Pilot scale production and isolation of recombinant NAD(+)- and NADP(+)-specific formate dehydrogenases

The expression of the recombinant wild-type NAD(+)- and mutant NADP(+)-depe ndent 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 solubl e 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 purif ication process has been developed to produce technical grade NAD(+)- and N ADP(+)-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems. (C) 1999 John Wiley & Sons, Inc.