Advertisement

Biotechnology Letters

, Volume 32, Issue 2, pp 243–248 | Cite as

Screening for conditions of enhanced production of a recombinant β-glucanase secreted into the medium by Escherichia coli

  • Meike Spexard
  • Usama BeshayEmail author
  • Joe Max Risse
  • Gerhard Miksch
  • Erwin Flaschel
Original Research Paper

Abstract

The extracellular production of a hybrid bacterial β-glucanase using Escherichia coli was studied by using combinations of promoters of varying strength for both a β-glucanase as the target protein and the Kil protein as the releasing factor. Four strains with different combinations of promoter strengths were cultivated in shake-flasks on four different media to assess the cross-influence of promoter and medium in a general manner. Promoters were taken from natural as well as synthetic sequences known to exhibit either weak or strong promoter strength. By far the highest extracellular glucanase activity (>200 U ml−1) was achieved when a strain harbouring the kil gene under control of a strong synthetic stationary-phase promoter and the glucanase gene under control of a strong synthetic constitutive promoter was cultivated on a complex medium mainly composed of casein peptone, yeast extract, and glycerol.

Keywords

β-Glucanase Extracellular enzyme Fermentation E. coli Complex and synthetic media 

Notes

Acknowledgements

U Beshay is grateful to the Alexander von Humboldt-Foundation for the financial support in the form of a fellowship.

References

  1. Bass EJ, Meredith WOS, Anderson JA (1952) Enzymes that degrade barley gums. 1. Isolation from a bacterial source. Cereal Chem 29:262–272Google Scholar
  2. Beshay U, Miksch G, Flaschel E (2007a) Improvement of a β-glucanase activity test by taking into account the batch reactor balance of the test system. Bioprocess Biosyst Eng 30:251–259CrossRefPubMedGoogle Scholar
  3. Beshay U, Miksch G, Friehs K, Flaschel E (2007b) Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter. Biotechnol Lett 29:1893–1901CrossRefPubMedGoogle Scholar
  4. Borriss R, Olsen O, Thomson KK, Wettstein D (1989) Hybrid Bacillus endo-1,3-1,4-β-glucanases. Construction of recombinant genes and molecular properties of the gene product. Carlsberg Res Commun 54:41–54CrossRefPubMedGoogle Scholar
  5. Brockmeier U, Caspers M, Freudl R, Jockwer A, Noll T, Egger T (2006) Systematic screening of all signal peptides from Bacillus subtilis: a powerful strategy in optimizing heterologous protein secretion in gram-positive bacteria. J Mol Biol 362:393–402CrossRefPubMedGoogle Scholar
  6. Campbell GL, Bedford MR (1992) Enzyme applications for monogastric feeds: a review. Can J Anim Sci 72:449–466CrossRefGoogle Scholar
  7. Chao L, Levin B (1981) Structured habitats and the evolution of anti-competitor toxins in Bacteria. PNAS 78:6324–6328CrossRefPubMedGoogle Scholar
  8. Jensen PR, Hammer K (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 64:82–87PubMedGoogle Scholar
  9. Klahorst S, Kumar A, Mullins MM (1994) Optimising the use of cellulase enzymes. Text Chem Color 26:13–18Google Scholar
  10. Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer W-D (1995) Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol 39:59–65CrossRefPubMedGoogle Scholar
  11. Manners DJ, Wilson G (1974) Purification and properties of an endo (1,3)-β-d-glucanase from malted barley. Carbohydr Res 37:9–22CrossRefPubMedGoogle Scholar
  12. Miksch G, Fiedler E, Dobrowolski P, Friehs K (1997) The kil gene of the ColE1 plasmid of Escherichia coli controlled by a growth phase-dependent promoter mediates the secretion of a heterologous plasmatic protein during the stationary phase. Arch Microbiol 167:143–150CrossRefGoogle Scholar
  13. Miksch G, Bettenworth F, Friehs K, Flaschel E (2005a) The sequence upstream of the −10 consensus sequence modulates the strength and induction time of the stationary phase promoters in Escherichia coli. Appl Microbiol Biotechnol 69:312–320CrossRefPubMedGoogle Scholar
  14. Miksch G, Bettenworth F, Friehs K, Flaschel E, Saalbach A, Twellmann T, Nattkemper TW (2005b) Libraries of synthetic stationary-phase promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli. J Biotechnol 120:25–37CrossRefPubMedGoogle Scholar
  15. Wood PJ (1984) Physicochemical properties and technological and nutritional significance of cereal β-glucans. In: Rasper CF (Hrsg.) Cereal polysaccharides in technology and nutrition. American Association of Cereal Chemists, St. Paul, pp 35–78Google Scholar
  16. Young DH, Pegg CF (1981) Purification and characterization of 1,3-β-glucan hydrolases from healthy and Verticillium alboatrum infected tomato plants. Physiol Plant Pathol 19:391–417Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Meike Spexard
    • 1
  • Usama Beshay
    • 2
    Email author
  • Joe Max Risse
    • 1
  • Gerhard Miksch
    • 1
  • Erwin Flaschel
    • 1
  1. 1.Fermentation Engineering, Faculty of TechnologyBielefeld UniversityBielefeldGermany
  2. 2.Bioprocess Development DepartmentGenetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research and Technology ApplicationsNew Borg El-Arab City, AlexandriaEgypt

Personalised recommendations