Applied Microbiology and Biotechnology

, Volume 74, Issue 5, pp 1062–1073 | Cite as

Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium

  • Rebekka Biedendieck
  • Rafael Beine
  • Martin Gamer
  • Eva Jordan
  • Klaus Buchholz
  • Jürgen Seibel
  • Lubbert Dijkhuizen
  • Marco Malten
  • Dieter Jahn
Applied Genetics and Molecular Biotechnology

Abstract

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His6- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His6-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru.

Keywords

Levansucrase Bacillus megaterium Secretion Affinity tag Fructosyloligosaccharide 

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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Rebekka Biedendieck
    • 1
  • Rafael Beine
    • 3
  • Martin Gamer
    • 1
  • Eva Jordan
    • 2
  • Klaus Buchholz
    • 3
  • Jürgen Seibel
    • 3
  • Lubbert Dijkhuizen
    • 4
  • Marco Malten
    • 1
  • Dieter Jahn
    • 1
  1. 1.Institute of MicrobiologyTechnical University BraunschweigBraunschweigGermany
  2. 2.Institute of Biochemistry and BiotechnologyTechnical University BraunschweigBraunschweigGermany
  3. 3.Institute for Technical ChemistryTechnology of CarbohydratesBraunschweigGermany
  4. 4.Microbial Physiology and Centre for Carbohydrate Bioprocessing (CCB, TNO Quality of Life-University of Groningen), Groningen Biomolecular Sciences and Biotechnology Institute (GBB)University of GroningenHarenThe Netherlands

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