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1

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Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae (1994)

Dyrset, N. ; Lystad, K. Q. ; Levine, D. W.

Springer

Applied microbiology and biotechnology 41 (1994), S. 523-530

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract A high-density-cell fermentation process for production of an exracellular alginat lyase from Klebseilla pneumoniae on a defined medium has been developed. The process employs a strategy using two carbon sources. One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubililty is used as the main carbons source for growth, while the high-molecular-mass and viscoous alginate in low concentration is used as an inducer for enzyme synthesis. The repression of algiante lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources (succrose and alginate) and by supplying nitrogen source as ammonia in the pH control. No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose 1−1) was found both for the maximum final concentration of enzyme (1330 U 1−1) and for the maximum volumetric rate of enzyme production (67 U 1−1 h−1). The enzyme production dependes of the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested. The final enzyme concentration shows a fiveflod increase compare with previously reproted daata where alginate was used as a carbon source. In addition, the ratio of alginate lyase by a factor of apporximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purification of the enzyme. On the othr hand, the final dry cell weight concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight)−1 h−1.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00178483

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2

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Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae (1994)

Dyrset, N. ; Lystad, K. Q. ; Levine, D. W.

Springer

Applied microbiology and biotechnology 41 (1994), S. 523-530

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract A high-cell-density fermentation process for production of an extracellular alginate lyase from Klebsiella pneumoniae on a defined medium has been developed. The process employs a strategy using two carbon sources. One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubility is used as the main carbon source for growth, while the high-molecular-mass and viscous alginate in low concentration is used as an inducer for enzyme synthesis. The repression of alginate lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources (sucrose and alginate) and by supplying nitrogen sources as ammonia in the pH control. No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose l-1) was found both for the maximum final concentration of enzyme (1330 U l-1) and for the maximum volumetric rate of enzyme production (67 U l-1 h-1). The enzyme production depends on the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested. The final enzyme concentration shows a fivefold increase compared with previously reported data where alginate was used as a sole carbon source. In addition, the ratio of alginate lyase produced to alginate substrate consumed increased by a factor of approximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purifiction of the enzyme. On the other hand, the final dry cell weigth concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight)-1 h -1.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002530050184

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3

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Uptake of lactose and continuous lactic acid fermentation by entrapped non-growing Lactobacillus helveticus in whey permeate (1996)

Øyaas, J. ; Storrø, I. ; Levine, D. W.

Springer

Applied microbiology and biotechnology 46 (1996), S. 240-249

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Continuous production of lactic acid from lactose has been carried out in a stirred-tank reactor with non-growing Lactobacillus helveticus entrapped in calcium alginate beads. A considerably longer operation half-life was obtained in a continuously operated reactor than in a batch-operated reactor. It is possible to simulate the action of entrapped non-growing cells on the basis of information from diffusion and kinetic experiments with suspended free cells. The simulation fit the experimental data over a broad range of substrate concentrations if the specific lactic acid production rate, q P, was used as a variable parameter in the model. The dynamic mathematical model used is divided into three parts: the reactor model, which describes the mass balance in a continuously operated stirred-tank reactor with immobilized biomass, the mass-transfer model including both external diffusion and internal mass transfer, and the kinetic model for uptake of substrate on the basis of a Michaelis-Menten-type mechanism. From kinetic data obtained for free biomass experiments it was found, with the use of non-linear parameter estimation techniques, that the conversion rate of lactose by L. helveticus followed a Michaelis-Menten-type mechanism with K S at half-saturation=0.22±0.01 g/l. The maximum specific lactose uptake rate for growing cells, q S,max, varied between 4.32±0.02 g lactose g cells-1 h-1 and 4.89 ±0.02 g lactose g cells-1 h-1. The initial specific lactose uptake rate for non-growing cells, q S,0, was found to be approximately 40% of the maximum specific lactose uptake rate for growing cells.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002530050811

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4

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Changes in intracellular composition in response to hyperosmotic stress of NaCl, sucrose or glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum (1996)

Skjerdal, O. T. ; Sletta, H. ; Flenstad, S. G. ; [et al.]

Springer

Applied microbiology and biotechnology 44 (1996), S. 635-642

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Changes in intracellular composition after hyperosmotic shock were studied in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032. Both strains accumulated betaine, proline, glutamic acid, glutamine and trehalose in response to stress. The accumulated amino acids were synthesized by the cells, while betaine and trehalose were taken up from the medium. The contribution of synthesized osmoregulators was highest in C. glutamicum. In a sucrose-limited continuous culture, the increased outer osmotic pressure was balanced within 15 min for C. glutamicum and somewhat later in B. lactofermentum. The rapid regulation was due to both accumulation of osmoregulators, and shrinkage of cell and cytoplasmic volume. Immediately after shock, glutamine and glutamic acid were the dominating osmolytes. During the adaptation process, glutamine was replaced by the better osmoprotectant proline. In betaine-enriched cultures, betaine accumulation increased at the expense of glutamic acid, glutamine and trehalose. The total intracellular concentration of osmolytes increased linearly with increasing stress for all stress factors.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00172497

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5

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Changes in intracellular composition in response to hyperosmotic stress of NaCl, sucrose or glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum (1996)

Skjerdal, O. T. ; Sletta, H. ; Flenstad, S. G. ; [et al.]

Springer

Applied microbiology and biotechnology 44 (1996), S. 635-642

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Changes in intracellular composition after hyperosmotic shock were studied in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032. Both strains accumulated betaine, proline, glutamic acid, glutamine and trehalose in response to stress. The accumulated amino acids were synthesized by the cells, while betaine and trehalose were taken up from the medium. The contribution of synthesized osmoregulators was highest in C. glutamicum. In a sucrose-limited continuous culture, the increased outer osmotic pressure was balanced within 15 min for C. glutamicum and somewhat later in B. lactofermentum. The rapid regulation was due to both accumulation of osmoregulators, and shrinkage of cell and cytoplasmic volume. Immediately after shock, glutamine and glutamic acid were the dominating osmolytes. During the adaptation process, glutamine was replaced by the better osmoprotectant proline. In betaine-enriched cultures, betaine accumulation increased at the expense of glutamic acid, glutamine and trehalose. The total intracellular concentration of osmolytes increased linearly with increasing stress for all stress factors.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002530050611

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6

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Changes in cell volume, growth and respiration rate in response to hyperosmotic stress of NaCl, sucrose and glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum (1995)

Skjerdal, O. T. ; Sletta, H. ; Flenstad, S. G. ; [et al.]

Springer

Applied microbiology and biotechnology 43 (1995), S. 1099-1106

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Responses to hyperosmotic shock in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032 were studied in batch and continuous culture. The strains were chosen because they are used in commercial production of lysine and glutamic acid. Both strains, as well as the wild type of B. lactofermentum, were able to grow at high osmotic stress, at least 3 osmol/kg, from NaCl, sucrose, glutamic acid or lysine. The specific growth rate decreased in a nearly linear fashion with increasing stress. However, low stress from glutamic acid stimulated growth, especially in the wild type of B. lactofermentum. Both cell and cytoplasmic volume decreased spontaneously after hyperosmotic shock and no plasmolysis was observed. Addition of betaine stimulated the subsequent increase in the volumes. The volumes decreased linearly with increasing stress, except at low glutamic acid stress, which caused a volume increase. The respiration rate, measured as CO2 evolution, decreased immediately after shock, but increased again and stabilized within 2 h. Betaine stimulated the respiration recovery rate.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00166932

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7

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Human interferon production with diploid fibroblast cells grown on microcarriers (1979)

Giard, Donald J. ; Loeb, David H. ; Thilly, W. G. ; [et al.]

New York, NY [u.a.] : Wiley-Blackwell

Biotechnology and Bioengineering 21 (1979), S. 433-442

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ISSN: 0006-3592

Keywords: Chemistry ; Biochemistry and Biotechnology

Source: Wiley InterScience Backfile Collection 1832-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: A variety of diploid human fibroblast lines have been successfully grown to high densities (〉106 cell/ml) on recently developed microcarriers. Interferon induction using poly I·poly C and a superinduction procedure resulted in yields greater than 10,000 units/ml with one cell line. A direct comparison of microcarrier cultures to roller bottle cultures showed equivalent interferon yields on a per cell basis and some apparent differences relating to optimum inducer concentrations and kinetics of interferon accumulation.

Additional Material: 2 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/bit.260210306

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8

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Transport of osmoprotective compounds in hybridoma cells exposed to hyperosmotic stress (1995)

Øyaas, K. ; Ellingsen, T. E. ; Dyrset, N. ; [et al.]

Springer

Cytotechnology 17 (1995), S. 143-151

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ISSN: 1573-0778

Keywords: Hybridoma cells ; hyperosmotic stress ; amino acids ; amino acid analogs ; transport

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Addition of osmoprotective compounds has a positive effect on growth and monoclonal antibody production in hyperosmotic hybridoma cell cultures. In order to better understand the processes involved in the osmoprotective response, uptake of the osmoprotective compounds glycine betaine, proline, sarcosine and glycine in mouse hybridoma cell line 6H11 during exposure to hyperosmotic stress was studied. Hyperosmotic stress (510 mOsmol/kg) was introduced through the addition of NaCl (100 mM) to the growth medium, and amino acid transport activity was measured immediately after transfer of the cells to the hyperosmotic medium. The osmoprotective capability of the four osmoprotectants tested was negatively affected if methylaminosobutyric acid (MeAiB), a specific substrate for amino acid transport system A, was simultaneously included in the hyperosmotic medium in equimolar amounts with one of the osmoprotective compounds. This was due to accumulation of MeAiB in the stressed cells, giving a significant reduction in the concentration of the osmoprotective compound inside the cells. Furthermore, addition of excess meAiB gave approx. 905 reduction in the initial rate of uptake of glycine betaine, while 40–50% reduction in the initial rate of uptake of proline, glycine and sarcosine. Similarly, addition of proline, glycine or sarcosine also gave a significant reduction in the initial rate of glycine betaine uptake. These results suggest that the four osmoprotective compounds share, at least in part, a common, MeAiB inhibitable carrier for transport into osmotically stressed hybridoma cells. This carrier is probably equal to amino acid transport system A.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00749652

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