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  • Biochemistry and Biotechnology  (13,095)
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  • 1
    ISSN: 0006-3592
    Keywords: phosphoglucomutase ; site-directed mutagenesis ; kinetic constants ; Pm promoter ; metabolic engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Mutants of Escherichia coli deficient in phosphoglucomutase accumulate amylose when the cells are grown on maltose or galactose as carbon source. In the presence of physiological levels of phosphoglucomutase, most of the sugar is catabolized, leading to strongly reduced levels of amylose accumulation. By varying the expression level of heterologous phosphoglucomutase, we show that the minimum level needed to block amylose accumulation corresponds to a phosphoglucomutase activity of 150-600 nmole substrate transformed per min per mg of total soluble protein. Mutant phosphoglucomutases with strongly reduced Vmax values and increased Km values for the substrate glucose-1-phosphate or the co-substrate glucose-1,6-diphosphate, could also reduce amylose accumulation, but much higher enzyme expression levels were required. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:299-302, 1998.
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  • 2
    ISSN: 0006-3592
    Keywords: glycerol ; Enterobacter agglomerans ; 3-hydroxypropionaldehyde ; catabolic limitation ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Batch fermentation of glycerol to 1,3-propanediol (1,3PPD) by Enterobacter agglomerans CNCM 1210 showed the lethal accumulation of 3-hydroxypropionaldehyde (3-HPA) when performed under initial substrate content higher than 40 g/L. Assigned to the inhibition by the NAD/NADH ratio of the 3-HPA converting enzyme: 1,3PPD dehydrogenase, intracellular assays were conducted in an attempt to identify the metabolic mechanisms involved in the increase of that ratio. An overflow metabolism through the 1,3PPD formation pathway was established, while a catabolic limitation in the oxidative branch at the level of glyceraldehyde-3-phosphate dehydrogenase occurred. Uncoupled activities of synthesis and consumption of reducing equivalents are thus suspected to provoke the increase of the NAD/NADH ratio and the subsequent accumulation of 3-HPA. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:303-305, 1998.
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  • 3
    ISSN: 0006-3592
    Keywords: lycopene ; Candida utilis ; carotenoids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The Erwinia uredovora crtE, crtB, and crtI genes, which are responsible for the synthesis of carotenoid lycopene from farnesyl pyrophosphate, were expressed in Candida utilis under the control of the promoters and terminators derived from the C. utilis GAP, PGK, and PMA genes, respectively. The yeast transformant carrying the carotenoid biosynthesis genes produced 758 μg/g dry weight of lycopene along with 407 μg/g dry weight of phytoene in the stationary phase. It was observed in the C. utilis transformant that ergosterol content was decreased to 65% of that in the parent strain that accumulated 6.04 mg/g dry weight of ergosterol. It is therefore possible that the carbon flux for the ergosterol biosynthesis has been branched at farnesyl pyrophosphate to generate a new pathway for the lycopene production in this yeast transformant. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:306-308, 1998.
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  • 4
    ISSN: 0006-3592
    Keywords: l-ascorbic acid ; vitamin C ; 2-keto-l-gulonic acid ; l-sorbose dehydrogenase ; l-sorbosone dehydrogenase ; Gluconobacter ; chemical mutation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We isolated Gluconobacter oxydans T-100 that had an activity to produce 2-KLGA from d-sorbitol; however, the yield of 2-KLGA was quite insufficient. Therefore, enzymes involved in the biosynthesis of l-sorbosone and 2-KLGA, l-sorbose dehydrogenase (SDH) and l-sorbosone dehydrogenase (SNDH), respectively, were purified from G. oxydans T-100. A genomic library of G. oxydans T-100 was screened to clone both genes for SDH and SNDH based on their amino acid sequences. SNDH and SDH were encoded in sequential open reading frames with 1497 and 1596 nucleotides, respectively, which were verified by the expression in Escherichia coli. The amino acid sequence of SDH and SNDH showed close similarity with E. coli choline dehydrogenase (CDH) and betaine-aldehyde dehydrogenase (BADH), respectively, which cooperatively play a key role for conferring osmotic tolerance. Because the yield of 2-KLGA by G. oxydans introduced with the genes for SDH and SNDH were insufficient, replacement of the promoter with that of Escherichia coli tufB1 in combination with chemical mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine resulted in improvement of the production level. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:309-315, 1998.
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  • 5
    ISSN: 0006-3592
    Keywords: ATP allocation ; celluloytic microorganisms ; consolidated bioprocessing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Under anaerobic, carbon limited conditions, celluloytic fermentative microorganisms face a metabolic choice with respect to the allocation of relatively scarce ATP: to invest it in cells or in hydrolytic enzymes. A model is proposed that defines an allocation parameter reflecting the fractional expenditure of ATP on cell synthesis relative to the total ATP available (gross ATP synthesized less maintenance). This parameter is then incorporated into an ATP-centered model of anaerobic cellulose fermentation based on the ethanol fermentation of yeast and the cellulase system of Trichoderma reesei. Results indicate that high processing rates are possible via a consolidated bioprocessing strategy, especially at high cellulase specific activities, and that cell/cellulase allocation represents an interesting system in which to study, and perhaps exploit, microbial evolution and metabolic control. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:316-320, 1998.
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  • 6
    ISSN: 0006-3592
    Keywords: yeast cell wall porosity and permeability ; β-1,3-glucanase ; selective protein release ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, we consider the impact on downstream process design resulting from the use of metabolically engineered yeast strains. We address the issue of how manipulation of cell wall permeability can improve the release and subsequent recovery of heterologous products produced in yeast. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:321-324, 1998.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 52-61 
    ISSN: 0006-3592
    Keywords: denitritification ; denitratification ; anoxic filter ; kinetic model ; distributed fraction of reductase ; parametric sensitivity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Denitritification and denitratification in anoxic filters were performed to generate experimental data. Also, a kinetic model of denitratification that accounts for intrinsic biokinetics and hydrodynamic behavior of the biofilter is proposed. In denitritification, the simulated results are in good agreement with the experimental data; and a higher nitrite influent concentration gives a higher nitrite reduction efficiency if the denitrifying loading is kept the same. In denitratification, the intermediate nitrite tends to accumulate, and a higher denitrifying loading results in a higher nitrite effluent concentration. By inserting biological and physical parameter values into the kinetic model, the variations in distributed fractions of nitrate-reductase (f) and nitrite-reductase (1-f) with different denitrifying loadings can be estimated by fitting in experimental data. The estimated f increased with an increase in denitrifying loading, implying that a higher denitrifying loading results in a higher nitrite effluent concentration. From parametric sensitivity analyses, the parameter f is more sensitive than other biological and physical parameters. Accordingly, the proposed kinetic model of denitratification can be used to predict the treatment performance of anoxic filters appropriately. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:52-61, 1998.
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  • 8
    ISSN: 0006-3592
    Keywords: enzymatic ; solid-to-solid conversion ; peptide synthesis ; proteases ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We have studied a thermolysin-catalyzed solid-to-solid dipeptide synthesis using equimolar amounts of Z-Gln-OH and H-Leu-NH2 as model substrates. The high substrate concentrations make this an effective alternative to enzymatic peptide synthesis in organic solvents. Water content was varied in the range of 0 to 600 mL water per mol substrate and enzyme concentration in the range of 0.5 to 10 g/mol of substrates. High yields around 80% conversion and initial rates from 5 to 20 mmol s-1 kg-1 were achieved. The initial rate increases 10-fold on reducing the water content, to reach a pronounced optimum at 40 mL water per mol substrate. Below this, the rate falls to much lower values in a system with no added water, and to zero in a rigorously dried system. This behavior is discussed in terms of two factors: At higher water contents the system is mass transfer limited (as shown by varying enzyme content), and the diffusion distances required vary. At low water levels, effects reflect the stimulation of the enzymatic activity by water. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:68-72, 1998.
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  • 9
    ISSN: 0006-3592
    Keywords: apoptosis ; necrosis ; bcl-2 ; amino acids ; cell culture ; cell death ; hybridoma ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The transfection of murine hybridomas with the apoptosis suppressor gene bcl-2 has been reported to result in the extension of batch culture duration, leading to significant improvements in culture productivity. In the present study, the effect of deprivation, individually, of each amino acid found in culture medium was examined to characterize the chemical environment of the culture in terms of its propensity to induce apoptosis. When cells were deprived of each amino acid, individually for 48 h, the majority of cell deaths in each case occurred by apoptosis, with essential amino acids being clearly most effective. For nearly all the amino acids, the viability of the bcl-2 cell line cultures was greater than 70% after 48 h, representing a substantial improvement in viability over control cell line cultures. Time course studies revealed that the induction of death could be divided into two phases. Initially, following the deprivation of a single essential amino acid, there was a period of time during which all the control cell line cultures retained high viability. The duration of this phase varied from 15 h in the case of lysine deprivation, through to 40 h in the case methionine deprivation. In the second phase of deprivation, the cultures exhibited an abrupt and rapid collapse in viability. The time taken for the viability to fall to 50% was similar for each amino acid. In every case, the duration of both phases of the bcl-2 cultures was considerably extended. Specific utilization rates were increased during the control cultures relative to the bcl-2 cultures for both the growth phase (ranging between 2% and 57% higher than the bcl-2 cultures) and the death phase (ranging between 172% to 1900% higher than the bcl-2 culture). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:90-98, 1998.
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  • 10
    ISSN: 0006-3592
    Keywords: Monod kinetics ; mixed substrate growth ; continuous culture ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In natural environments, heterotrophic microorganisms encounter complex mixtures of carbon sources, each of which is present only at very low concentrations. Under such conditions no significant growth could be expected if cells utilized only one of the available carbon compounds as suggested by the principle of diauxic growth. Indeed, there is much evidence that microbial cells utilize many carbon sources simultaneously. In order to predict bacterial growth under such conditions we developed a model describing the specific growth rate as a function of the individual concentrations of several simultaneously utilized carbon substrates. Together with multisubstrate models previously published, this model was evaluated for its ability to describe growth of Escherichia coli during the simultaneous utilization of mixtures of sugars in carbon-limited continuous culture. Using the μmax and Ks constants determined for single substrate growth with six different sugars, the model was able for most experiments to adequately describe the specific growth rate of the culture, i.e., the experimentally set dilution rate, from the measured concentrations of the individual sugars. The model provides an explanation why bacteria can still grow relatively fast under environmental conditions where the concentrations of carbon substrates are usually extremely low. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:99-107, 1998.
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  • 11
    ISSN: 0006-3592
    Keywords: thermophilic β-glycosidase ; catalytic membranes ; nonisothermal bioreactors ; thermodialysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Catalytic membranes, obtained by immobilizing thermophilic β-glycosidase onto nylon supports, were used in a nonisothermal bioreactor to study the effect of temperature gradients on the rate of enzyme reaction. Two experimental approaches were carried out to explain the molecular mechanisms by which the temperature gradients affect enzyme activity. The results showed that the thermophilic enzyme behaved as the mesophilic β-galactosidase, exhibiting an activity increase which was linearly proportional to the transmembrane temperature difference. The efficiency of the system proposed was determined by calculating two constants, α and β, which represent respectively the percentage increase of enzyme activity when a temperature difference of 1°C or a temperature gradient of 1°C cm-1 were applied across the catalytic membrane. The increase of enzyme activity in nonisothermal bioreactors entailed a proportional reduction of production times. The advantages in using thermophilic enzymes immobilized in nonisothermal bioreactors are also discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:108-115, 1998.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 116-121 
    ISSN: 0006-3592
    Keywords: mixed-substrate growth ; mathematical model ; competing species ; dynamical analogy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: There is a similarity between the metabolic dynamics of a microbial species growing on a mixture of two substrates and the dynamics of growth of two competing populations. Specifically, the enzymes catalyzing the uptake and catabolism of substrates exhibit phenomena analogous to extinction and coexistence.“Extinction” of the enzymes associated with one of the substrates results in sequential utilization of the substrates (diauxie) (Monod, 1942). “Coexistence” of the enzymes associated with the substrates results in simultaneous utilization of the substrates (Egli, 1995). Here, we formulate a simple model that shows the basis for this dynamical similarity: The equations describing the evolution of the enzyme levels are dynamical analogs of the Lotka-Volterra model for two competing species. The analogy suggests ways of capturing the experimentally observed preculture-dependent growth patterns, i.e., growth patterns that vary depending on the physiological state of the preculture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:116-121, 1998.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 128-128 
    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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  • 14
    ISSN: 0006-3592
    Keywords: lipase ; organic solvent ; flavour esters ; interfacial activation ; enzyme conformers ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In order to improve the lipase-catalyzed synthesis of flavour esters, we have used the reported strategy of interfacial activation-based molecular (bio)imprinting [Mingarro et al. 1995. Proc. Natl. Acad. Sci. U.S.A. 92: 3308], later called trapping in the presence of amphiphile interfaces (TPI) [Mingarro et al. 1996. Biochemistry 35: 9935]. Five lipases of fungal and mammalian origin typically used for esterification process have been explored to improve production by TPI treatment. A marked enhancement of enzymatic activity has been observed in all TPI-treated lipases assayed and the activation factor obtained was up to 90-fold. The dependence on chain length of acyl donors in the esterification of geranyl alcohol has been investigated, showing clear differences between activated and nonactivated lipase. The results indicate that this rational approach leads to conversion yields that are remarkably higher, not only than its counterpart pH-optimized control lipase, but also the “protected” lipase by conventional methods (lyoprotectans or salts). We propose this strategy as a promising tool to be used in more industrial biotransformations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:122-127, 1998.
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  • 15
    ISSN: 0006-3592
    Keywords: smooth muscle ; polyglycolic acid ; biodegradable ; tissue engineering ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 × 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 ± 0.8 × 108 cells/cm3 after 5 weeks, compared to 2.0 ± 1.1 × 108 cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 ± 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 46-54, 1998.
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  • 16
    ISSN: 0006-3592
    Keywords: Escherichia coli ; protein production ; secretion ; plasmid stability ; fed-batch ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Periplasmic secretion of overexpressed Bacillus stearothermophilus α-amylase was analyzed in batch and fed-batch cultivations of Escherichia coli MG1655:pCSS4-p and the mutant strain CWML2:pCSS4-p. Under all conditions investigated, growth and product formation of MG1655:pCSS4-p were severely impaired by heterologous protein expression and/or processing, while E. coli CWML2:pCSS4-p was found to be more robust and to accumulate 2- to 3-fold higher maximum α-amylase levels. While this strain is itself potentially interesting for applications, its properties also illustrate the potential of the selection procedure that was employed to obtain it from its progenitor MG1655 (Weikert, C., Sauer, U., Bailey, J. E., 1997. Microbiol. 143: 1567-1574. Application of this procedure to existing industrial strains may lead to significantly improved process organisms. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:386-391, 1998.
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  • 17
    ISSN: 0006-3592
    Keywords: denitrification ; biodegradation ; kinetics ; 1,1,1-trichloroethane ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A denitrifying consortium capable of degrading carbon tetrachloride (CT) was shown to also degrade 1,1,1-trichloroethane (TCA). Fed-batch experiments demonstrated that the specific rate of TCA degradation by the consortium was comparable to the specific rate of CT degradation (approximately 0.01 L/gmol/min) and was independent of the limiting nutrient. Although previous work demonstrated that 4-50% of CT transformed by the consortium was converted to chloroform (CF), no reductive dechlorination products were detected during TCA degradation, regardless of the limiting nutrient. The lack of chlorinated TCA degradation products implies that the denitrifying consortium possesses an alternate pathway for the degradation of chlorinated solvents which does not involve reductive dechlorination. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:393-399, 1998.
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  • 18
    ISSN: 0006-3592
    Keywords: nar promoter ; oxygen-dependent inducible promoter ; anaerobic/microaerobic conditions ; recombinant E. coli ; fed-batch cultivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The nar promoter of Escherichia coli is maximally induced under anaerobic or microaerobic conditions in the presence of nitrate. We previously demonstrated in batch experiments that the intact nar promoter of E. coli cloned into a pBR322-based plasmid serves as a high-level expression system in a nar mutant of E. coli (Lee et al., 1996b). In this study, we extend characterization of the nar promoter expression system to the fed-batch culture mode, which is widely used in industrial-scale fermentation. From these experiments, it was found that the specific β-galactosidase activity expressed from the lacZ gene fused to the nar promoter was maximal when host cells were grown under aerobic conditions [dissolved oxygen, (DO) = 80%] to absorbance at 600 nm (OD600) = 35 before induction of the nar promoter by lowering DO to 1-2% with alternating microaerobic and aerobic conditions. Approximately 15 h after induction, the OD600 of the culture reached 135 and the specific β-galactosidase activity increased to 40,000 Miller units, equivalent to approximately 35% of the total cellular proteins. The specific β-galactosidase activity before induction was approximately 1,000 Miller units, giving an induction ratio of approximately 40. Based on these results, we conclude that the nar promoter provides a convenient and effective high level expression system under conditions of fed-batch culture. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:400-406, 1998.
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  • 19
    ISSN: 0006-3592
    Keywords: Alcaligenes eutrophus ; hydrogenase ; NADH regeneration ; HLADH ; organic solvent ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A soluble NAD-dependent hydrogenase contained in Alcaligenes eutrophus was evaluated as a coenzyme regenerating catalyst in an organic-aqueous two-phase (predominantly organic) system. The horse-liver alcohol-dehydrogenase (HLADH) catalyzed reduction of cyclohexanone to cyclohexanol was used as a model reaction. The impact of different solvents (selected to span a large variety of principal properties) on the stability and activity of the HLADH, using substrate-driven regeneration, was studied. Solvents suitable for the HLADH were then selected for an evaluation of the hydrogenase-driven coenzyme regeneration. Hydrophobic solvents such as heptane, toluene, and 1,1,1-trichloroethane were found to be suitable for the coupled reactions catalyzed by HLADH and hydrogenase. Nonimmobilized cells, permeabilized with cetyl-trimethyl-ammonium bromide, were the most efficient preparation for the regeneration of NADH. The use of this preparation in heptane (10% water) was optimized with respect to the yield obtained in the HLADH-catalyzed reduction of cyclohexanone. Using the optimized conditions, yields of 99% cyclohexanol were obtained. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 79-86, 1988.
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  • 20
    ISSN: 0006-3592
    Keywords: CHO cells ; glycosylation engineering ; antisense ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:445-450, 1998.
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  • 21
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 461-470 
    ISSN: 0006-3592
    Keywords: aqueous two-phase separation ; protein partitioning ; T4 lysozyme ; electrochemical partitioning ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Protein partitioning in aqueous two-phase systems based on phase-forming polymers is strongly affected by the net charge of the protein, but a thermodynamic description of the charge effects has been hindered by conflicting results. Many of the difficulties could be because of problems in isolating electrochemical effects from other interactions of phase components.We explored charge effects on protein partitioning in poly(ethylene glycol)-dextran two-phase systems by using two series of genetically engineered charge modifications of bacteriophage T4 lysozyme produced in Escherichia coli. The two series, one in the form of charged-fusion tails and the other in the form of charge-change point mutations, provided matching net charges but very different polarity. Partition coefficients of both series were obtained and interfacial potential differences of the phase systems were measured. Multi-angle laser light scattering measurements were also performed to determine second virial coefficients. A semi-empirical model accounting for the roles of both charge and non-charge effects on protein partitioning behavior is proposed, and the results predicted from the model are compared to the results from the experiments. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:461-470, 1998.
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  • 22
    ISSN: 0006-3592
    Keywords: polycyclic aromatic hydrocarbon ; biodegradation ; surfactants ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objectives of this study were to isolate and evaluate microorganisms with the ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) in the presence of synthetic surfactants. Stenotrophomonas maltophilia VUN 10,010, isolated from PAH-contaminated soil, utilized pyrene as a sole carbon and energy source and also degraded other high molecular weight PAHs containing up to seven benzene rings. Various synthetic surfactants were tested for their ability to improve the PAH degradation rate of strain VUN 10,010. Anionic and cationic surfactants were highly toxic to this strain, and the Tween series was used as a growth substrate. Five nonionic surfactants (Brij 35, Igepal CA-630, Triton X-100, Tergitol NP-10, and Tyloxapol) were not utilized by, and were less toxic to, strain VUN 10,010. MSR and log Km values were determined for fluoranthene, pyrene, and benzo[a]pyrene in the presence of these nonionic surfactants and their apparent solubility was increased by a minimum of 250-fold in the presence of 10 g L-1 of all surfactants. The rate of pyrene degradation by strain VUN 10,010 was enhanced by the addition of four of the nonionic surfactants (5-10 g L-1); however, 5 g L-1 Igepal CA-630 inhibited pyrene degradation and microbial growth. The specific growth rate of VUN 10,010 on pyrene was increased by 67% in the presence of 10 g L-1 Brij 35 or Tergitol NP-10. The addition of Brij 35 and Tergitol NP-10 to media containing a single high molecular weight PAH (four and five benzene rings) as the sole carbon source increased the maximum specific PAH degradation rate and decreased the lag period normally seen for PAH degradation. The addition of Tergitol NP-10 to VUN 10,010 cultures which contained a PAH mixture (three to seven benzene rings) substantially improved the overall degradation rate of each PAH and increased the specific growth rate of VUN 10,010 by 30%. Evaluation of the use of VUN 10,010 for degrading high molecular weight PAHs in leachates from surfactant-flushed, weathered, PAH-contaminated sites is warranted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:482-494, 1998.
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  • 23
    ISSN: 0006-3592
    Keywords: biocompatibility ; microfabrication ; biohybrid organs ; immunoisolation ; Islets of Langerhans ; silicon ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A microfabricated silicon-based biocapsule for the immunoisolation of cell transplants is presented. The biocapsule-forming process employs bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes. The membranes are surface micromachined to present a high density of uniform pores, thus affording sufficient permeability to oxygen, glucose, and insulin. The pore dimensions, as small as 20 nm, are designed to impede the passage of immune molecules and graft-borne viruses. The underlying filter-membrane nanotechnology has been successfully applied in controlled cell culture systems (Ferrari et al., 1995), and is under study for viral elimination in plasma fractionation protocols. Here we report the encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule, and demonstrate that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks. These results appear to warrant further investigations on the potential of cell xenografts encapsulated within microfabricated, immunoisolating environments for the treatment of insulin-dependent diabetes. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 118-120, 1998.
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  • 24
    ISSN: 0006-3592
    Keywords: sucrose monoester synthesis ; lipase-catalyzed acylation ; water activity (a w) ; regioselectivity ; salt hydrate pair ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Sucrose monoesters of a fatty acid were synthesized by using lipase in a solvent-free system. When lipase from Mucor miehei was used as a catalyst with capric acid as the donor and sugar as the acceptor, sucrose 6-monocaprate was predominantly produced in a yield of 25.3%. The yield of product was significantly increased by the direct addition of a suitable pair of solid salt hydrates to the reaction mixture to control the water activity (aw). Among the salt hydrate pairs investigated, the barium hydroxide, 8/1H2O pair resulted in the highest yield of the product. This salt addition method was also successfully employed for acylation of primary hydroxyl groups in various unprotected mono- and disaccharides such as glucose, galactose, fructose, trehalose, mannose, maltose, and lactose. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 121-125, 1998.
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  • 25
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    Biotechnology and Bioengineering 57 (1998), S. 127-135 
    ISSN: 0006-3592
    Keywords: membrane mass spectrometer ; kinetic measurements ; anaerobic biofilm ; acetate ; inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A small, stirred, 14.4-mL tank reactor was designed to serve as a measurement cell for short-term investigation of microbial kinetics. A mass spectrometer membrane probe allowed the measurement of the dissolved gases of hydrogen, methane, oxygen, and carbon dioxide. pH was measured by an electrode and controlled by addition of acid or alkali. The highly sensitive measurement of gases with low solubility allowed rapid measurements at very low conversion. In kinetic experiments, a stepwise increase of substrate concentration (method A) and continuous feed of substrate (method B) were used, allowing quick estimation of substrate kinetics. Acetate conversion in mixed culture biofilms from a fluidized bed reactor was investigated. Substrate inhibition was found to be negligible in the concentration range studied. Experiments at various pH values showed that the undissociated acid form was the kinetic determinant. Kinetic parameters for Haldane kinetics of protons were KSH = 1.3 × 10-5 mol m-3 and KIH = 8.1 × 10-3 mol m-3. With free acid (HAc) as the rate determining species, the kinetic parameters for method A were KSHAc = 0.005 mol m-3 and KIHAc = 100 mol m-3 and for method B were KSHAc = 0.2 mol m-3 and KIHAc = 50 mol m-3. The maximum biomass activity occurred at around pH 6.5. Acetate was exclusively converted to methane and CO2 at pH > 6. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 127-135, 1998.
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  • 26
    ISSN: 0006-3592
    Keywords: down-flow fluidization ; bed expansion ; biofilm ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: This article describes the bed expansion characteristics of a down-flow anaerobic fluidized bed reactor treating a synthetic wastewater. Experiments were carried out in a 0.08 m diameter and 1 m length PVC column. The carrier used was ground perlite (an expanded volcanic rock). Particles characteristics were 0.968 mm in diameter, specific density of 213 kg · m-3 and Umf (minimal fluidization velocity): 2.3 m · h-1. Experimental data of terminal velocities and bed expansion parameters at several biofilm thicknesses were compared to different models predicting the bed expansion of up-flow and down-flow fluidized beds.Measured bed porosities at different liquid superficial velocities for the different biofilm thicknesses were in agreement with the Richardson-Zaki model, when Ut (particle terminal velocity) and n (expansion coefficient) were calculated by linear regression of the experimental data. Terminal velocities of particles at different biofilm thicknesses calculated from experimental bed expansion data, were found to be much smaller than those obtained when Cd (drag coefficient) is determined from the standard drag curve (Lapple and Sheperd, 1940) or with others' correlations (Karamanev and Nikolov, 1992a,b). This difference could be explained by the fact that free-rising particles do not obey Newton's law for free-settling, as proposed by Karamanev and Nikolov (1992a,b) and Karamanev et al. (1996). In the present study, the same free-rising behavior was observed for all particles (densities between 213 and 490 kg · m-3). © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 136-144, 1998.
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  • 27
    ISSN: 0006-3592
    Keywords: bioavailability ; PAH ; biodegradation ; dissolution ; hydrodynamic ; mixing ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The influence of hydrodynamic conditions on the dissolution rate of crystalline naphthalene as a model polycyclic aromatic hydrocarbon (PAH) was studied in stirred batch reactors with varying impeller speeds. Mass transfer from naphthalene melts of different surface areas to the aqueous phase was measured and results were modeled according to the film theory. Results were generalized using dimensionless numbers (Reynolds, Schmidt, and Sherwood). In combined mass transfer and biodegradation experiments, the effect of hydrodynamic conditions on the degradation rate of naphthalene by Pseudomonas 8909N was studied. Experimental results were mathematically described using mass-transfer and microbiological models. The experiments allowed determination of mass-transfer and microbiological parameters separately in a single run. The biomass formation rate under mass transfer limited conditions, which is related to the naphthalene biodegradation rate, was correlated to the dimensionless Reynolds number, indicating increased bioavailability at increased mixing in the reactor liquid. The methodology presented in which mass transfer processes are quantified under sterile conditions followed by a biodegradation experiment can also be adapted to more complex and realistic systems, such as particulate, suspended PAH solids or soils with intrapartically sorbed contaminants when the appropriate mass-transfer equations are incorporated. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 145-154, 1998.
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  • 28
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    Biotechnology and Bioengineering 57 (1998), S. 155-163 
    ISSN: 0006-3592
    Keywords: endogenous respiration ; activated sludge ; multi-time scales ; identifiability ; observability ; model reduction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In this article, an autonomous four-compartment model that describes the endogenous respiration in an aerobic biodegradation process is proposed and analyzed theoretically. First, the multi-time scale of the system's behavior, to be taken into account in subsequent analyses, is emphasized. Then, an identifiability and observability study, given measurements of MLVSS (mixed liquor volatile suspended solids) and respiration rate, is performed for use under practical circumstances, such as in state and parameter estimation. It appears that the process is observable, but not fully identifiable. Hence, for the identification of some of the model parameters, additional measurements or experiments, also indicated here, have to be performed. Furthermore, it is shown that, under quasi-steady state conditions which, in general, appear shortly after initialization of an endogenous respiration experiment, the model can be reduced significantly. Finally, results of parameter estimation from available data are presented and discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 155-163, 1998.
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  • 29
    ISSN: 0006-3592
    Keywords: cell death ; apoptosis ; bcl -2 ; cell culture ; cell viability ; growth factors ; survival factors ; abortive proliferation ; hybridomas ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Cultures of the CRL-1606 hybridoma (ATCC) have been reported to undergo continuous proliferation with simultaneous death during nutrient limited fed-batch fermentations. The bcl-2 proto-oncogene has been shown to prevent cell death under a variety of otherwise death inducing conditions. We were interested in elucidating the nature of the massive death observed in cultures of CRL-1606, specifically with respect to the possible environmental causes, and the ability of overexpressed human bcl-2 (hbcl-2) to mitigate cell death. Abortive proliferation, or continuous proliferation in the presence of continuous death, could be induced in serum free cultures of CRL-1606 through the withdrawal of insulin provided the culture was competent for cell proliferation. Culture competency for proliferation was found to be solely determined by the presence of cell culture nutrients. Abortive proliferation was defective in cultures transfected with hbcl-2 and the enhanced viability observed resulted from an increased viable cell population and at the expense of the nonviable cell population normally found in untransfected cultures. Abortive proliferation was also observed in serum containing cultures upon serum shiftdowns. Like the insulin-supplemented serum free culture system, hbcl-2 transfected cultures exhibited defects in the abortive proliferation process. These results suggest that the massive death observed during nutrient-limited fed-batch fermentation originate, in part, from growth or survival factor limitations. Hence, approaches to design cell culture media that account for the cell's proliferation requirements without accounting for the cell's survival requirements may represent a cell death sentence. Given the transformed nature of the hybridomas, we conclude that the abortive proliferation of CRL-1606 is a consequence of inappropriate cell cycle entry in a survival factor limited environment. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 164-171, 1998.
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  • 30
    ISSN: 0006-3592
    Keywords: hybridoma ; futile cycling ; hollow fiber bioreactor ; glutamine ; NMR ; C-13 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of changes in extracellular glutamine level on metabolism of a murine hybridoma was examined with in vivo nuclear magnetic resonance (NMR) spectroscopy. Cells were cultured in a hollow-fiber bioreactor at high cell density to allow intracellular metabolite levels to be determined on a metabolically relevant time scale. Steady infusions of [1-13C] glucose were used to label glycolytic and tricarboxylic acid cycle intermediates, which permitted continuous monitoring with NMR spectroscopy during changes in environmental glutamine level. Samples of the extracellular medium were also analyzed to determine the effect of glutamine on other metabolites associated with primary and secondary metabolism. The changes in glutamine concentration had several effects on primary and secondary metabolism, depending on the rate the changes were made. For a brief reduction in feed glutamine concentration from 4 to 0 mM (which produced a rapid change from 0.67 to ∼0 mM in residual glutamine), large changes were observed in the rate of consumption of metabolites normally associated with energy production. Antibody synthesis was strongly stimulated and nitrogen metabolism was significantly altered. For a more prolonged reduction from 2.4 to 1.2 mM (which produced a slower reduction from 0.30 to 0.08 mM in residual glutamine), much smaller changes were observed even though the concentration of glutamine at the reduced feed level was very low. Energy metabolism did not appear to be limited by glutamine at 0.08 mM, which suggests that significant futile cycling may occur in energy producing pathways when excess glucose and glutamine are available. However, this concentration of extracellular glutamine appeared to affect some anabolic pathways, which require amino groups from glutamine. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 172-186, 1998.
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  • 31
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    Biotechnology and Bioengineering 57 (1998), S. 187-197 
    ISSN: 0006-3592
    Keywords: algal cultures ; photosynthetic efficiency ; light saturation effect ; spatial dilution of light ; Arthrospira (Spirulina) platensis ; tubular and flat photobioreactors ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The light saturation effect imposes a serious limitation on the efficiency with which solar energy can be utilized in outdoor algal cultures. One solution proposed to reduce the intensity of incident solar radiation and overcome the light saturation effect is “spatial dilution of light” (i.e., distribution of the impinging photon flux on a greater photosynthetic surface area), but consistent experimental data supporting a significant positive influence of spatial light dilution on the productivity and the photosynthetic efficiency of outdoor algal cultures have never been reported. We used a coiled tubular reactor and compared a near-horizontal straight tubular reactor and a near-horizontal flat panel in outdoor cultivation of the cyanobacterium Arthrospira (Spirulina) platensis under defined operating conditions for optimum productivity. The photosynthetic efficiency achieved in the tubular systems was significantly higher because their curved surface “diluted” the impinging solar radiation and thus reduced the light saturation effect. This interpretation was supported by the results of experiments carried out in the laboratory under continuous artificial illumination using both a flat and a curved chamber reactor. The study also showed that, when the effect of light saturation is eliminated or reduced, productivity and solar irradiance are linearly correlated even at very high diurnal irradiance values, and supported findings that outdoor algal cultures are light-limited even during bright summer days. It was also observed that, besides improving the photosynthetic efficiency of the culture, spatial dilution of light also leads to higher growth rates and lowers the cellular content of accessory pigments; that is, it reduces mutual shading in the culture. The inadequacy of using volumetric productivity as the sole criterion for comparing reactors of different surface-to-volume ratio and of the areal productivity for evaluating the performance of elevated photobioreactors operated outdoors is stressed; it is furthermore suggested that the photosynthetic efficiency achieved by the culture also be calculated to provide a suitable parameter for comparison of different algal cultivation systems operated under similar climatic conditions. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 187-197, 1998.
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  • 32
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    Biotechnology and Bioengineering 57 (1998), S. 198-210 
    ISSN: 0006-3592
    Keywords: Xanthan fermentation ; agitator speed ; caverns ; dissolved oxygen ; specific oxygen uptake rate ; specific Xanthan production rate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Agitation speed affects both the extent of motion in Xanthan fermentation broths because of their rheological complexity and the rate of oxygen transfer. The combination of these two effects causes the dissolved oxygen concentration and its spatial uniformity also to change with agitator speed. Separating these complex interactions has been achieved in this study in the following way. First, the influence of agitation speeds of 500 and 1000 rpm has been investigated at a constant nonlimiting dissolved oxygen concentration of 20% of air saturation using gas blending. Under these controlled dissolved oxygen conditions, the results demonstrate that the biological performance of the culture was independent of agitation speed as long as broth homogeneity could be ensured. With the development of increasing rheological complexity lending to stagnant regions at Xanthan concentrations >20 g/L, it is shown that the superior bulk mixing achieved at 1000 rpm, compared with 500 rpm, leading to an increased proportion of the cells in the fermentor to be metabolically active and hence higher microbial oxygen uptake rates, was responsible for the enhanced performance. Second, the effects of varying dissolved oxygen are compared with a control in each case with an agitator speed of 1000 rpm to ensure full motion, but with a fixed, nonlimiting dissolved oxygen of 20% air saturation. The specific oxygen uptake rate of the culture in the exponential phase, determined using steady-state gas analysis data, was found to be independent of dissolved oxygen above 6% air saturation, whereas the specific growth rate of the culture was not influenced by dissolved oxygen, even at levels as low as 3%, although a decrease in Xanthan production rate could be measured. In the production phase, the critical oxygen level was determined to be 6% to 10%, so that, below this value, both specific Xanthan production rate as well as specific oxygen uptake rate decreased significantly. In addition, it is shown that the dynamic method of oxygen uptake determination is unsuitable even for moderately viscous Xanthan broths. © 1998 John Wiley & Sons, Inc. Biotechnol. Bioeng. 57: 198-210, 1998.
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  • 33
    ISSN: 0006-3592
    Keywords: protein ; conformational memory ; organic solvent ; molecular imprinting ; enzyme ; catalysis ; transition state analogue ; bovine serum albumin ; β-lactoglobulin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The induction of catalytic activity in proteins by lyophilization in the presence of a transition state analogue (biomolecular imprinting) has been attempted. It was shown that proteins which were freeze-dried with n-isopropyl-4-nitrobenzyl-amine (a transition state analogue for the reaction of dehydrofluorination of 4-fluoro-4-[p-nitrophenyl] butan-2-one) displayed higher β-elimination activity as compared to their-non-imprinted counterparts. It was also found that native bovine serum albumin has a high dehydrofluorination activity towards the above substrate with kinetic parameters rather similar to those of a catalytic antibody prepared by Shokat et al. (1989). A comparison of the kinetic parameters determined in this study with those obtained for analogous catalytic antibodies and imprinted polymers was made. © 1998 John Wiley & Sons, Inc. Biotechnol. Bioeng. 57: 211-215, 1998.
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  • 34
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    Biotechnology and Bioengineering 57 (1998), S. 216-219 
    ISSN: 0006-3592
    Keywords: liposomes ; vesicles ; microreactor ; permeability ; chymotrypsin ; enzyme ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Liposomes were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), which contained the water soluble proteinase α-chymotrypsin. This liposome entrapped enzyme showed selectivity for externally added substrates in that only small substrates (benzoyl-l-Tyr-p-nitroanilide or acetyl-l-Phe-p-nitro-anilide) - for which the liposome bilayer was permeable - were transformed into products. Large substrates (succinyl-l-Ala-l-Ala-l-Pro-l-Phe-p-nitroanilide or casein) could not penetrate from the external aqueous phase into the liposomes, and were not hydrolyzed. This substrate selectivity is entirely based on the compartimentation and permeability properties of the liposome microreactor. © 1998 John Wiley & Sons, Inc.Biotechnol. Bioeng. 57: 216-219, 1998.
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  • 35
    ISSN: 0006-3592
    Keywords: PAH degradation ; white rot fungus ; Bjerkandera sp. ; surfactant ; toxicity ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of nonionic surfactants on the polycyclic aromatic hydrocarbon (PAH) oxidation rates by the extracellular ligninolytic enzyme system of the white-rot fungus Bjerkandera sp. strain BOS55 was investigated. Various surfactants increased the rate of anthracene, pyrene, and benzo[a]pyrene oxidation by two to fivefold. The stimulating effect of surfactants was found to be solely due to the increased bioavailability of PAH, indicating that the oxidation of PAH by the extracellular ligninolytic enzymes is limited by low compound bioavailability. The surfactants were shown to improve PAH dissolution rates by increasing their aqueous solubility and by decreasing the PAH precipitate particle size. The surfactant Tween 80 was mineralized by Bjerkandera sp. strain BOS55; as a result both the PAH solubilizing activity of Tween 80 and its stimulatory effect on anthracene and pyrene oxidation rates were lost within 24 h after addition to 6-day-old cultures. It was observed that the surfactant dispersed anthracene precipitates recrystallized into larger particles after Tween 80 was metabolized. However, benzo[a]pyrene precipitates remained dispersed, accounting for a prolonged enhancement of the benzo[a]pyrene oxidation rates. Because the endogenous production of H2O2 is also known to be rate limiting for PAH oxidation, the combined effect of adding surfactants and glucose oxidase was studied. The combined treatment resulted in anthracene and benzo[a]pyrene oxidation rates as high as 1450 and 450 mg L-1 d-1, respectively, by the extracellular fluid of 6-day-old fungal cultures. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 220-227, 1998.
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  • 36
    ISSN: 0006-3592
    Keywords: AlkB ; Pseudomonas oleovorans ; alkane hydroxylase ; iron ; Escherichia coli ; alk + recombinants ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The alk genes enable Pseudomonas oleovorans to utilize alkanes as sole carbon and energy source. Expression of the alk genes in P. oleovorans and in two Escherichia coli recombinants induced iron limitation in minimal medium cultures. Further investigation showed that the expression of the alkB gene, encoding the integral cytoplasmic membrane protein AlkB, was responsible for the increase of the iron requirement of E. coli W3110 (pGEc47).AlkB is the non-heme iron monooxygenase component of the alkane hydroxylase system, and can be synthesized to levels up to 10% (w/w) of total cell protein in E. coli W3110 (pGEc47). Its synthesis is, however, strictly dependent on the presence of sufficient iron in the medium. Our results show that a glucose-grown E. coli alk+ strain can reach alkane hydroxylase activities of about 25 U/g cdw, and are consistent with the recent finding that catalytically active AlkB contains two, rather than one iron atom per polypeptide chain. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 228-237, 1998.
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  • 37
    ISSN: 0006-3592
    Keywords: expression ; membrane protein ; glycophorin ; neomycin resistance ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gene for the integral membrane protein glycophorin A (GPA) was cloned in frame to the 5′ end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT). Protein expression was achieved in Escherichia coli as well as in mammalian cells. In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks after transfection; the amount of GPA-NPT fusion protein produced was constant from experiment to experiment. Neomycin resistance was directly correlated with GPA expression, thus allowing the direct selection for a stable GPA-expressing cell population without the need of a cloning step. The amount of GPA-NPT produced was further increased by weakening the specific NPT enzymatic activity via site-directed mutagenesis. Detection was simplified by the fact that all different fusion proteins could be detected by the same anti-NPT antibody. This approach may be also applicable to other membrane proteins. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 238-244, 1998.
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  • 38
    ISSN: 0006-3592
    Keywords: human parathyroid hormone ; proteolysis ; L-arginine ; secretory production ; KEX2 endoproteinase ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor α pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (≥0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases. © 1998 John Wiley & Sons, Inc. Biotechnol. Bioeng. 57: 245-249, 1998.
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  • 39
    ISSN: 0006-3592
    Keywords: continuous culture ; metabolic overflow ; multiplicity ; stability analysis ; dynamics ; growth inhibition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic overflow (enhanced uptake of substrate and secretion of intermediates) is a phenomenon often observed for cells grown under substrate excess. Growth inhibition by substrate and/or product is also normally found for this kind of culture. An effort is made in this work to analyze the dynamic behavior of a continuous culture subject to metabolic overflow and growth inhibition by substrate and/or product. Analysis of a model system shows that in a certain range of operating conditions three nonwashout steady state solutions are possible. Local stability analysis indicates that only two of them are stable thus leading to multiplicity and hysteresis. Further analysis of the intrinsic effects of different terms describing the metabolic overflow and growth inhibitions reveals that for the model system and the parameters considered, the combined effects of product inhibition and an enhanced formation rate of product under substrate excess cause the multiplicity and hysteresis. Growth inhibition by substrate and/or an enhanced substrate uptake appear not to be necessary conditions. The combined effects of enhanced product formation and product inhibition can also lead to unusual dynamic behavior such as a prolonged time period to reach a steady state, oscillatory transition from one steady state to another, and sustained oscillations. Using the occurrence of multiplicity and oscillation as criteria, the operating regime of a continuous culture can be divided into four domains: one with multiplicity and oscillation, one with unique steady state but possible oscillatory behavior, the other two with unique and stable steady state. The model predictions are in accordance with recent experimental results. The results presented in this work may be used as guidelines for choosing proper operating conditions of similar culture systems to avoid undesired instability and multiplicity. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 57: 251-261, 1998.
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  • 40
    ISSN: 0006-3592
    Keywords: directed evolution ; esterase ; epothilon ; Pseudomonas fluorescens ; stereoselectivity ; mutator strain ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The directed evolution of an esterase from Pseudomonas fluorescens using the mutator strain Epicurian coli XL1-Red was investigated. Mutants were assayed for their ability to hydrolyze a sterically hindered 3-hydroxy ester, which can serve as a building block in the synthesis of epothilones. Screening was performed by plating esterase producing colonies derived from mutation cycles onto minimal media agar plates containing indicator substances (neutral red and crystal violet). Esterase-catalyzed hydrolysis of the 3-hydroxy ester (ethyl or glycerol ester) was detected by the formation of a red color due to a pH decrease caused by the released acid. Esterases isolated from positive clones were used in preparative biotransformations of the ethyl ester. One variant containing two mutations (A209D and L181V) stereoselectively hydrolyzed the ethyl ester resulting in 25% ee for the remaining ester. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 554-559, 1998.
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  • 41
    ISSN: 0006-3592
    Keywords: fungal morphology ; Aspergillus oryzae ; on-line image analysis ; growth kinetics and branching pattern ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A flow-through cell is designed to measure the growth kinetics of hyphae of Aspergillus oryzae grown submerged in a well controlled environment. The different stages of the growth process are characterized, from the spore to the fully developed hyphal element with up to 60 branches and a total length lt up to 10,000 μm. Spore swelling is found to occur without change in the form of the spore (circularity index constant at about 1.06) and the spore volume probably increases exponentially. The germ tube appears after about 4 h. The branching frequency and the rate of germ tube extension is determined. After about 10 h growth at a glucose concentration of 250 mg L-1, 6-7 branches have been set, and both the total hyphal length lt and the number of tips increase exponentially with time. The specific growth rate of the hyphae is 0.33 h-1 while the average rate of the extension of the growing tips approaches 55 μm h-1.The growth kinetics for all the branches on the main hypha have also been found. The main hypha and all the branches grow at a rate which can be modeled by saturation kinetics with respect to the branch length and with nearly equal final tip speeds (160 μm h-1). Branches set near the apical tip of the main hypha attain their final tip speed in the shortest time, i.e., the value of the saturation parameter is small.Finally, the influence of substrate (glucose) concentration cs on the values of the morphological parameters has been determined. It is found that saturation type kinetics can be used to describe the influence of cs on the growth.Experiments with recirculation of effluent from the cell back to the inlet strongly suggest that the fungus secretes an inducer for growth and branching. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 541-553, 1998.
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  • 42
    ISSN: 0006-3592
    Keywords: fluoroether surfactants ; liquid CO2 ; high pressure ; emulsion ; solubilization ; subtilisin Carlsberg ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Carbon dioxide is a naturally abundant, environmentally benign solvent whose use, like water, in a process is not regulated by either EPA or FDA. Unfortunately, polar compounds such as amino acids and proteins are essentially insoluble in carbon dioxide. Further, alkyl-functional surfactants, which have been shown to allow extraction of proteins into conventional organic solvents, exhibit very poor or negligible solubility in CO2 at pressures below 50 MPa. Consequently, highly CO2-soluble fluoroether-functional surfactants have been generated and used to solubilize subtilisin Carlsberg from aqueous buffer and cell culture medium into CO2, with recovery accomplished by depressurization. Both the amount of protein solubilized in the emulsion and the extent of activity retention by the protein following recovery are functions of the initial protein concentration in the buffer. This, plus the observation that the presence of protein affects the stability of the emulsion, suggests that some of the protein is sacrificed to act as a stabilizer in these systems. In addition to solubilization via an inverse emulsion, it has also been shown that one can strip protein-surfactant aggregates from a middle phase emulsion using pure CO2, suggesting an ion-pairing type mechanism. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 572-580, 1998.
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  • 43
    ISSN: 0006-3592
    Keywords: metabolism analysis ; AB fermentation equations ; on-line physiological state diagnosis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Fermentation equations for acetone-butanol (AB) were applied in a metabolic analysis of the reaction network under various conditions; that is, at different pHs and a high NADH2 turnover rate using methyl viologen, in a Clostridium acetobutylicum culture. The results disclosed variations in the pattern of rate changes that reflected changes in the physiological state. A linear relationship was found to exist between NADH2 generation and butanol production rate. By coupling an automated measurement system with the fermentation model, on-line estimation of the culture state was accomplished. Based on the AB fermentation model, new parameters were defined for on-line diagnosis of the physiological state and determination of the best timing for amplifying NADH2 generation by the addition of methyl viologen to obtain a high level of butanol productivity. A potential means of achieving optimal control for a high level of solvent production, involving the correlation of certain rates, is proposed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58: 561-571, 1998.
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  • 44
    ISSN: 0006-3592
    Keywords: cyclone design and configuration ; receiving vessel ; spray drying ; system design ; production yield ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The objective of this work was to improve a bench-top spray dryer's efficiency in both production recovery and throughput for preparing protein aerosol powders. A Büchi mini-spray dryer was used to prepare the powders of recombinant humanized anti-IgE antibody. The resulting powder's physical properties such as particle size, residual moisture, and morphology, along with its recovery and production rate was the basis of this development work. Mass balance suggests that approximately 10-20% of powder was lost in the exhaust air, consisting primarily of particles less than 2 μm. Also, significant loss (20-30%) occurred in the cyclone. Attempts were made to improve product recovery in the receiving vessel using dual-cyclone configurations, different cyclone designs, cyclones with anti-static treatment, and different receiver designs. System modifications such as replacing the original bag-filter unit with a vacuum system effectively reduced drying air flow resistance, allowing the protein to be dried at a lower inlet air temperature and the production scale to be increased. We concluded that the modified spray-drying system is advantageous over the original bench-top spray dryer. This improvement will be beneficial to early-stage research and development involving high-valued protein powders. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 301-309, 1998.
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  • 45
    ISSN: 0006-3592
    Keywords: enzyme inactivation ; modeling ; avocado ; polyphenoloxidase ; pressure stability ; thermostability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Irreversible combined pressure-temperature inactivation of the food quality related enzyme polyphenoloxidase was investigated. Inactivation rate constants (k) were obtained for about one hundred combinations of constant pressure (0.1-900 MPa) and temperature (25-77.5°C). According to the Eyring and Arrhenius equation, activation volumes and activation energies, respectively, representing pressure and temperature dependence of the inactivation rate constant, were calculated for all temperatures and pressures studied. In this way, temperature and pressure dependence of activation volume and activation energy, respectively, could be considered. Moreover, for the first time, a mathematical model describing the inactivation rate constant of a food quality-related enzyme as a function of pressure and temperature is formulated. Such pressure-temperature inactivation models for food quality-related aspects (e.g., the spoilage enzyme polyphenoloxidase) form the engineering basis for design, evaluation, and optimization of new preservation processes based on the combined effect of temperature and pressure. Furthermore, the generated methodology can be used to develop analogous kinetic models for microbiological aspects, which are needed from a safety and legislative point of view, and other quality aspects, e.g., nutritional factors, with a view of optimal quality and consumer acceptance. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 292-300, 1998.
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  • 46
    ISSN: 0006-3592
    Keywords: Penicillium chrysogenum ; phenylacetic acid ; transport ; metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Uptake of phenylacetic acid, the side-chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54-1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high-yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid-derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250-3500 μM), and in the high-yielding strain at low phenylacetic acid concentrations (2.8-100 μM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC-analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]-labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport-assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high-affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54-1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 310-316, 1998.
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  • 47
    ISSN: 0006-3592
    Keywords: yeast ; inulin ; inulase ; fructose ; secretion ; hexokinases ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The gene encoding inulase of the yeast Kluyveromyces marxianus (INU1Km) was cloned and expressed in the inulin-negative yeast Saccharomyces cerevisiae. Cells of S. cerevisiae transformed with the INU1Km gene have acquired extracellular inulase activity and were able to grow in the medium with inulin as a sole carbon source. The S. cerevisiae strain was constructed that is capable of heterologous expression of secreted K. marxianus inulase and is defective in fructose uptake due to null-mutations of the hexokinase structural genes HXK1 and HXK2. When grown in inulin-containing media, this strain is capable of accumulating at least 10% glucose-free fructose in the culture liquid. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 492-497, 1998.
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  • 48
    ISSN: 0006-3592
    Keywords: biofiltration ; toluene ; start-up ; nitrogen limitation ; heat and carbon balances ; water evaporation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Biotechnological techniques, including biofilters and biotrickling filters are increasingly used to treat air polluted with VOCs (Volatile Organic Compounds). In this work, the start-up, the effect of the gaseous ammonia addition on the toluene removal rate, and the problems of the heat accumulation on the performance of a laboratory scale biofilter were studied. The packing material was sterilized peat enriched with a mineral medium and inoculated with an adapted consortium (two yeast and five bacteria). Start-up showed a short adaptation period and an increased toluene elimination capacity (EC) up to a maximum of 190 g/m3/h. This was related to increased CO2 outlet concentration and temperature gradients between the packed bed and the inlet (Tm-Tin). These events were associated with the growth of the microbial population. The biofilter EC decreased thereafter, to attain a steady state of 8 g/m3/h. At this point, gaseous ammonia was added. EC increased up to 80 g/m3/h, with simultaneous increases on the CO2 concentration and (Tm-Tin). Two weeks after the ammonia addition, the new steady state was 30 g/m3/h. In a second ammonia addition, the maximum EC attained was 40 g/m3/h, and the biofilter was in steady state at 25 g/m3/h. Carbon, heat, and water balances were made through 88 d of biofilter operation. Emitted CO2 was about 44.5% of the theoretical value relative to the total toluene oxidation, but accumulated carbon was found as biomass, easily biodegradable material, and carbonates. Heat and water balances showed strong variations depending on EC. For 88 d the total metabolic heat was -181.2 × 103 Kcal/m3, and water evaporation was found to be 56.5 kg/m3. Evidence of nitrogen limitation, drying, and heterogeneities were found in this study. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 483-491, 1998.
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  • 49
    ISSN: 0006-3592
    Keywords: acetic acid fermentation ; whey ; fibrous-bed bioreactor ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 498-507, 1998.
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  • 50
    ISSN: 0006-3592
    Keywords: glutamine metabolism ; glucose ; fructose ; 1H/15N NMR ; glutamate dehydrogenase ; glutaminase ; ammonium ions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The glutamine metabolism was studied in glucose-starved and glucose-sufficient hybridoma and Sp2/0-Ag14 myeloma cells. Glucose starvation was attained by cultivating the hybridoma cells with fructose instead of glucose, and the myeloma cells with a low initial glucose concentration which was rapidly exhausted. Glutamine used in the experiments was labeled with 15N, either in the amine or in the amide position. The fate of the label was monitored by 1H/15N NMR analysis of released 15NH4+ and 15N-alanine. Thus, NH4+ formed via glutaminase (GLNase) could be distinguished from NH4+ formed via glutamate dehydrogenase (GDH). In the glucose-sufficient cells a small but measurable amount of 15NH4+ released by GDH could be detected in both cell lines (0.75 and 0.31 μmole/106 cells for hybridoma and myeloma cells, respectively). The uptake of glutamine and the total production of NH4+ was significantly increased in both fructose-grown hybridoma and glucose-starved myeloma cells, as compared to the glucose-sufficient cells. The increased NH4+ production was due to an increased throughput via GLNase (1.6 -1.9-fold in the hybridoma, and 2.7-fold in the myeloma cell line) and an even further increased metabolism via GDH (4.8-7.9-fold in the hybridoma cells, and 3.1-fold in the myeloma cells). The data indicate that both GLNase and GDH are down-regulated when glucose is in excess, but up-regulated in glucose-starved cells. It was calculated that the maximum potential ATP production from glutamine could increase by 35-40 % in the fructose-grown hybridoma cells, mainly due to the increased metabolism via GDH. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 508-517, 1998.
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  • 51
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 525-526 
    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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  • 52
    ISSN: 0006-3592
    Keywords: protein-protein affinity chromatography ; dextrans as spacer arms ; adsorption of immunoglobulins on protein A ; hydrolysis of casein by rennin ; immobilized protein A ; immobilized rennin ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: New dextran-agarose supports, suitable for covalent immobilization of enzymes and proteins acting on macromolecular substrates, were prepared. The thick internal fibers of agarose gels were covered by a low-density layer of long, flexible, hydrophilic, and inert dextran molecules. Rennin and protein A were immobilized on these novel supports and the resulting derivatives exhibited a very high capacity for biological recognition of soluble macromolecular substrates. Caseinolytic activity of this immobilized enzyme was 15-fold higher than activity of directly immobilized rennin, through short spacer arms, on agarose gels. Similarly, the new derivatives of immobilized protein A were able to adsorb up to 2 molecules of immunoglobulin per each molecule of immobilized protein A. When the immobilized proteins were secluded away from the support surface by using these new long and hydrophilic spacer arms, they exhibit minimal steric hindrances that could be promoted by the proximity of the support surface. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 518-523, 1998.
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  • 53
    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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  • 54
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 534-540 
    ISSN: 0006-3592
    Keywords: ω-transaminase ; kinetic modeling ; kinetic resolution ; product inhibition ; α-methylbenzylamine ; sensitivity analysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A kinetic model for ω-transaminase from Bacillus thuringiensis JS64 was developed by using the King-Altman method to simulate the kinetic resolution of α-methylbenzylamine (α-MBA). Starting from a ping-pong bi-bi mechanism, a complete kinetic model including substrate inhibition only in the reverse reaction (i.e., transamination between acetophenone and L-alanine) was developed. The asymmetric synthesis of (S)-α-MBA proved to be difficult due to a much lower maximum reverse reaction rate than the maximum forward reaction rate, thermodynamically exergonic forward reaction (i.e., transamination between (S)-α-MBA and pyruvate), and the severe product and substrate inhibition of the reverse reaction. Experimental values for kinetic parameters show that the product inhibition constant of (S)-α-MBA is the most important parameter on determining the resolution reaction rate, suggesting that the resolution reaction rate will be very low unless (S)-α-MBA strongly inhibits the reverse reaction. Using the kinetic model, the kinetic resolution of α-MBA in aqueous buffer was simulated, and the simulation results showed a high degree of consistency with experimental data over a range of reaction conditions. Various simulation results suggest that the crucial bottleneck in the kinetic resolution of α-MBA lies mainly in the accumulation of acetophenone in reaction media as the reaction proceeds, whereas L-alanine exerts a little inhibitory effect on the reaction. The model predicts that removing acetophenone produced during the reaction can enhance the reaction rate dramatically. Indeed, the biphasic reaction system is capable of extracting acetophenone from the aqueous phase, showing a much higher reaction rate compared to a monophasic reaction system. The kinetic model was also useful in predicting the properties of other, better enzymes as well as the optimal concentrations of amino acceptor and enzyme in the resolution reaction. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 534-540, 1998.
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  • 55
    ISSN: 0006-3592
    Keywords: Escherichia coli ; SOS ; DNA repair ; recombinant proteins ; promoter ; proteolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The production of several non-related heterologous proteins in recombinant Escherichia coli cells promotes a significant transcription of recA and sfiA SOS DNA repair genes. The activation of the SOS system occurs when the expression of plasmid-encoded genes is directed by the strong lambda lytic promoters, but not by IPTG-controlled promoters either at 37 or at 42°C, and it is linked to an extensive degradation of the proteins after their synthesis. The triggering signal for the SOS response could be an important arrest of cell DNA replication observed within the first hour after the induction of recombinant gene expression. The stimulation of this DNA repair system can partially account for the toxicity exhibited by recombinant proteins on actively producing E. coli cells. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 551-559, 1998.
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  • 56
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 60 (1998), S. 541-550 
    ISSN: 0006-3592
    Keywords: biofilm ; dual substrate limitation ; cometabolism ; secondary substrate ; biofilm modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A dynamic model was developed to describe the behaviour of primary and secondary substrates in a biofilm reactor. The model incorporates structured kinetics to describe the generation and consumption of reducing power in the catabolic and respiratory subsystems, respectively. Secondary substrate transformation through oxygenolytic or reductive mechanisms can be modelled under either single or dual limitation of the electron donor and electron acceptor substrates. An example simulation of a theoretical biofilm system was performed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 541-550, 1998.
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  • 57
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 58 (1998), S. 125-132 
    ISSN: 0006-3592
    Keywords: metabolic engineering ; metabolic flux analysis ; metabolic control analysis ; thermokinetics ; Saccharomyces cerevisiae ; Penicillium chrysogenum ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic engineering has been defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. With this definition metabolic engineering includes: (1) inserting new pathways in microorganisms with the aim of producing novel metabolites, e.g., production of polyketides by Streptomyces; (2) production of heterologous peptides, e.g., production of human insulin, erythropoitin, and tPA; and (3) improvement of both new and existing processes, e.g., production of antibiotics and industrial enzymes. Metabolic engineering is a multidisciplinary approach, which involves input from chemical engineers, molecular biologists, biochemists, physiologists, and analytical chemists. Obviously, molecular biology is central in the production of novel products, as well as in the improvement of existing processes. However, in the latter case, input from other disciplines is pivotal in order to target the genetic modifications; with the rapid developments in molecular biology, progress in the field is likely to be limited by procedures to identify the optimal genetic changes. Identification of the optimal genetic changes often requires a meticulous mapping of the cellular metabolism at different operating conditions, and the application of metabolic engineering to process optimization is, therefore, expected mainly to have an impact on the improvement of processes where yield, productivity, and titer are important design factors, i.e., in the production of metabolites and industrial enzymes. Despite the prospect of obtaining major improvement through metabolic engineering, this approach is, however, not expected to completely replace the classical approach to strain improvement - random mutagenesis followed by screening. Identification of the optimal genetic changes for improvement of a given process requires analysis of the underlying mechanisms, at best, at the molecular level. To reveal these mechanisms a number of different techniques may be applied: (1) detailed physiological studies, (2) metabolic flux analysis (MFA), (3) metabolic control analysis (MCA), (4) thermodynamic analysis of pathways, and (5) kinetic modeling. In this article, these different techniques are discussed and their applications to the analysis of different processes are illustrated. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:125-132, 1998.
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  • 58
    ISSN: 0006-3592
    Keywords: metabolic modeling