Optimized Production of Active α‐Glucosidase by Recombinant Escherichia coli . Evaluation of Processes Using in Vivo Reactivation from Inclusion Bodies

During fast production in recombinant Escherichia coli , the enzyme α‐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 °C and reactivation at 30 °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 α‐glucosidase without affecting the total amount of α‐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 °C and with a set growth rate of 0.12 h ‐ 1 controlled by feeding was most efficient for production of active α‐glucosidase. Compared to production under optimal conditions on complex medium, the specific and volumetric activities obtained were 3 and 45 times higher, respectively.