Continuous production of a chiral amine in a packed bed reactor with co-immobilized amine dehydrogenase and formate dehydrogenase

A continuous flow packed bed reactor system was developed for the asymmetric reductive amination of 5-methyl-2-hexanone with a chimeric amine dehydrogenase (AmDH) and the formate dehydrogenase (FDH) from Candida boidinii. Both enzymes were co-immobilized onto the Nuvia (R) IMAC resin from Bio-Rad which employs the well-established binding chemistry between 6xHis-tagged proteins and immobilized Ni2+. Binding was robust under reaction conditions and continuous operation in a packed-bed flow column for more than five days was achieved. At 40 degrees C, conversion of 20 mM 5-methyl-2-hexanone ranged between 19 and 48%, with volumetric productivities between 443 and 107 g/L.day. The apparent biocatalyst half-life was 159 h and the absence of both internal and external mass transfer limitations was confirmed. Conversion was limited by modest competitive product inhibition of (R)-amine on ketone and an excess of enzyme over cofactor concentration, which precludes the use of Michaelis-Menten kinetics and results in a rate decrease of 80% compared to conditions where [S] >> [E]. The relationship between flow rate, conversion, and productivity was examined, with important lessons for reaction engineering. The reported work represents the first stable application of AmDHs in a continuous flow reactor for the production of chiral amines.