Biotransformations with Whole-Cell Catalysts

In the following section, a summary about selected examples of applications of whole-cell-catalyzed biotransformations in organic synthesis will be given. In particular, synthetic applications in the presence of recombinant whole-cell catalysts will be described. Examples of transformations utilizing wild-type whole cells will be provided, in particular when those biotransformations have resulted in technical applications. 

---

Chiral alcohols are valuable products mainly as building blocks for pharmaceuticals or agro chemicals or as part of chiral catalysts. Cheap biotransformation methods for the selective reduction of particular ketone compounds are known for many years rather catalyzed by fermentation than with isolated enzymes. Products prepared with whole cells such as baker s yeast often lack high enantioselectivity and there were several attemps to use isolated enzymes. Resolution of racemates with hydrolases are known in some cases but very often the reduction of the prochiral ketone using alcohol dehydrogenases are much more attractive. 

Enzymes with different stereochemical preferences for 3-oxo esters and 2-alkyl 3-0X0 esters have been isolated [57,62,63,68]. They are NADPH-dependent enzymes and are able to catalyze the reduction of oxo esters of different type. However, they are not available for enzyme-catalyzed reaction in substitution of the whole-cell catalyst. Synthetic applications make use of whole-cell biocatalysts. Valuable intermediates in synthesis are keto esters possessing additional functionality. Thus 5 -4-chloro-3-hydroxybutanoic acid 33 (Scheme 12) has been obtained by reduction with suspended cells from cultures of G. candidum. The compound is the intermediate in the synthesis of the cholesterol antagonist 34. In the biotransformation process a reaction yield of 95% and optical purity of 96% were obtained at 10 g/L. The optical purity was increased to 99% by heat treatment of cell suspensions prior to conducting the bioreduction [69]. 

Biocatalysis. Biocatalysis, also termed biotransformation and bioconversion, makes use of natural or modified isolated enzymes, enzyme extracts, or whole-cell systems for the production of small molecules. A starting material is converted by the biocatalyst in the desired product. Enzymes are differentiated from chemical catalysts particularly with regard to stereoselectivity. 

There are different possibilities for performing whole cell biotransformations with several enzymes. Each enzyme can be produced from another strain. To carry out the desired reaction, these strains are combined in different amounts which depend on the particular enzyme activities. Disadvantages of this method are diffusion problems and the membrane barrier [126]. Alternatively, all enzymes can be expressed in one single host strain. This can be reached by means of recombinant DNA techniques in several ways The genes can be expressed under one single promoter or under different promoters. Furthermore, it is possible to express all genes from the same promoter but with different copy numbers. Using these methods it is possible to create tailor-made catalysts for manifold purposes. 

Numerous enzymes have been used to hydrolyze N-acyl amino acid esters, the most versatile and thus very popular catalyst being a-chymotrypsin isolated from bovine pancreas (Scheme 2.12) [113-115]. Since it is one of the early examples of a pure enzyme which became available for biotransformations, its mode of action is well understood. A useful and quite reliable model of its active site has been proposed in order to rationalize the stereochemical outcome of resolutions performed with a-chymotrypsin [116, 117]. Alternatively, other proteases, such as subtUisin [118, 119], thermolysin [120], and alkaline protease [121] are also commonly used for the resolution of amino acid esters. Even whole microorganisms such as lyophihzed cells of baker s yeast, possessing unspecific proteases, can be used as biocatalysts for this type of transformation [122],... 

---