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Pilot scale production and isolation of recombinant NAD+- and NADP+-specific formate dehydrogenases
V I Tishkov1, A G Galkin, V V Fedorchuk
1Department of Chemical Enzymology, Faculty of Chemistry, M. V. Lomonosov Moscow State University, 119899 Moscow, Russian Federation. tishkov@ibt022.ibt.kfa-juelich.de
Abstract:
The expression of the recombinant wild-type NAD+- and mutant NADP+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from the methanol-utilizing bacterium Pseudomonas sp. 101 in Escherichia coli cells has been improved to produce active and soluble enzyme up to the level of 50% of total soluble proteins. The cultivation process for E. coli/pFDH8a and E. coli/pFDH8aNP cells was optimized and scaled up to a volume of 100 L. A downstream purification process has been developed to produce technical grade NAD+- and NADP+-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems.