mounted in a port of a bioreactor viewing directly into the agitated broth. Still images from cells are generated using pulsed illumination. They are directly visualized on a monitor and used for automatic image analysis. The cell concentration and morphological information are determined by counting and evaluating the cell images with respect to their depth from focus characteristic. An in situ microscope was successfully tested during yeast fermentations and yielded results which correlated well with results from a hemocytometer. © 1995 John Wiley & Sons, Inc.
    Additional Material: 10 Ill.
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    URL: http://dx.doi.org/10.1002/bit.260470113
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  • 8
    Electronic Resource
    Electronic Resource
    Effects of methocel A15LV, polyethylene glycol, and polyvinyl alcohol on CD13 and CD33 receptor surface content and metabolism of HL60 cells cultured in stirred tank bioreactors (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 251-258 
    Details
    ISSN: 0006-3592
    Keywords: HL60 cells ; CD13 ; CD33 ; media additives ; hydrodynamic effects ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Flow cytometry was used to examine the effect of hydrodynamic forces in a stirred tank bioreactor on the CD13 and CD33 receptor surface content of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 400 rpm reduced the HL60 cell apparent growth rate and increased the CD13 receptor surface content per cell, on average, by 95%. In contrast, this step increase in agitation rate to 400 rpm decreased the CD33 receptor surface content per cell, on average, by 10%. The protective effects of 0.1% Methocel A15LV, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) on CD13 and CD33 receptor surface content were examined under agitation at 300 rpm in parallel 2 L bioreactor runs. The average CD33 receptor surface content was unaffected by the presence of Methocel A15LV or PEG, while PVA had a slight protective effect. In contrast, in terms of CD13 receptor content, HL60 cells agitated at 300 rpm with Methocel A15LV, PEG, or PVA behaved like cells agitated at 80 rpm with no media additives (McDowell and Papoutsakis, 1998). That is, Methocel A15LV, PEG, and PVA prevented the transduction of mechanical forces which affect CD13 cell content. HL60 cells cultured with 0.1% A15LV, PEG or PVA under conditions of mild agitation (60 rpm) in spinner flasks exhibited glucose consumption and lactate production rates that were approximately 20% lower than values of cultures containing no additive. Under conditions of agitation at 300 rpm in the 2 L bioreactor, the presence of A15LV, PEG, and PVA reduced the HL60 glucose consumption and lactate production rates by approximately 50%. Thus, media additives can dramatically reduce lactate accumulation in agitated bioreactors due to cell growth, in addition to providing protection from cellular injury. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 251-258, 1998.
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  • 9
    Electronic Resource
    Electronic Resource
    CHO DUKX cell lineages preadapted to growth in serum-free suspension culture enable rapid development of cell culture processes for the manufacture of recombinant proteins (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 518-528 
    Details
    ISSN: 0006-3592
    Keywords: CHO cells ; serum-free medium ; adaptation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. © 1996 John Wiley & Sons, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    Tissue engineering lamb heart valve leaflets (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 562-567 
    Details
    ISSN: 0006-3592
    Keywords: tissue engineering ; synthetic biodegradable matrix ; polyglycolic acid ; polylactic acid ; endothelial cell ; heart valve ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Tissue engineered lamb heart valve leaflets (N - 3) were constructed by repeatedly seeding a concentrated suspension of autologous myofibroblasts onto a biodegradable synthetic polymeric scaffold composed of fibers made from polyglycolic acid and polylactic acid. Over a 2-week period the cells attached to the polymer fibers, multiplied, and formed a tissue core in the shape of the matrix. The tissue core was seeded with autologous large-vessel endothelial cells that formed a monolayer which coated the outer surface of the leaflet. The tissue engineered leaflets were surgically implanted in place of the right posterior pulmonary valve leaflet of the donor lamb while on cardiopulmonary bypass. Pulmonary valve function was evaluated by two-dimensional echocardiography with color Doppler which demonstrated valve function without evidence of stenosis and with only trivial regurgitation under normal physiologic conditions. Histologically, the tissue engineered heart valve leaflets resembled native valve leaflet tissue. © 1996 John Wiley & Sons, Inc.
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