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Anthony P. J. Trinci - One of the best experts on this subject based on the ideXlab platform.
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Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in Chemostat Culture
Biotechnology and bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.
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evolution of a recombinant gucoamylase producing strain of fusarium venenatum a3 5 in Chemostat Culture
Biotechnology and Bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.
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Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in Chemostat Culture.
Biotechnology and bioengineering, 1998Co-Authors: Julie M. Withers, Marilyn G. Wiebe, Geoffrey D. Robson, Richard J. Swift, P.j. Punt, C. A. M. J. J. Van Den Hondel, Anthony P. J. TrinciAbstract:When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch Culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited Chemostat Culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (q(p)) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited Chemostat Cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow Culture system operated at a high dilution rate is an efficient way of producing this enzyme. In Chemostat Cultures grown at high dilution rates, GAM production in Chemostat Cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (q(p), 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For Chemostat Cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in Chemostat Culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged Chemostat Culture were shown to have selective advantage in the Chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask Cultures carried out with the various morphological mutants revealed that in batch Culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. The predominant morphological mutants occurring after prolonged Chemostat Culture were shown to have selective advantage in the Chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar glucoamylase (GAM) production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of GAM gene copies in the later strain was reduced, which could explain its reduced GAM production. Shake-flask Cultures of morphological mutants revealed that in batch Culture all three strains produced considerably less GAM than their parent strains. The physiological changes in these morphological mutants which contribute to this decreased level of GAM production were shown
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Extracellular proteases produced by the Quorn® myco-protein fungus Fusarium graminearum in batch and Chemostat Culture.
Microbiology (Reading England), 1997Co-Authors: Alison M. Griffen, Marilyn G. Wiebe, Geoffrey D. Robson, Anthony P. J. TrinciAbstract:Summary: Fusarium graminearum was grown in batch and continuous (Chemostat) Culture on a glucose-mineral salts medium in the presence and absence of casein. In the absence of casein no protease activity was detected in the Culture filtrate from either batch or Chemostat Culture. For batch Cultures grown on medium containing casein, most of the proteolytic activity detected in the supernatant during exponential growth had an optimum at ca pH 5.0. However, as the Cultures passed from late exponential into stationary phase, the pH profile of the protease activity broadened until most of it was in the alkaline pH region. For glucose-limited Chemostat Cultures grown on media containing casein, protease activity had a narrow pH optimum with maximum activity at pH 5.0. For all concentrations of casein examined, protease activity was greater in Chemostat Culture than in batch Culture. Extracellular proteases from batch and Chemostat Cultures were purified by bacitracin-Sepharose affinity chromatography. At least seven proteins were purified from batch Cultures but Chemostat Cultures contained only a single aspartic protease with a molecular mass of 40 kDa.
Marilyn G. Wiebe - One of the best experts on this subject based on the ideXlab platform.
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Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in Chemostat Culture
Biotechnology and bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.
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evolution of a recombinant gucoamylase producing strain of fusarium venenatum a3 5 in Chemostat Culture
Biotechnology and Bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.
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Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in Chemostat Culture.
Biotechnology and bioengineering, 1998Co-Authors: Julie M. Withers, Marilyn G. Wiebe, Geoffrey D. Robson, Richard J. Swift, P.j. Punt, C. A. M. J. J. Van Den Hondel, Anthony P. J. TrinciAbstract:When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch Culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited Chemostat Culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (q(p)) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited Chemostat Cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow Culture system operated at a high dilution rate is an efficient way of producing this enzyme. In Chemostat Cultures grown at high dilution rates, GAM production in Chemostat Cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (q(p), 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For Chemostat Cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in Chemostat Culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged Chemostat Culture were shown to have selective advantage in the Chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask Cultures carried out with the various morphological mutants revealed that in batch Culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. The predominant morphological mutants occurring after prolonged Chemostat Culture were shown to have selective advantage in the Chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar glucoamylase (GAM) production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of GAM gene copies in the later strain was reduced, which could explain its reduced GAM production. Shake-flask Cultures of morphological mutants revealed that in batch Culture all three strains produced considerably less GAM than their parent strains. The physiological changes in these morphological mutants which contribute to this decreased level of GAM production were shown
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Extracellular proteases produced by the Quorn® myco-protein fungus Fusarium graminearum in batch and Chemostat Culture.
Microbiology (Reading England), 1997Co-Authors: Alison M. Griffen, Marilyn G. Wiebe, Geoffrey D. Robson, Anthony P. J. TrinciAbstract:Summary: Fusarium graminearum was grown in batch and continuous (Chemostat) Culture on a glucose-mineral salts medium in the presence and absence of casein. In the absence of casein no protease activity was detected in the Culture filtrate from either batch or Chemostat Culture. For batch Cultures grown on medium containing casein, most of the proteolytic activity detected in the supernatant during exponential growth had an optimum at ca pH 5.0. However, as the Cultures passed from late exponential into stationary phase, the pH profile of the protease activity broadened until most of it was in the alkaline pH region. For glucose-limited Chemostat Cultures grown on media containing casein, protease activity had a narrow pH optimum with maximum activity at pH 5.0. For all concentrations of casein examined, protease activity was greater in Chemostat Culture than in batch Culture. Extracellular proteases from batch and Chemostat Cultures were purified by bacitracin-Sepharose affinity chromatography. At least seven proteins were purified from batch Cultures but Chemostat Cultures contained only a single aspartic protease with a molecular mass of 40 kDa.
Geoffrey D. Robson - One of the best experts on this subject based on the ideXlab platform.
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Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in Chemostat Culture
Biotechnology and bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.
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evolution of a recombinant gucoamylase producing strain of fusarium venenatum a3 5 in Chemostat Culture
Biotechnology and Bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.
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Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in Chemostat Culture.
Biotechnology and bioengineering, 1998Co-Authors: Julie M. Withers, Marilyn G. Wiebe, Geoffrey D. Robson, Richard J. Swift, P.j. Punt, C. A. M. J. J. Van Den Hondel, Anthony P. J. TrinciAbstract:When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 ± 8 mg (mean ± S.E.) glucoamylase (GAM) L-1 in batch Culture and 373 ± 9 mg GAM L-1 in maltodextrin- limited Chemostat Culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (q(p)) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited Chemostat Cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow Culture system operated at a high dilution rate is an efficient way of producing this enzyme. In Chemostat Cultures grown at high dilution rates, GAM production in Chemostat Cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (q(p), 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For Chemostat Cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 ± 12 to 496 ± 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 ± 0.4 to 16.4 ± 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in Chemostat Culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged Chemostat Culture were shown to have selective advantage in the Chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask Cultures carried out with the various morphological mutants revealed that in batch Culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production. The predominant morphological mutants occurring after prolonged Chemostat Culture were shown to have selective advantage in the Chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar glucoamylase (GAM) production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of GAM gene copies in the later strain was reduced, which could explain its reduced GAM production. Shake-flask Cultures of morphological mutants revealed that in batch Culture all three strains produced considerably less GAM than their parent strains. The physiological changes in these morphological mutants which contribute to this decreased level of GAM production were shown
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Extracellular proteases produced by the Quorn® myco-protein fungus Fusarium graminearum in batch and Chemostat Culture.
Microbiology (Reading England), 1997Co-Authors: Alison M. Griffen, Marilyn G. Wiebe, Geoffrey D. Robson, Anthony P. J. TrinciAbstract:Summary: Fusarium graminearum was grown in batch and continuous (Chemostat) Culture on a glucose-mineral salts medium in the presence and absence of casein. In the absence of casein no protease activity was detected in the Culture filtrate from either batch or Chemostat Culture. For batch Cultures grown on medium containing casein, most of the proteolytic activity detected in the supernatant during exponential growth had an optimum at ca pH 5.0. However, as the Cultures passed from late exponential into stationary phase, the pH profile of the protease activity broadened until most of it was in the alkaline pH region. For glucose-limited Chemostat Cultures grown on media containing casein, protease activity had a narrow pH optimum with maximum activity at pH 5.0. For all concentrations of casein examined, protease activity was greater in Chemostat Culture than in batch Culture. Extracellular proteases from batch and Chemostat Cultures were purified by bacitracin-Sepharose affinity chromatography. At least seven proteins were purified from batch Cultures but Chemostat Cultures contained only a single aspartic protease with a molecular mass of 40 kDa.
Alan T. Bull - One of the best experts on this subject based on the ideXlab platform.
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Vancomycin production is enhanced in Chemostat Culture with biomass-recycle.
Biotechnology and bioengineering, 1999Co-Authors: J.j. Mcintyre, Alan William Bunch, Alan T. BullAbstract:Production of the glycopeptide antibiotic vancomycin by Amycolatopsis orientalis ATCC 19795 was examined in phosphate-limited Chemostat Cultures with biomass-recycle, employing an oscillating membrane separator, at a constant dilution rate (D = 0.14 h(-1)). Experiments made under low agitation conditions (600 rpm) showed that the biomass concentration could be increased 3.9-fold with vancomycin production kinetics very similar to that of Chemostat Culture without biomass-recycle. The specific production rate (q(vancomycin)) was maximal when the biomass-recycle ratio (R) was 0.13 (D = 0.087 h(-1)). When the dissolved oxygen tension dropped below 20% (air saturation), the biomass and vancomycin concentrations decreased and an unidentified red metabolite was released into the Culture medium. Using increased agitation (850 rpm), used to maintain the dissolved oxygen tension above 20% air saturation, maximum increases in biomass concentration (7.9-fold) and vancomcyin production 1.6-fold (0.6 mg/g dry weight/h) were obtained when R was 0.44(D = 0.056 h(-1)) compared to Chemostat Culture without biomass-recycle. Moreover, at this latter recycle ratio the volumetric vancomycin production rate was 14.7 mg/L/h (a 7-fold increase compared to Chemostat Culture without biomass-recycle). These observations encourage further research on biomass-recycling as a means of optimising the production of antibiotics. (C) 1999 John Wiley & Sons, Inc.
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the effect of the dilution rate on cho cell physiology and recombinant interferon gamma production in glucose limited Chemostat Culture
Biotechnology and Bioengineering, 1993Co-Authors: Paul M Hayter, Elisabeth M A Curling, Malcolm L Gould, Anthony J Baines, Nigel Jenkins, Ian Salmon, Philip G Strange, Alan T. BullAbstract:The physiology of a recombinant Chinese hamster ovary cell line in glucose-limited Chemostat Culture was studied over a range of dilution rates (D = 0.008 to 0.20 h(-1)). The specific growth rate (micro) deviated from D at low dilution rates due to an increased specific death rate. Extrapolation of these data suggested a minimum specific growth rate of 0.011 h(-1) (micro(max) = 0.025 h(-1)) The metabolism at each steady state was characterized by determining the metabolic quotients for glucose, lactate, ammonia, amino acids, and interferon-gamma (IFN-gamma). The specific rate of glucose uptake increased linearly with mu, and the saturation constant for glucose (K(s)) was calculated to be 59.6 microM. There was a linear increase in the rate of lactate production with a higher yield of lactate from glucose at high growth rates. The decline in the rate of production of lactate, alanine, and serine at low growth rate was consistent with the limitation of the glycolytic pathway by glucose. The specific rate of IFN-gamma production increased with mu in a manner indicative of a growth-related product. Despite changes in the IFN-gamma production rate and cell physiology, the pattern of IFN-gamma glycosylation was similar at all except the lowest growth rates where there was increased production of nonglycosylated IFN-gamma.
Jeff Shuster - One of the best experts on this subject based on the ideXlab platform.
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Evolution of a recombinant (gucoamylase‐producing) strain of Fusarium venenatum A3/5 in Chemostat Culture
Biotechnology and bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.
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evolution of a recombinant gucoamylase producing strain of fusarium venenatum a3 5 in Chemostat Culture
Biotechnology and Bioengineering, 2001Co-Authors: Marilyn G. Wiebe, Geoffrey D. Robson, Jeff Shuster, Anthony P. J. TrinciAbstract:Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited Chemostat Cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 Chemostat. The highly branched mutants detected in this Chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 Chemostat complemented representative strains previously isolated from glucose-limited Chemostat Cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 Chemostat Culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 Chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 73: 146–156, 2001.