Influence of temperature on the production of archaeal thermoactive alcohol dehydrogenases from Pyrococcus furiosus with recombinant E. coli

J. Kube, C. Brokamp, M.P. Machielsen, J. van der Oost, H. Markl

Research output: Contribution to journalArticleAcademicpeer-review

9 Citations (Scopus)

Abstract

The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD600 of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD600 of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD600 of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular
LanguageEnglish
Pages221-227
JournalExtremophiles
Volume10
Issue number3
DOIs
Publication statusPublished - 2006

Fingerprint

Pyrococcus furiosus
Alcohol Dehydrogenase
Escherichia coli
Temperature
Shock
Hot Temperature
Isopropyl Thiogalactoside
Proteins
Inclusion Bodies
Bioreactors
Cell Count
Weights and Measures

Keywords

  • high-cell-density
  • hyperthermophilic archaea
  • biochemical-characterization
  • sulfolobus-solfataricus
  • expression
  • cloning
  • growth
  • gene
  • fermentation
  • overexpression

Cite this

@article{ad55732f7fb445bdb476ed46ef3b9a26,
title = "Influence of temperature on the production of archaeal thermoactive alcohol dehydrogenases from Pyrococcus furiosus with recombinant E. coli",
abstract = "The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD600 of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD600 of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD600 of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular",
keywords = "high-cell-density, hyperthermophilic archaea, biochemical-characterization, sulfolobus-solfataricus, expression, cloning, growth, gene, fermentation, overexpression",
author = "J. Kube and C. Brokamp and M.P. Machielsen and {van der Oost}, J. and H. Markl",
year = "2006",
doi = "10.1007/s00792-005-0490-z",
language = "English",
volume = "10",
pages = "221--227",
journal = "Extremophiles",
issn = "1431-0651",
publisher = "Springer Verlag",
number = "3",

}

Influence of temperature on the production of archaeal thermoactive alcohol dehydrogenases from Pyrococcus furiosus with recombinant E. coli. / Kube, J.; Brokamp, C.; Machielsen, M.P.; van der Oost, J.; Markl, H.

In: Extremophiles, Vol. 10, No. 3, 2006, p. 221-227.

Research output: Contribution to journalArticleAcademicpeer-review

TY - JOUR

T1 - Influence of temperature on the production of archaeal thermoactive alcohol dehydrogenases from Pyrococcus furiosus with recombinant E. coli

AU - Kube, J.

AU - Brokamp, C.

AU - Machielsen, M.P.

AU - van der Oost, J.

AU - Markl, H.

PY - 2006

Y1 - 2006

N2 - The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD600 of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD600 of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD600 of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular

AB - The heterologous production of a thermoactive alcohol dehydrogenase (AdhC) from Pyrococcus furiosus in Escherichia coli was investigated. E. coli was grown in a fed-batch bioreactor in minimal medium to high cell densities (cell dry weight 76 g/l, OD600 of 150). Different cultivation strategies were applied to optimize the production of active AdhC, such as lowering the cultivation temperature from 37 to 28°C, heat shock of the culture from 37 to 42°C and from 37 to 45°C, and variation of time of induction (induction at an OD600 of 40, 80 and 120). In addition to the production of active intracellular protein, inclusion bodies were always observed. The maximal activity of 30 U/l (corresponding to 6 mg/l active protein) was obtained after a heat shock from 37 to 42°C, and IPTG induction of the adhC expression at an OD600 of 120. Although no general rules can be provided, some of the here presented variations may be applicable for the optimization of the heterologous production of proteins in general, and of thermozymes in particular

KW - high-cell-density

KW - hyperthermophilic archaea

KW - biochemical-characterization

KW - sulfolobus-solfataricus

KW - expression

KW - cloning

KW - growth

KW - gene

KW - fermentation

KW - overexpression

U2 - 10.1007/s00792-005-0490-z

DO - 10.1007/s00792-005-0490-z

M3 - Article

VL - 10

SP - 221

EP - 227

JO - Extremophiles

T2 - Extremophiles

JF - Extremophiles

SN - 1431-0651

IS - 3

ER -