Applied Microbiology and Biotechnology

, Volume 60, Issue 4, pp 408–416

Production of heterologous thermostable glycoside hydrolases and the presence of host-cell proteases in substrate limited fed-batch cultures of Escherichia coli BL21(DE3)

  •  S. Ramchuran
  •  E. Nordberg Karlsson
  •  S. Velut
  •  L. de Maré
  •  P. Hagander
  •  O. Holst
Original Paper

DOI: 10.1007/s00253-002-1132-3

Cite this article as:
Ramchuran, S., Nordberg Karlsson, E., Velut, S. et al. Appl Microbiol Biotechnol (2002) 60: 408. doi:10.1007/s00253-002-1132-3

Abstract.

Metabolic stress is a phenomenon often discussed in conjunction with recombinant protein production in Escherichia coli. This investigation shows how heterologous protein production and the presence of host cell proteases is related to: (1) Isopropyl-β-D-thiogalactopyranoside (IPTG) induction, (2) cell-mass concentration at the time of induction, and (3) the presence of metabolites (glutamic acid or those from tryptone soy broth) during the post-induction phase of high cell density fed-batch cultivations. Two thermostable xylanase variants and one thermostable cellulase, all originating from Rhodothermus marinus, were expressed in E. coli strain BL21 (DE3). A three-fold difference in the specific activity of both xylanase variants [between 7,000 and 21,000 U/(g cell dry weight)], was observed under the different conditions tested. Upon induction at high cell-mass concentrations employing a nutrient feed devoid of the metabolites above, the specific activity of the xylanase variants, was initially higher but decreased 2–3 h into the post-induction phase and simultaneously protease activity was detected. Furthermore, protease activity was detected in all induced cultivations employing this nutrient feed, but was undetected in uninduced control cultivations (final cell-mass concentration of 40 g/l–1), as well as in induced cultivations employing metabolite-supplemented nutrient feeds. By contrast, maximum specific cellulase activity [between 700 and 900 U/(g cell dry weight)] remained relatively unaffected in all cases. The results demonstrate that detectable host cell proteases was not the primary reason for the decrease in post-induction activity observed under certain conditions, and possible causes for the differing production levels of heterologous proteins are discussed.

Copyright information

© Springer-Verlag 2002

Authors and Affiliations

  •  S. Ramchuran
    • 1
  •  E. Nordberg Karlsson
    • 1
  •  S. Velut
    • 2
  •  L. de Maré
    • 2
  •  P. Hagander
    • 2
  •  O. Holst
    • 1
  1. 1.Dept. Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O.Box 124, SE-22100 Lund, Sweden
  2. 2.Automatic Control, Lund University, Lund, Sweden

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