title: Optimized production of active alpha-glucosidase by recombinant Escherichia coli. evaluation of processes using in vivo reactivation from inclusion bodies.
abstract: During fast production in recombinant Escherichia coli, the enzyme alpha-glucosidase from Saccharomyces cerevisiae accumulates partially as inclusion bodies. The inclusion bodies are reactivated inside the cell upon temperature downshift. This in vivo reactivation was most efficient on complex medium with inclusion body production at 42 degrees C and reactivation at 30 degrees C, yielding volumetric activities 85% higher than those of extended isothermal production at low temperature. On defined medium, however, in vivo reactivation was slow. In fed-batch cultivations, feeding controls the specific growth rate independent of the temperature. Here, high growth rates fostered inclusion body formation even at low temperature. Intermediate growth rates permitted accumulation of active alpha-glucosidase without affecting the total amount of alpha-glucosidase. Low growth rates yielded similar activities and additionally prevented inclusion body formation. Moreover, high growth rates during production forestalled subsequent in vivo reactivation. Accumulation of activity after temperature reduction was possible with intermediate growth rates. The best time for temperature shift was concomitant to induction. Thus, in fed-batch culture, isothermal production at 30 degrees C and with a set growth rate of 0.12 h(-)(1) controlled by feeding was most efficient for production of active alpha-glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.
authors.affiliation: Department of Biochemistry/Biotechnology, Institute for Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, D-06120 Halle, Germany.
authors.forename: Ha
authors.initials: H
authors.lastname: Le Thanh
authors.forename: Frank
authors.initials: F
authors.lastname: Hoffmann
mesh_terms: D019149:Bioreactors; D003470:Culture Media; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D002479:Inclusion Bodies / Q000235:genetics*; D007218:Industrial Microbiology / Q000295:instrumentation / Q000379:methods*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D000520:alpha-Glucosidases