(55d) E. coli Pilot-Scale Fermentation and One-Step Purification of Amine Dehydrogenases Employing an Aqueous Two-Phase Extraction System

Authors: 
Bommarius, B., Georgia Institute of Technology
Bommarius, A. S., Georgia Institute of Technology
Wu, R., Georgia Institute of Technology
Au, S., University of Kentucky
Jaeger, K. E., Heinrich-Heine-University Düsseldorf

We have recently developed several amine dehydrogenases (AmDHs) that are of interest for production of enantiomerically pure amines as precursors for active pharmaceutical ingredients (Abrahamson et al. 2012 and 2013, Bommarius B et al, submitted). Their potential as synthetically interesting enzymes led us to develop a scalable, cost-effective pilot-scale fermentation and purification strategy.

Fermentations were carried out on 15 L scale with a mostly defined high-density medium and continuous glucose feed controlled by both the pO2 value and the coupled growth rate of the E. coli host.  This fermentation protocol led to 1 g/L pure amine dehydrogenase with an overall yield of 140 g/L wet cells.

To achieve simple purification on large scale, we have developed a one-step purification using an aqueous two-phase system (ATPS) with 9% PEG 6000 in the top phase and 16 % ammonium citrate in the lower phase. After homogenization the whole cell lysate was applied onto this ATPS and purification of AmDH was achieved through separation into the top layer, whereas cell debris was concentrated at the interface and contaminating proteins remained in the bottom phase.  This ATPS is scalable and ammonium citrate is both cheap and environmentally friendly, even on large scale. We currently work on a method to recycle PEG 6000.

In summary, we present a straightforward and affordable protocol to produce and purify AmDH for industrial scale.

References:

M.J. Abrahamson, E. Vazquez-Figueroa, N.B. Woodall, J.C. Moore, A.S. Bommarius, “Development of a Novel Amine Dehydrogenase for Synthesis of Chiral Amines“, Angew. Chem. Intl. Ed. 2012, 51, 3969-3972

M.J. Abrahamson, J.W. Wong, A.S. Bommarius, “The rapid evolution of an amine dehydrogenase biocatalyst for the asymmetric production of chiral amines“ , Adv. Synth. Catal., 2013, 355, 1780-1786

B. Bommarius, M. Schürmann, A.S. Bommarius, “A chimeric amine dehydrogenase offers surprising properties not found in either parent”, submitted