Wild-type whole cells

The reduction of several ketones, which were transformed by the wild-type lyophilized cells of Rhodococcus ruber DSM 44541 with moderate stereoselectivity, was reinvestigated employing lyophilized cells of Escherichia coli containing the overexpressed alcohol dehydrogenase (ADH- A ) from Rhodococcus ruber DSM 44541. The recombinant whole-cell biocatalyst significantly increased the activity and enantioselectivity [41]. For example, the enantiomeric excess of (R)-2-chloro-l-phenylethanol increased from 43 to >99%. This study clearly demonstrated the advantages of the recombinant whole cell biocatalysts over the wild-type whole cells. 

In summary, ketoreductases have emerged as valuable catalysts for asymmetric ketone reductions and are preparing to enter the mainstream of synthetic chemistry of chiral alcohols. These biocatalysts are used in three forms wild-type whole-cell microorganism, recombinant... 

To circumvent these issues, instead of wild-type whole cells, the use of cloned and overexpressed enoate reductases together with suitable redox enzymes for cofactor recycling - the so-called designer bugs - appears as an excellent ahemative to overcome the lack of selectivity observed when whole (fermenting) cell systems are applied, due to the existence of enzymes that can catalyze side reactions, and so on (Scheme 2.4) [18,19]. Thus, when enoate reductases are overexpressed, the enhancement of its concentration leads to higher selectivity and yields in the desired products. This area is clearly expanding nowadays, and therefore further innovations are ejqjected to appear. 

Prior to the widespread awdlabdity of recombiant carbonyl reductases enzymes, the use of microbial reductions using either actively growing or dormant cells was commonplace Bakers yeast in particular, was a readily available source of stereoselective carbonyl reductases enzymes. Even with the widespread knowledge of the power of recombinant CRED biocatalysts, the literature is still rife with wild-type whole-cell microbial reductions. The reductions presented have advanced well beyond the early Bakers yeast reduction and have an apphcation even today. When the whole-cell fermentation is developed and finely tuned, high titers of product alcohol are possible and Scheme 6.4 shows m example of a keto-amide 12 bioreduction performing at 100 g/L with more than 98% ee with multi-kg isolation [12]. The bioprocess was performed over 8 days at pH 7 using the yeast Candida sorbophila. 

This section gives the organic chemist examples of how to perform the bioreductions in the laboratory using both wild-type whole cell and recombinant systems. 

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. 

A further hydrolytic process in which whole-cell catalysis turned out to be very suitable is the transformation of a racemic nitrile into the corresponding acid exemplified for the dynamic kinetic resolution of mandelonitrile into (R)-mandelic acid, (R)-IO. This reaction is catalyzed by means of a nitrilase, which is known as highly enantioselective enzyme. As early as 1991, researchers from Asahi Chemical Industry Ltd. reported such a reaction utilizing wild-type whole cells from Alcaligenes faeccdis bearing a suitable nitrilase [32]. When starting from racemic mandelonitrile, rac-7. 

Synthesis of (8)-mandelic acid via dynamic kinetic resolution of mandelonitrile with a wild-type whole-cell catalyst from Alcaligenes faecalis containing a nitrilase. 

Synthesis of octanol regiosio-mers via asymmetric hydroxylation of n-octane with a wild-type whole-cell catalyst containing a P450-monooxygenase. 

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