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Industrial Applications of Whole-Cell Biocatalysis
Biocatalytic processes for producing enantiomerically pure pharmaceutical intermediates or active ingredients are of growing importance. Recent advances in molecular biological methods such as recombinant enzyme expression, high-throughput DNA sequencing, and enzyme-evolution technologies, make biocatalysis a viable option for producing single enantiomers. Even two- and three-step biotransformations can be accomplished by combining and adapting enzymes from different sources. Recombinant microbial whole-cell biocatalysts, or so-called "designer cells," which provide each enzyme at the optimum amount, is a particularly efficient approach. Under this approach, all of the required enzymes can be produced by one fermentation. Cell disruption, clarification, and concentration of the enzyme solution are dispensable. The separation of the biocatalyst after biotransformation can easily be done by flocculation and filtration of the biomass. Substantial substrate and product-transfer limitations through the cell membranes have not been observed using frozen or dried biocatalysts.
Conversions typically are > 90% in enantiomeric excess (ee) and enantioselectivities are at least > 90% ee, and are normally > 99% ee. Biotransformations have been run under two-phase conditions without the addition of a cosolvent at substrate concentrations of > 100 g/L, and in some cases even > 200 g/L, typically within < 30 h reaction time. Fermentation of the biocatalyst, biotransformation, and downstream processing (consisting of filtration of the biocatalyst and extraction of the alcohol) are applied on a production scale.
Amino acid dehydrogenases
Evonik (formerly Degussa) started its L-tert-leucine production more than 20 years ago. The first generation process is based on purified leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH) for cofactor regeneration using the enzyme-membrane reactor concept (5). Using this enzyme combination, a broad range of amino acids are accessible in excellent yields and enantioselectivities. This approach can be a superior approach, especially for extremely bulky amino acids such as L-tert-leucine, other 2,2,2-trialkylamino acids, and L-neopentylglycine (6).
To meet future growth for these amino acids, Evonik developed a second-generation whole-cell biocatalytic process. LeuDH whole-cell catalysts with FDH and also glucose dehydrogenase (GDH) for cofactor regeneration have been constructed and tested. Because of the approximately 50-fold specific activity of GDH, this second approach has turned out to be much more efficient. A pilot production of L-neopentylglycine was run on multi-10-kg scale at an approximately 50 g/L substrate concentration with a whole-cell LeuDH/GDH biocatalyst reaching > 95% conversion within one day and an optical purity > 99.8% ee. The starting material a-ketoacid is easily accessible through condensation of pivalaldehyde with hydantoin and successive hydrolysis.
The hydantoinase platform
Therefore, Evonik expanded its hydantoinase platform for producing L-amino acids by focusing on strain development and process optimization by biochemical engineering (14, 15). Despite significant progress in reducing the biocatalyst production cost, increasing the activity of the biocatalyst and improving the space-time-yield process economics were still prohibitive for commercialization of this process.
To address these problems, Evonik developed a new generation of an L-hydantoinase process based on a tailor-made recombinant whole-cell biocatalyst. The biocatalyst costs have been reduced by designing recombinant Escherichia coli cells overexpressing a hydantoinase, carbamoylase, and hydantoin racemase from Arthrobacter sp. DSM 9771. Despite this progress, the D-selectivity of the hydantoinase for D,L-methylthioethylhydantoin was significantly limiting the space-time yield of the L-hydantoinase process (16, 17). As screening did not provide us with better hydantoinases, the authors intended to invert the enantioselectivity of the hydantoinase by directed evolution (18). The productivity of the process could be dramatically improved using the recombinant E. coli coexpressing the newly designed L-selective hydantoinase mutant with an L-carbamoylase and a hydantoin racemase. These improvements have been confirmed at industrial scale and resulted in a process for producing various natural and nonnatural L-amino acids.
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).
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).
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.
The examples show the successful development of biocatalytic technologies for producing optically pure amino acids and other fine chemicals. Evonik has developed two different dynamic kinetic resolution processes for producing natural and nonnatural L-amino acids that allow a broad range of relevant substrates. The first process is based on the resolution of 5'-monosubstituted hydantoins, and the second process is based on the resolution of N-acetyl amino acids using an acylase in combination with a racemase.
These technologies have been recently expanded by a process using oxido-reductases and a cofactor regeneration system for producing amino acids and chiral molecules starting from prochiral ketones. Whether the oxidoreductase platform, the L-hydantoinase process or the combination of an L-acylase with an N-acetyl-amino acid racemase is superior, is strongly dependent on the specific product in demand and influenced by the biocatalyst properties as well as the cheapest access to the substrate.
All approaches together provide a high degree of flexibility producing a number of different L-amino acids, which is especially important for fast-changing product demands typical of the fine-chemicals and pharmaceutical industries.
Wolfgang Wienand is director of the industry segment photovoltaics of the inorganic materials business unit and Kai Doderer and Steffen Osswald* are project managers at the Service Center Biocatalysis in the health and nutrition business unit at Evonik Degussa GmbH,
Rodenbacher Chaussee 4, 63457 Hanau-Wolfgang, Germany, tel. +49 6181.59.6748, fax +49.6181.59.76748, email@example.com
*To whom all correspondence should be addressed.
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