Kerstin Spornhauer (geb. Würges), Katerina Petruevska, Stefanie Serci, Susanne Wilhelm, Christian Wandrey, Andreas Seidel-Morgenstern, Martin Peter Elsner, Stephan Lütz:
Enzyme-assisted physicochemical enantioseparation processes - part I: Production and characterization of a recombinant amino acid racemase
Journal of Molecular Catalysis B: Enzymatic, 2009, 58, 10-16
The demand for enantiopure substances, e.g. for pharmaceutical applications or fine chemical production, continues to increase. This has led to the development of numerous stereoselective synthesis methods. Nevertheless a large number of chemical syntheses still result in racemic mixtures making a subsequent enantioseparation step necessary and thus are restricted to a maximum yield of 50%. Our work focuses on strategies to overcome this limitation by combining physicochemical separation processes with enzymatic racemization of the unwanted enantiomer in order to produce enantiopure amino acids. This paper deals with the production and characterization of a suitable amino acid racemase with broad substrate specificity (EC 22.214.171.124) from Pseudomonas putida which we cloned into Escherichia coli. Two enzyme lyophilizates of different purity were obtained from which the crude (CL) was sufficient for the racemization of methionine (Met) and the pure (PL) was used for asparagine (Asn). Racemization reactions of D-/L-Asn inH2O and D-/L-Met in 95 vol.% 100mMKPi-buffer, 5 vol.%methanol (MeOH) at different pH values and temperatures were characterized. The studied range of reaction parameters was chosen in dependency on planned enantioseparation processes. We found increasing Vmax values when temperature was risen stepwise from 20 to 40 ◦C for both systems and when pH was shifted from 6 to 8 for the Met system. The presented results provide the basis for engineering enzyme-assisted physicochemical enantioseparation processes.
Amino acid racemase, Enzyme purification, Integrated enantioseparation process, Chiral chromatography, Preferential crystallization