Stefan Verseck

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. 

The cofactor-dependent asymmetric reduction of ketones catalyzed by alcohol dehydrogenases represents a valuable methodology for the synthesis of optically active alcohols (1–3). For the 

 regeneration of the expensive cofactor (NADH or NADPH, which is normally sufficiently present within the whole-cell catalyst), the enzyme-coupled approach is the most efficient method because of the irreversibility of the overall reaction and the high specific activity of the glucose dehydrogenases. This process is suitable for preparing a wide range of functionalized (

)-alcohols from aliphatic and aromatic ketones and keto esters with different substitution patterns by the application of whole-cell catalysts with several complementary alcohol dehydrogenases and the appropriate glucose dehydrogenases (see Table I) (4).  

Table I (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.) 

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. 

-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-

-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 process was introduced in the 1970s for producing D-amino acids such as D-phenylglycine and 

-OH-phenylglycine (7). Today, > 1000 tons of each of these amino acids are produced annually. They are used as side chains for the  -lactame antibiotics ampicillin and amoxicillin. 

Figure 1 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.) 

The D-hydantoinase process (see Figure 1) is an excellent example of a dynamic kinetic resolution process. As 5'-monosubstituted hydantoins racemize spontaneously or enzyme-catalyzed under conditions used for biotransformation, a 100% yield of optically pure D- or L-amino acid can be reached (8, 9). Another advantage of the hydantoinase route is that racemic 5'-monosubstituted hydantoins can be easily synthesized from cheap starting materials through the reactions shown in Figure 2. In addition, if the decarbamoylation step is done enzymatically,  carbamoylases waste and by-product formation is extremely low (CO

 are the only by-products), which is also advantageous in the product-isolation step. All these features make the hydantoinase route very attractive for the industrial production of optically pure artificial amino acids (10). To date, low space-time yields and high biocatalyst costs prevent the production of L-amino acids based on the hydantoinase process (11–13). 

Figure 2 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.) 

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 

 cells overexpressing a hydantoinase, carbamoylase, and hydantoin racemase from 

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 

 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 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).  

The starting materials in the acylase process are  

-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  

-acetyl-L-amino acid is deacetylated. This reaction is catalyzed by a stereospecific L-acylase from 

e and produces the L-amino acid, acetic acid and 

-acetyl-D-amino acid. After separation of the L-amino acid by crystallization, the remaining  

-acetyl-D-amino acid has to be racemized by physical or chemical means under severe conditions (high temperature, low pH) to form the 

-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. 

Figure 3 (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.) 

-acetylamino acids could be selectively racemized by an enzyme in the presence of an optically active amino acid, then 

-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 

-acetylamino acid racemase (AAR) activity was found by Tokuyama et al. in various actinomycetes strains (22). The gene for the 

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). 

-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 

-acylamino acid racemase catalyzed the racemization of various industrial important 

-acylamino acids, which are listed in Table II. Besides 

-acyl derivatives of aromatic amino acids such as 

-chloroacetyl-L-phenylalanine were effective substrates. The derivatives 

-benzyloxycarbonyl-L-phenylalanine were not racemized. In contrast, L-methionine was no substrate for 

Table II (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.) 

An important exception is that the AAR from 

 exhibited substrate inhibition at concentrations of 

-acetyl-D-methionine exceeding 200 mM in contrast to 50 mM for the racemase from A

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 

-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 

-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. 

 is director of the industry segment photovoltaics of the inorganic materials business unit and 

 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, 

 is a pilot-plant manager at Evonik's Service Center Biocatalysis. 

 is head of biotechnology at Cognis GmbH. 

 *To whom all correspondence should be addressed. 

 1. K. Nakamura and T. Matsuda, "Enzyme Catalysis" in 

, K. Drauz and H. Waldmann Eds., (Wiley-VCH,  Weinheim, Vol. 3, 2nd ed., 2002), pp. 991–1047.   

 2. W. Hummel, "New Alcohol Dehydrogenases for the Synthesis of Chiral Compounds," 

 (Springer-Verlag, Berlin, 5th ed., 2004), pp. 192–197. 

 4. H. Gröger et al., "Enantioselective Reduction of Ketones with Designer Cells at High Substrate Concentrations: Highly Efficient Access to Functionalized Optically Active Alcohols," 

 5. M.R. Kula and C. Wandrey, "Continuous Enzymatic Transformation in an Enzyme-Membrane Reactor with Simultaneous NADH Regeneration," in 

 K. Mosback, Ed. (Vol. 136, 1987), pp.  9-21. 

 6. A. Bommarius, M. Schwarm, and K.H. Drauz, "Biocatalysis to Amino Acid-based Chiral Pharmaceuticals—Examples and Perspectives," 

 7. F. Cecere et al., "D-Carbamylamino Acids and Corresponding D-Amino Acids," Ger. Offen,  2,621, 076, Addn. to Ger. Offen. 2,422,737, Dec. 1976.  

 8. M. Kato, H. Kitagawa, and T. Miyoshi, "Microbial Racemization of Optically Active 5-Substituted Hydantions,"  Japanese Patent 62122591, 1987.  

 9. M. Pietzsch, C. Syldatk, and F. Wagner, "Isolation and Characterization of a New, Nonpyrioxal-5'-Phosphate Dependent Hydantion Racemase,"

 10.  C. Syldatk et al., "Microbial Hydantoinases—Industrial Enzymes from the Origin of Life?," 

 11. C. Gross, C. Syldatk, and F. Wagner, "Screening Method for Microorganisms Producing L-Amino Acids from D,L-5-Monosubstituted Hydantoins," 

 12. Y. Nishida et al., "Enzymatic Production of L-tryptophan from D,L-5-Indolylmethylhydantoin by

 13. D. Cotoras and F. Wagner,  "Stereospecific Hydrolysis of 5-Monosubstituted Hydantoins," 

 14. F. Wagner et al., "Microorganisms Producing L- Amino  Acids, " German Patent DE 19519717, 1996 

  15. T. Wagner, B. Hantke, and F.J. Wagner, "Production of L-methionine from D,L-5-(2-methylthioethyl) Hydantoin by Resting Cells of a New Mutant Strain of 

 16. O. May et al., "Substrate-Dependent Enantioselectivity of a Novel Hydantoinase from 

DSM 3745: Purification and Characterization as New Member of Cyclic Amidases,"  

 17.  D. Volkel and F. Wagner, "Reaction Mechanism for the Conversion of 5-Monosubstituted Hydantoins to Enantiomerically Pure L-Amino Acids," 

 18. O. May, P.T. Nguyen, and F.H. Arnold, "Inverting Enantioselectivity by Directed Evolution of Hydantoinase for Improved Production of L-Methionine," 

 19. A.S. Bommarius et al., "Operational Stability of Enzymes. Acylase-Catalyzed Resolution of 

-Acetyl Amino Acids to Enantiomerically Pure L-Amino Acids," 

Articles

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