Enzyme-assisted physicochemical enantioseparation processes - part III: Overcoming yield limitations by dynamic kinetic resolution of asparagine via preferential crystallization and enzymatic racemization

The application of enantioseparation methods alone can only yield up to 50% of the desired chiral product. Thus enantioseparation becomes more attractive when accompanied by the racemization of the counter-enantiomer. Here we present first results of dynamic kinetic resolution of L-asparagine (L-Asn) via preferential crystallization and enzymatic racemization from a racemic, supersaturated solution on a 20 mL scale. An enzyme lyophilisate (WT amino acid racemase from P. putida KT2440 (E.C. 5.1.1.10), overexpressed in E. coli BL21(DE3)) was used for in situ racemization (enzyme concentrations varying from 0 to 1 mg/mL). When preferential crystallization was applied without any enzyme, a total of 31 mg of L-Asn monohydrate could be crystallized, before crystal formation of d-Asn started. Crystallization experiments accompanied by enzymatic racemization led to a significant increase of crystallized L-Asn (198 mg L-Asn monohydrate; >92%ee) giving the first experimental proof for this new process concept of dynamic kinetic resolution via preferential crystallization and enzymatic racemization. Measurements of the racemase activity before and after the crystallization process showed no significant differences, which would allow for enzyme recovery and recycling

Enzyme supported preferential crystallization of enantiopure amino acids

The production of enantiopure substances has drawn a lot of interest during the last decades. Especially intermediates for the pharmaceutical industry, fine chemicals and feed additives like L-amino acids are of great scientific and economic relevance [1, 2]. Despite an increasing number of stereoselective synthesis there are still a lot of reactions that lead to racemic mixtures of the desired homochiral products. Numerous separation techniques have been developed [3-5], but a major drawback of them is the principal yield limitation to a maximum of 50 %. We present a new approach for the production of enantiopure amino acids (AA) with a theoretical yield of 100 % that integrates preferential crystallization (PC) of a conglomerate and enzymatic in situ racemization (Fig. 1). Starting from a racemic oversaturated AA solution one enantiomer can be crystallized by seeding with homochiral crystals, which leads to an increasing enantiomeric excess (ee) of the counter enantiomer in the crystallization medium. PC is only possible as long as the medium composition remains in the metastable zone of the ternary phase system, thus crystallization of the counter enantiomer will start at a certain point. In order to prevent this, enzymatic in situ racemization can be applied which keeps the ee in the medium at a minimum. By continuous feeding of racemic AA into the system a stationary process can be obtained

Enzyme-assisted physicochemical enantioseparation processes - part II: Solid-liquid equilibria, preferential crystallization, chromatography and reacemization reaction

This contribution addresses the design and investigation of two hybrid enantioseparation processes including an enzymatic racemization step in order to enhance the overall performance. Complementary to part I where the manufacturing and the characterization of an amino acid racemase (EC 5.1.1.10) was emphasized [Wurges, K., Petrusevska, K., Serci, S., Wilhelm, S., Wandrey, C., Seidel-Morgenstern, A., Elsner, M.P.. Lutz, S., 2009. Enzyme-assisted physicochemical enantioseparation processes-part I: production and characterization of a recombinant amino acid racemase. J. Mol. Cat. B (in print). online available: doi:10.1016/j.molcatb.2008.10.006], the work presented in this paper tends more towards developing a data base for potential process schemes for the manufacture of selected amino acids.The first proposed process concept (P-I) couples preferential crystallization (PC) and racemization for the production of I-asparagine (I-Asn) using racemic mixture of dl-asparagine (conglomerate-forming system) as a starting material, while the second concept (P-II) integrates chromatography and racemization for the preparation of I-methionine (I-Met) starting with racemic mixture of dl-methionine (compound-forming system). As mentioned in part I, a racemization unit, where the unwanted enantiomer will be converted into racemate, is incorporated into the hybrid processes for the sake of 100% yield, theoretically. Besides the basic investigation according to the solid-liquid equilibria, PC and chromatography, the focus of this paper is mainly on the kinetic studies of the racemization reaction. Initially, the solubility ternary phase diagrams of both examined systems were determined, leading into the idea of combination of the proposed process schemes. For P-I the concept of PC of I-Asn was experimentally proven and the kinetics of the racemization was examined for d- and I-Asn in water using purified lyophilizate (PL). Concerning P-II, for the chromatographic unit the impact on the separation of dl-Met on eremomycin based stationary phase using KPi buffer and MeOH as mobile phase was evaluated in terms of resolution and selectivity at three different temperatures by varying the content of methanol (MeOH) in the mobile phase and the pH. The experiments for determination of the racemization kinetics were done for a compromised parameter set using crude lyophilizate (CL). In both cases a Michaelis-Menten three-step model was used to describe the enzymatic reaction. (C) 2009 Elsevier Ltd. All rights reserved