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J. J. Heijnen - One of the best experts on this subject based on the ideXlab platform.
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influence of temperature on growth and Ajmalicine production by catharantus roseus suspension cultures
Enzyme and Microbial Technology, 2002Co-Authors: Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, J. J. HeijnenAbstract:Industrial production of valuable secondary metabolites by plant cell cultures is generally hampered by low productivity. This productivity is controlled by several factors, one of these is temperature. Secondary metabolites are in most cases produced in a two-stage process: biomass growth, followed by secondary metabolite production. In part I of this study the optimal temperature for biomass growth was investigated aiming at: maximal formation of biosynthetic active biomass, minimal formation of useless by-products. These processes each had their characteristic temperature dependence. The growth of Catharanthus roseus biomass for the production of Ajmalicine was found to be optimal at 27.5°C. The effects of oxygen limitation are discussed. In part II the temperature effect on Ajmalicine production was investigated. The productivity was governed by two processes: induction and production. Induction and production were both optimal at 27.5°C. The length of the induction period was easily estimated from a rapid concentration decrease of the precursor tryptamine.
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a simple structured model for maintenance biomass formation and Ajmalicine production by nondividing catharanthus roseus cells
Biotechnology and Bioengineering, 1999Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. J. HeijnenAbstract:The stoichiometry of maintenance and carbo- hydrate storage as well as Ajmalicine production kinetics of non-dividing Catharanthus roseus cells in the second stage of a two-stage batch process were investigated. For the mathematical description of these processes, a simple structured model with 5 parameters is proposed. In the model the biomass is divided in two compart- ments: active biomass and storage carbohydrates. In in- duction medium (standard medium without phosphate, nitrogen and hormones), biomass formation, glucose consumption, and CO2 production appeared to be con- stant in time. Therefore, it is assumed that the active biomass level is constant. The maintenance coefficient mS, and the yield of storage carbohydrates on glucose YSC were optimized by fitting the model on experimental data: 0.003 C-mol/C-mol/h and 0.82 C-mol/C-mol, respec- tively. Production kinetics were incorporated in this model and related to the active biomass fraction. The maximum specific Ajmalicine production rate qp max was fitted on the data: 7.5 µmol/C-mol/h. The model was tested at several different experimental conditions, and proved to describe the experimental results adequately. An independent experiment at a very high cell density in order to obtain maximum product formation was used to validate the model. It provided a satisfactory description of the results, but the final Ajmalicine concentration (198 µmol/L after 18 days) was lower than the calculated maximum, due to accumulation of inhibiting gaseous metabolites. © 1999 John Wiley & Sons, Inc. Biotechnol Bio- eng 66: 147-157, 1999.
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the role of glucose in Ajmalicine production by catharanthus roseus cell cultures
Biotechnology and Bioengineering, 1995Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. L. Vinke, C M A Koolhaas, J. J. HeijnenAbstract:The role of glucose in Ajmalicine production by Catharanthus roseus was investigated in the second stage of a two-stage batch process. Activities of tryptophan decar-boxylate (TDC) and anthranilate synthase (AS), two enzymes In the pathway leading to Ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support Ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific Ajmalicine production rate, q(p), was investigated in seven (fed-)batch cultures with constant glucose concentrations: 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) the q(p) was 2.7-times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the Ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration and q(p). Differences in enzyme induction and Ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch. (c) 1995 John Wiley & Sons Inc.
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Two-stage batch process for the production of Ajmalicine by Catharanthus roseus: The link between growth and production stage.
Biotechnology and bioengineering, 1995Co-Authors: Jurriaan E. Schlatmann, Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, M. Sellés, J. J. HeijnenAbstract:The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, Ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the Ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated Ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high Ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. © 1995 John Wiley & Sons, Inc.
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relation between dissolved oxygen concentration and Ajmalicine production rate in high density cultures of catharanthus roseus
Biotechnology and Bioengineering, 1995Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. L. Vinke, J. J. HeijnenAbstract:The relation between dissolved oxygen (DO) and the Ajmalicine production rate of Catharanthus roseus was investigated in 15-L tank reactors at constant stirrer speed and gas flow rate. Below a DO concentration of 29% of air saturation the Ajmalicine production rate was less than 0.06 μmol/g/d. Above a DO of 43% the Ajmalicine production rate was constant at 0.21 μmol/g/d. Between a DO of 29% and 43% there was a strong relation between the Ajmalicine production rate and the DO concentration. After a period of at least 12 days at DO ⩽29% the culture lacked the ability to adapt to a DO ⩾57%. A kinetic equation is proposed for the relation between DO and the specific Ajmalicine production rate. © 1995 John Wiley & Sons, Inc.
Hens J.g. Ten Hoopen - One of the best experts on this subject based on the ideXlab platform.
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influence of temperature on growth and Ajmalicine production by catharantus roseus suspension cultures
Enzyme and Microbial Technology, 2002Co-Authors: Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, J. J. HeijnenAbstract:Industrial production of valuable secondary metabolites by plant cell cultures is generally hampered by low productivity. This productivity is controlled by several factors, one of these is temperature. Secondary metabolites are in most cases produced in a two-stage process: biomass growth, followed by secondary metabolite production. In part I of this study the optimal temperature for biomass growth was investigated aiming at: maximal formation of biosynthetic active biomass, minimal formation of useless by-products. These processes each had their characteristic temperature dependence. The growth of Catharanthus roseus biomass for the production of Ajmalicine was found to be optimal at 27.5°C. The effects of oxygen limitation are discussed. In part II the temperature effect on Ajmalicine production was investigated. The productivity was governed by two processes: induction and production. Induction and production were both optimal at 27.5°C. The length of the induction period was easily estimated from a rapid concentration decrease of the precursor tryptamine.
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a simple structured model for maintenance biomass formation and Ajmalicine production by nondividing catharanthus roseus cells
Biotechnology and Bioengineering, 1999Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. J. HeijnenAbstract:The stoichiometry of maintenance and carbo- hydrate storage as well as Ajmalicine production kinetics of non-dividing Catharanthus roseus cells in the second stage of a two-stage batch process were investigated. For the mathematical description of these processes, a simple structured model with 5 parameters is proposed. In the model the biomass is divided in two compart- ments: active biomass and storage carbohydrates. In in- duction medium (standard medium without phosphate, nitrogen and hormones), biomass formation, glucose consumption, and CO2 production appeared to be con- stant in time. Therefore, it is assumed that the active biomass level is constant. The maintenance coefficient mS, and the yield of storage carbohydrates on glucose YSC were optimized by fitting the model on experimental data: 0.003 C-mol/C-mol/h and 0.82 C-mol/C-mol, respec- tively. Production kinetics were incorporated in this model and related to the active biomass fraction. The maximum specific Ajmalicine production rate qp max was fitted on the data: 7.5 µmol/C-mol/h. The model was tested at several different experimental conditions, and proved to describe the experimental results adequately. An independent experiment at a very high cell density in order to obtain maximum product formation was used to validate the model. It provided a satisfactory description of the results, but the final Ajmalicine concentration (198 µmol/L after 18 days) was lower than the calculated maximum, due to accumulation of inhibiting gaseous metabolites. © 1999 John Wiley & Sons, Inc. Biotechnol Bio- eng 66: 147-157, 1999.
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the role of glucose in Ajmalicine production by catharanthus roseus cell cultures
Biotechnology and Bioengineering, 1995Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. L. Vinke, C M A Koolhaas, J. J. HeijnenAbstract:The role of glucose in Ajmalicine production by Catharanthus roseus was investigated in the second stage of a two-stage batch process. Activities of tryptophan decar-boxylate (TDC) and anthranilate synthase (AS), two enzymes In the pathway leading to Ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support Ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific Ajmalicine production rate, q(p), was investigated in seven (fed-)batch cultures with constant glucose concentrations: 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) the q(p) was 2.7-times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the Ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration and q(p). Differences in enzyme induction and Ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch. (c) 1995 John Wiley & Sons Inc.
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Two-stage batch process for the production of Ajmalicine by Catharanthus roseus: The link between growth and production stage.
Biotechnology and bioengineering, 1995Co-Authors: Jurriaan E. Schlatmann, Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, M. Sellés, J. J. HeijnenAbstract:The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, Ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the Ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated Ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high Ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. © 1995 John Wiley & Sons, Inc.
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relation between dissolved oxygen concentration and Ajmalicine production rate in high density cultures of catharanthus roseus
Biotechnology and Bioengineering, 1995Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. L. Vinke, J. J. HeijnenAbstract:The relation between dissolved oxygen (DO) and the Ajmalicine production rate of Catharanthus roseus was investigated in 15-L tank reactors at constant stirrer speed and gas flow rate. Below a DO concentration of 29% of air saturation the Ajmalicine production rate was less than 0.06 μmol/g/d. Above a DO of 43% the Ajmalicine production rate was constant at 0.21 μmol/g/d. Between a DO of 29% and 43% there was a strong relation between the Ajmalicine production rate and the DO concentration. After a period of at least 12 days at DO ⩽29% the culture lacked the ability to adapt to a DO ⩾57%. A kinetic equation is proposed for the relation between DO and the specific Ajmalicine production rate. © 1995 John Wiley & Sons, Inc.
Michael L Shuler - One of the best experts on this subject based on the ideXlab platform.
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Sparge gas composition affects biomass and Ajmalicine production from immobilized cell cultures of Catharanthus roseus
Enzyme and Microbial Technology, 2005Co-Authors: Carolyn W. T. Lee-parsons, Michael L ShulerAbstract:Abstract Despite their low solubility in aqueous medium, dissolved gases play important roles in the cultivation and successful scale-up of plant cell cultures. In this paper, the effects of O 2 and CO 2 on growth and secondary metabolism were investigated using the production of Ajmalicine from Catharanthus roseus cultures. The effects of gas composition were investigated using shear-protected alginate-immobilized cells (diameter 2 and CO 2 . A wider range of concentrations (10–95% O 2 , 0.03–10% CO 2 by mole) was studied to explore potential benefits or drawbacks. Sparge gas composition significantly altered growth and Ajmalicine production. Low and high O 2 concentrations (10, 90, 95% O 2 ) were either inhibitory or toxic to growth and Ajmalicine production. The effects of CO 2 depended on O 2 concentration. At lower O 2 concentrations (21% O 2 ), increasing the CO 2 concentration decreased both growth and specific Ajmalicine production. At higher O 2 concentrations (78.4% O 2 ), increasing the CO 2 concentration decreased growth while specific Ajmalicine production was not affected. In these studies, extracellular Ajmalicine concentration was maximized with a gas mixture of 50% O 2 + 0.03% CO 2 .
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the effect of Ajmalicine spiking and resin addition timing on the production of indole alkaloids from catharanthus roseus cell cultures
Biotechnology and Bioengineering, 2002Co-Authors: Carolyn W T Leeparsons, Michael L ShulerAbstract:The potential for the feedback inhibition of indole alkaloid synthesis was investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or 18 mg/L Ajmalicine on day 0. The production of Ajmalicine, catharanthine, and serpentine were inhibited in a dose-dependent manner. The inhibition was transient as the exogenous Ajmalicine was ultimately either metabolized in the medium or within the cell. The addition of neutral resin has previously been shown to enhance Ajmalicine production. To minimize product inhibition and product metabolism, Amberlite XAD-7 resin was added to immobilized cultures of C. roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for spent resin every 5 days. The addition of resin did not decrease the viability of the culture. Growth was reduced only in cultures with resin added on day 0. Alkaloid production was enhanced to different extents by the timing of resin addition, suggesting that feedback inhibition or product metabolism was present throughout the culture period. Ajmalicine recovery was nearly 100% when the resin was added initially either on day 0 or day 5. Ajmalicine recovery was reduced to 55% when the resin was added later in the culture period starting on day 15, presumably because of resin saturation or the inaccessibility of alkaloids trapped in the vacuole. Delaying the addition of XAD-7 resin until 5 days after the start of the culture resulted in the highest improvement in Ajmalicine production, i.e approximately 70% and also resulted in the complete recovery of Ajmalicine from the cell.
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The effect of inoculum density and conditioned medium on the production of Ajmalicine and catharanthine from immobilized Catharanthus roseus cells
Biotechnology and bioengineering, 2000Co-Authors: Carolyn W. T. Lee, Michael L ShulerAbstract:The effect of the cell-inoculum size and the addition of conditioned medium on Ajmalicine and catharanthine production were studied using immobilized Catharanthus roseus cells. Higher specific-uptake rates of ammonium, nitrate, and sugars were observed in the low-inoculum-density cultures (50 g FW/L) compared to the high-inoculum-density cultures (100 g FW/L). Alkaloid production was not correlated with the exhaustion of a particular nutrient from the medium. The high-inoculum-density cultures produced higher Ajmalicine concentrations throughout the experiment. Catharanthine production was similar between the two inoculum-density cultures. The addition of conditioned medium to MS-production medium dramatically improved the production of Ajmalicine and catharanthine. The addition of conditioned medium enhanced Ajmalicine production from immobilized Catharanthus roseus cultures on day 15 by at least two- to fourfold compared to media without the conditioning factors. Catharanthine production was increased by nearly fivefold in cultures with conditioned medium compared to those without conditioned medium. The enhancing effects of conditioned medium on alkaloid production were attributed to an unidentified factor produced and secreted by suspension cultures of C. roseus. The presence of conditioned medium also decreased the sucrose hydrolysis rate. The Ajmalicine concentration in these immobilized cell cultures was found to be a function of the fresh-weight concentration, irrespective of the inoculum density or the culture medium. The medium choice and the inoculum density determined how rapidly fresh weight was accumulated and thus, how quickly Ajmalicine was produced. Ajmalicine production correlated positively with fresh-weight concentration, but catharanthine production was not correlated with fresh-weight concentration.
Robert Verpoorte - One of the best experts on this subject based on the ideXlab platform.
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influence of temperature on growth and Ajmalicine production by catharantus roseus suspension cultures
Enzyme and Microbial Technology, 2002Co-Authors: Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, J. J. HeijnenAbstract:Industrial production of valuable secondary metabolites by plant cell cultures is generally hampered by low productivity. This productivity is controlled by several factors, one of these is temperature. Secondary metabolites are in most cases produced in a two-stage process: biomass growth, followed by secondary metabolite production. In part I of this study the optimal temperature for biomass growth was investigated aiming at: maximal formation of biosynthetic active biomass, minimal formation of useless by-products. These processes each had their characteristic temperature dependence. The growth of Catharanthus roseus biomass for the production of Ajmalicine was found to be optimal at 27.5°C. The effects of oxygen limitation are discussed. In part II the temperature effect on Ajmalicine production was investigated. The productivity was governed by two processes: induction and production. Induction and production were both optimal at 27.5°C. The length of the induction period was easily estimated from a rapid concentration decrease of the precursor tryptamine.
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Two-stage batch process for the production of Ajmalicine by Catharanthus roseus: The link between growth and production stage.
Biotechnology and bioengineering, 1995Co-Authors: Jurriaan E. Schlatmann, Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, M. Sellés, J. J. HeijnenAbstract:The link between the growth stage and the production stage in a two-stage batch process was investigated using (filtered) inocula from different periods of the stationary phase of the growth cycle. In the production stage, Ajmalicine production by Catharanthus roseus in a 3-L stirred tank reactor was induced with a high glucose concentration (80 g/L). Ajmalicine production in cultures started with cells from the late stationary phase was five times higher than in cultures started with cells from the early stationary phase. After transfer to the production stage, cells from the early stationary phase showed a transient increase in respiration and enzyme induction, followed by culture browning. In contrast, cells in the late stationary phase showed a typical induction pattern: constant respiration, and permanent enzyme induction. A striking similarity between the geraniol-10-hydroxylase (G10H) activity and the Ajmalicine accumulation profile could be observed in all cultures, suggesting that G 10H regulated Ajmalicine production in this investigation. The intracellular nitrate concentration was significantly higher in the inoculum showing a high Ajmalicine production than in the inoculum with a low production. Consequently, nitrate may act as a marker for the start of the production stage: as soon as the nitrate is depleted in the growth medium secondary metabolism can be induced. © 1995 John Wiley & Sons, Inc.
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effects of oxygen and nutrients limitation on Ajmalicine production and related enzyme activities in high density cultures of catharanthus roseus
Biotechnology and Bioengineering, 1994Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, J. J. HeijnenAbstract:Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific Ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on Ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in Ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. (c) 1994 John Wiley & Sons, Inc.
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Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to bioreactor
Primary and Secondary Metabolism of Plants and Cell Cultures III, 1994Co-Authors: Hens J.g. Ten Hoopen, Walter M. Van Gulik, Jurriaan E. Schlatmann, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, J. J. Heijnen, Robert VerpoorteAbstract:The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of Ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of Ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the Ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation.
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Ajmalicine metabolism in Catharanthus roseus cell cultures
Phytochemistry, 1994Co-Authors: Rosana I. Dos Santos, Jan Schripsema, Robert VerpoorteAbstract:Abstract Ajmalicine degradation was investigated in a low alkaloid-accumulating Catharanthus roseus cell culture, and compared with the chemical degradation which occurred in medium without cells. Degradation was found to be faster in the presence of cells. By adding [Me- 2 H 3 ] Ajmalicine to the cell culture the production and the degradation rates were estimated. Both were relatively high at the beginning of the growth phase while the degradation rate was also high in the stationary phase.
J E Schlatmann - One of the best experts on this subject based on the ideXlab platform.
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a simple structured model for maintenance biomass formation and Ajmalicine production by nondividing catharanthus roseus cells
Biotechnology and Bioengineering, 1999Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. J. HeijnenAbstract:The stoichiometry of maintenance and carbo- hydrate storage as well as Ajmalicine production kinetics of non-dividing Catharanthus roseus cells in the second stage of a two-stage batch process were investigated. For the mathematical description of these processes, a simple structured model with 5 parameters is proposed. In the model the biomass is divided in two compart- ments: active biomass and storage carbohydrates. In in- duction medium (standard medium without phosphate, nitrogen and hormones), biomass formation, glucose consumption, and CO2 production appeared to be con- stant in time. Therefore, it is assumed that the active biomass level is constant. The maintenance coefficient mS, and the yield of storage carbohydrates on glucose YSC were optimized by fitting the model on experimental data: 0.003 C-mol/C-mol/h and 0.82 C-mol/C-mol, respec- tively. Production kinetics were incorporated in this model and related to the active biomass fraction. The maximum specific Ajmalicine production rate qp max was fitted on the data: 7.5 µmol/C-mol/h. The model was tested at several different experimental conditions, and proved to describe the experimental results adequately. An independent experiment at a very high cell density in order to obtain maximum product formation was used to validate the model. It provided a satisfactory description of the results, but the final Ajmalicine concentration (198 µmol/L after 18 days) was lower than the calculated maximum, due to accumulation of inhibiting gaseous metabolites. © 1999 John Wiley & Sons, Inc. Biotechnol Bio- eng 66: 147-157, 1999.
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the role of glucose in Ajmalicine production by catharanthus roseus cell cultures
Biotechnology and Bioengineering, 1995Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. L. Vinke, C M A Koolhaas, J. J. HeijnenAbstract:The role of glucose in Ajmalicine production by Catharanthus roseus was investigated in the second stage of a two-stage batch process. Activities of tryptophan decar-boxylate (TDC) and anthranilate synthase (AS), two enzymes In the pathway leading to Ajmalicine, were higher after induction with 40 g/L glucose than after induction with 60 or 80 g/L glucose. Experiments with different media containing mixtures of glucose and the nonpermeating osmotic agent xylose, and using an already induced culture as inoculum, revealed that a minimum amount of glucose is required to support Ajmalicine production after enzyme induction. This requirement was not an osmotic effect. The relation between the glucose concentration and the specific Ajmalicine production rate, q(p), was investigated in seven (fed-)batch cultures with constant glucose concentrations: 23, 29, 35, 53, 57, 75, and 98 g/L. In the cultures with a low glucose concentration (23, 29, and 35 g/L) the q(p) was 2.7-times higher than the cultures with 53 and 57 g/L, and almost six times higher than the cultures with a high glucose concentration (75 and 98 g/L). A glucose perturbation experiment (from 53 to 32 g/L) demonstrated that the Ajmalicine production rate was adjusted without much delay. A kinetic equation is proposed for the relationship between the glucose concentration and q(p). Differences in enzyme induction and Ajmalicine production at different glucose levels could not be explained by the intracellular concentrations of glucose, fructose, sucrose, or starch. (c) 1995 John Wiley & Sons Inc.
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relation between dissolved oxygen concentration and Ajmalicine production rate in high density cultures of catharanthus roseus
Biotechnology and Bioengineering, 1995Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, J. L. Vinke, J. J. HeijnenAbstract:The relation between dissolved oxygen (DO) and the Ajmalicine production rate of Catharanthus roseus was investigated in 15-L tank reactors at constant stirrer speed and gas flow rate. Below a DO concentration of 29% of air saturation the Ajmalicine production rate was less than 0.06 μmol/g/d. Above a DO of 43% the Ajmalicine production rate was constant at 0.21 μmol/g/d. Between a DO of 29% and 43% there was a strong relation between the Ajmalicine production rate and the DO concentration. After a period of at least 12 days at DO ⩽29% the culture lacked the ability to adapt to a DO ⩾57%. A kinetic equation is proposed for the relation between DO and the specific Ajmalicine production rate. © 1995 John Wiley & Sons, Inc.
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the negligible role of carbon dioxide and ethylene in Ajmalicine production by catharanthus roseus cell suspensions
Plant Cell Reports, 1994Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, E Fonck, J. J. HeijnenAbstract:Removal of gaseous metabolites in an aerated fermenter affects Ajmalicine production by Catharanthus roseus negatively. Therefore, the role of CO2 and ethylene in Ajmalicine production by C. roseus was investigated in 3 l fermenters (working volume 1.8 l) with recirculation of a large part of the exhaust air. Removal of CO2, ethylene or both from the recirculation stream did not have an effect on Ajmalicine production. Inhibition of ethylene biosynthesis in shake flasks with Co2+, Ni2+ or aminooxyacetic acid did not affect Ajmalicine production. However, the removal of CO2 did enhance the amount of extracellular Ajmalicine.
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effects of oxygen and nutrients limitation on Ajmalicine production and related enzyme activities in high density cultures of catharanthus roseus
Biotechnology and Bioengineering, 1994Co-Authors: J E Schlatmann, Hens J.g. Ten Hoopen, Paulo Roberto Hrihorowitsch Moreno, J. L. Vinke, Robert Verpoorte, J. J. HeijnenAbstract:Oxygen and nutrient limitation was investigated in order to identify the origin of a lower specific Ajmalicine production in Catharanthus roseus cultures at high cell densities in an induction medium. The effect of oxygen limitation was explored by comparing two identically aerated and agitated high cell density bioreactor cultures with dissolved oxygen (DO) concentration of 15% and 85% of air saturation, with respect to alkaloid formation and related enzymes activities. Oxygen had an evident effect on Ajmalicine production: in the high DO cultures production was more than 5 times higher than in the low DO cultures. The difference in Ajmalicine production between high and low DO could not be explained by the enzyme activity profiles. Moreover, the productivity in the high density culture could not restored to the level of a low density culture (at a high DO) by increasing the DO alone. The effect of nutrient limitation was studied with response surface methodology in shake flask cultures. Nutrient limitation could not be demonstrated to be responsible for the productivity loss. Alkaloid and enzyme measurements in the shake flask cultures supported previous findings that the tryptamine pathway may regulate alkaloid production, provided that the terpenoid pathway is sufficiently active. (c) 1994 John Wiley & Sons, Inc.