Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli.
The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His(6) tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L(-1) could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.
Dresler
Karolin
K
Biochemical Engineering Group TU-BCE, GBF Braunschweig, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, 38124 Braunschweig, Germany.
van den Heuvel
Joop
J
Müller
Rolf-Joachim
RJ
Deckwer
Wolf-Dieter
WD
D000192:Actinomycetales / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011091:Polyesters / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry
D011091:Polyesters; D011994:Recombinant Proteins; D006867:Hydrolases