Biocatalytic with monooxygenases

Most biochemical and biocatalytic studies have been performed with type I B VMOs. This is partly because of the fact that they represent relatively uncomplicated monooxygenase systems. These monooxygenases are typically soluble and composed of only one polypeptide chain. Expression systems have been developed for a number of type I BVMOs while no recombinant expression has been reported for a type II BVMO. Cyclohexanone monooxygenase (CHMO) from an Acinetobacter sp. NCIMB9871 was the only recombinant available BVMO... 

Schmid, A., Hofstetter, K., Feiten, H.J., Holhnann, R, Witholt, B. (2001) Integrated Biocatalytic Synthesis on Gram Scale The Highly Enantio Selective Preparation of Chiral Oxiranes with Styrene Monooxygenase. Advanced Synthesis Catalysis, 343(6-7), I il-l il. 

A variety of monooxygenases (see above) can perform epoxidations. Some biocatalytic methods come into sight, which will become attractive for industrial use. In these cases chiral epoxides are the targeted products, and therefore this subject will be dealt with in Section 4.6. 

Fig. 4.106 Biocatalytic epoxidation with styrene monooxygenase including cofactor regeneration.

From a biocatalytic point of view the most interesting question to be answered is what enables these enzymes to catalyze the monooxygenation of even relatively inert chemical substrates, such as benzene derivatives, imder mild conditions. To answer this question several examples of monooxygenases, with different cofactors used to perform the job, will be discussed. 

A. Schmid, K. Hofstetter, H. J. Feiten, F. Hollmann, B. Witholt, Integrated biocatalytic synthesis on gram scale The highly enantioselective preparation of chiral oxiranes with styrene monooxygenase, Adv. Synth. Catal. 343 (2001) 732. 

Together with enantioselective hydrolysis/acylation reactions, enantioselective ketone reductions dominate biocatalytic reactions in the pharma industry [10], In addition, oxidases [11] have found synthetic applications, such as in enantioselective Baeyer-Villiger reactions [12] catalyzed by, for example, cyclohexanone monooxygenase (EC 1.14.13) or in the TEMPO-mediated oxidation of primary alcohols to aldehydes, catalyzed by laccases [13]. Hence, the class of oxidoreductases is receiving increased attention in the field of biocatalysis. Traditionally they have been perceived as difficult due to cofactor requirements etc, but recent examples with immobilization and cofactor regeneration seem to prove the opposite. 

Biocatalytic asymmetric epoxidation of alkenes catalyzed by monooxygenases cannot be performed on a preparative scale with isolated enzymes due to their complex nature and their dependence on a redox cofactor, such as NAD(P)H. Thus, whole microbial cells are used instead. Although the toxic effects of the epoxide formed, and its further (undesired) metabolism by the cells catalyzed by epoxide hydrolases (Sect. 2.1.5), can be reduced by employing biphasic media, this method is not trivial and requires bioengineering skills [1151]. Alternatively, the aUcene itself can constitute the organic phase into which the product is removed, away from the cells. However, the bulk apolar phase tends to damage the cell membranes, which reduces and eventually abolishes all enzyme activity [1152]. 

An important message from the scientific literature is that ISPR alone will not solve all the challenges to be met in scaling-up a bioreduction. In common with other complex biocatalytic reactions such as the transaminase-catalyzed synthesis of optically pure amines [65,66] or the cyclohexanone monooxygenase-catalyzed... 

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. 

Biocatalytic reactions with P450 monooxygenases are generally carried out using whole cells—of wild-type... 

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