Whole-cell enzymes, asymmetric oxidation

Apart from the asymmetric metal catalysis, enantioselective Baeyer-Villiger oxidations mediated by enzymes have been known for some time [32,33,34]. Both whole-cell cultures and isolated enzymes, usually flavin-dependent monooxygenases, can be used to oxidize ketones enantioselectively. For future improvements in the asymmetric Baeyer-VilHger oxidation the use of chiral Lewis acids in combination with an appropriate oxidant seems worthy of intensive investigation. 

Clearly, most biocatalytic reactions for the production of fine chemicals are used to obtain enantiopure or enantioemiched compoimds, and only a minor nimiber of syntheses lead to products without chiral centers. More than 65 ap-pHcations of immobilized enzymes or whole cells for industrial research and production have been treated in this review, and it can be stated that approximately 80% utilize the class of hydrolytic enzymes. This number reflects the ease of handling and the broad utility of these enzymes. The reported hydrolytic enzyme applications mainly involve lipases, whereas other hydrolases can only be found in fewer but nevertheless just as attractive cases. The broad field of asymmetric synthesis (e.g., asymmetric reduction/oxidation) is defi-... 

Reduction with isolated enzymes avoids difficulties associated with diffusion limitations and also avoids the presence of many different enzymes, present in the whole cell, which can cause side reactions or reduced enantioselectivity. The main drawback, however, is the instability of the isolated enzyme and the requirement for added co-factor NAD(H) or NADP(H), which are the oxidized (or reduced) forms of nicotinamide adenine diphosphate or its 2 -phosphate derivative. These co-factors are expensive, but can be used as catalysts in the presence of a co-reductant such as formate ion HCOO or an alcohol (e.g. isopropanol or ethanol). The reduction of ketones occurs by transfer of hydride from the C-4 position of the dihydropyridine ring of NADH or NADPH (7.105). Only one of the two hydrogen atoms is transferred and this process occurs within the active site of the enzyme to promote asymmetric reduction. 

Recently, the enzyme was expressed in baker s yeast and used for the asymmetric oxidation of alkyl cyclohexanones. The use of designer baker s yeast combined the advantages of using purified enzymes (single catalytic species, no overmetabolism) with the benefits of whole-cell reactions (experimentally simple, no cofactor regeneration necessary). The enantioselectivities observed with the recombinant enzyme were the same as those with the original, isolated enzyme. 

Based on the above observations, the employment of chiral metal catalysts for the asymmetric BV oxidation is still underdeveloped for practical appUcations on the scale of natural products and drugs synthesis. Only biocatalytic BV oxidations performed with isolated enzymes or whole cells containing cyclohexanone monooxygenases (CHMOs) or BV monooxygenases (BVMOs) shows practical conversions as weU as enantioselectivity above 95% ee with cyclohexanones as substrates. 

Recently, the first asymmetric cell-free application of styrene monooxygenase (StyAB) from Pseudomonas sp. VLB 120 was reported [294]. StyAB catalyses the enantiospecific epoxidation of styrene-type substrates and requires the presence of flavin and NADH as cofactor. This two-component system enzyme consists of the actual oxygenase subunit (StyA) and a reductase (StyB). In this case, the reaction could be made catalytic with respect to NADH when formate together with oxygen were used as the actual oxidant and sacrificial reductant respectively. The whole sequence is shown in Fig. 4.106. The total turnover number on StyA enzyme was around 2000, whereas the turnover number relative to NADH ranged from 66 to 87. Results for individual substrates are also given in Fig. 4.106. Excellent enantioselectivities are obtained for a- and -styrene derivatives. 

Membrane proteins have a variety of functions. Most, but not all, of the important functions of the membrane as a whole are those of the protein component. Transport proteins help move substances in and out of the cell, and receptor proteins are important in the transfer of extracellular signals, such as those carried by hormones or neurotransmitters, into the cell. In addition, some enzymes are tightly bound to membranes examples include many of the enzymes responsible for aerobic oxidation reactions, which are found in specific parts of mitochondrial membranes. Some of these enzymes are on the inner surface of the membrane, and some are on the outer surface. There is an uneven distribution of proteins of all types on the inner and outer layers of all cell membranes, just as there is an asymmetric distribution of lipids. 

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