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


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.

Alcohol dehydrogenases


Table I (ALL FIGURES AND TABLES ARE COURTESY OF EVONIK.)
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 in situ 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 (R)- and (S)-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).

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.


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