Industrial Applications of Whole-Cell Biocatalysis - Pharmaceutical Technology

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Industrial Applications of Whole-Cell Biocatalysis
Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure interemediates. The authors examine several groups of enzymes using this approach: dehydrogenases, hydantoinases, and acylases.


Pharmaceutical Technology


The acylase platform

The acylase process was established by Evonik in 1970 for producing L-methionine, but also other proteinogenic and nonproteinogenic L-amino acids could be produced by this method (e.g., L-valine, L-phenylalanine, or L-norvaline). Enantiomerically pure L-amino acids are interesting compounds in infusion solutions as feed and food additives, intermediates for pharmaceuticals, cosmetics, pesticides, and as chiral synthons in organic synthesis (19). More than 200 tons per year of L-methionine are produced by this enzymatic conversion with an enzyme membrane reactor (20).


Figure 3 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.)
The starting materials in the acylase process are N-acetyl-D,L-amino acids, which are chemically synthesized by acetylation of D,L-amino acids with acetyl chloride or acetic anhydride in alkali in a Schotten–Baumann reaction (21). The enantiomerically pure L-amino acids are formed by a kinetic resolution reaction of this racemic mixture wherein only the N-acetyl-L-amino acid is deacetylated. This reaction is catalyzed by a stereospecific L-acylase from Aspergillus oryzae and produces the L-amino acid, acetic acid and N-acetyl-D-amino acid. After separation of the L-amino acid by crystallization, the remaining N-acetyl-D-amino acid has to be racemized by physical or chemical means under severe conditions (high temperature, low pH) to form the N-acetyl-D,L-amino acids and will be used for a next cycle of the process (see Figure 3). The use of a D-specific acylase would also make D-amino acids accessible. It has to be pointed out that the enzyme catalyzed kinetic resolution produces a yield of only 50% referring to the staring material. The recycling procedure is a fairly energy- and chemical-consuming process, so there was still a need to enhance the performance of this process.

If N-acetylamino acids could be selectively racemized by an enzyme in the presence of an optically active amino acid, then N-acetyl-D,L-methionine could be converted completely into L-methionine by the combined action of a racemase with the L-aminoacylase without any intermittent separation step. Such a N-acetylamino acid racemase (AAR) activity was found by Tokuyama et al. in various actinomycetes strains (22). The gene for the N-acetylamino acid racemase from Amycolatopsis sp. TS-1-60 was cloned, in E. coli overexpressed, and the gene product characterized (23, 24). The requirement for a high concentration of divalent metal ions for enzyme activity, substrate inhibition at concentrations exceeding 50 mM and inhibition by L-methionine at less than 100 mM severely restrict the use of this enzyme in a commercial process (25).

To obtain N-acetylamino acid racemases with different properties, various actinomycetes strains were examined in a genetic screening. As a result of that screening, we obtained an AAR from Amycolatopsis orientalis subsp. lurida, which was successfully overexpressed in E. coli (26).


Table II (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.)
This N-acylamino acid racemase catalyzed the racemization of various industrial important N-acylamino acids, which are listed in Table II. Besides N-acetyl-D- and L-methionine, the N-acyl derivatives of aromatic amino acids such as N-acetyl-D- and L-phenylalanine, N-acetyl-L-tyrosine, and N-chloroacetyl-L-phenylalanine were effective substrates. The derivatives N-acetyl-D-naphthylalanine, N-acetyl-L-tert-leucine and N-benzyloxycarbonyl-L-phenylalanine were not racemized. In contrast, L-methionine was no substrate for N-acylamino acid racemase (26).

An important exception is that the AAR from A. orientalis subsp. lurida exhibited substrate inhibition at concentrations of N-acetyl-D-methionine exceeding 200 mM in contrast to 50 mM for the racemase from Amycolatopsis sp. TS-1-60 27. This fact is important for the use of AAR in an industrial racemization process because the reactor loading with the substrate could be much higher with this new enzyme.


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