Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium.
Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His(6)- 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 His(6)-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.
Biedendieck
Rebekka
R
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, 38106 Braunschweig, Germany.
Beine
Rafael
R
Gamer
Martin
M
Jordan
Eva
E
Buchholz
Klaus
K
Seibel
Jürgen
J
Dijkhuizen
Lubbert
L
Malten
Marco
M
Jahn
Dieter
D
D001410:Bacillus megaterium; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D006602:Hexosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D052200:Limosilactobacillus reuteri / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data
D006602:Hexosyltransferases; C021513:levansucrase