Biocatalytic Properties Of Recombinant Available Bvmos

The list of heterologously expressed type I BVMOs has grown significantly in recent years (Table 1). Except for BVMOs primarily acting on small cyclic ketones (cyclopentanone and cyclohexanone)1920, variants specific for larger cyclic ketones (cyclododecanone and cyclopentadecanone) have also been discovered.2122 Additionally, BVMOs that readily accept aromatic ketones (4-hydroxyacetophenone and phenylacetone derivatives) have been described.1723 Also, a BVMO acting on steroids has been reported.24 Substrate profiling studies suggest that BVMOs have a rather broad specificity and often display overlapping substrate specificities. For example, bicycloheptenone and aromatic sulfides have been shown to be converted by several BVMOs. Illustrative for the broad substrate specificity of BVMOs is the fact that in 2002 it was reported that for CHMO from Acinetobacter sp. over 100 different substrates had been reported.25 Since that time, a number of other ketones have been shown to be converted by this monooxygenase extending the impressive list of CHMO substrates even further.25-29

Except for exploring its catalytic potential, CHMO from Acinetobacter has also been used as a model system for upscaling BVMO-mediated biocatalysis.

Table 1

Recombinant available BVMOs



Primary substrate


Year of cloning

Cyclopentanone monooxygenase CPMO

Cyclohexanone monooxygenase CHMO

Cyclododecanone monooxygenase CDDMO

Cyclopentadecanone monooxygenase CPDMO

4-Hydroxyacetophenone monooxygenase

Phenylacetone monooxygenase

Ethionamide monooxygenase Steroid monooxygenase



Comamonas sp. Acinetobacter sp.

Rhodococcus ruber

Pseudomonas sp. Pseudomonas fluorescens

Thermobifida fusca

EtaA Physiological substrates unidentified Mycobacterium tuberculosis


Rhodococcus rhodochrous

200220'41 198813'19 200121

200622 200123


200447 199924

List of BVMOs that have been overexpressed in E. coli and of which the isolated enzyme has been characterized to some extend.

In recent years, the main focus has been on using whole cells expressing CHMO as biocatalyst. By this, the problem of coenzyme usage by the enzyme can be circumvented, as NADPH will be regenerated by the cellular machinery. During the years, problems related to, e.g., product/substrate inhibition and oxygen sensitivity have been tackled.3031 An efficient methodology has been developed that is applicable on kilogram scale.32 Using whole cells expressing CHMO in combination with resin-based in situ substrate feeding and product removal (SFPR) and a tuned oxygen supply, a highly productive process was developed yielding two nearly enantiopure (ee > 98%) regioisomeric lactones in good yield. This nicely illustrates that BVMOs can be applied on a scale that is relevant for synthesis of fine chemicals.

While the substrate acceptance of a specific BVMO is often relaxed, catalytic efficiencies and regio- and/or enantioselectivities can differ significantly when comparing BVMOs. A number of CHMO and CPMO homologs (>35% sequence identity) have recently been identified and explored concerning their biocatalytic potential. These comparative biocatalytic studies using highly similar enzymes have revealed that, as expected, all studied CHMOs and CPMOs cover a similar substrate range.29'33'34 However, regio- and/or enantioselectivi-ties differ significantly. It was observed that CPMOs and CHMOs often display opposite enantioselectivities.34 This illustrates the need for a large library of BVMOs to meet the demand for enantio- and/or regioselective oxidative biocatalysts.

Although BVMOs often display a broad substrate specificity, each BVMO has a certain preference for a specific type of substrate. While CHMO and CPMO are highly active with a range of (cyclic) aliphatic ketones, HAPMO and PAMO prefer aromatic substrates.17 23 35-37 Figure 3 illustrates the overlapping substrate specificities for several well-studied BVMOs displaying several typical substrates for each BVMO.

For several recombinant available BVMOs, no extensive substrate profiling studies have been performed. For steroid monooxygenase, only steroid substrates have been tested while cyclododecanone and cyclopentadecanone monooxyge-nases have only been recently identified and await further exploration. Based on the first biocatalytic data obtained with cyclopentadecanone monooxygenase, it appears that this novel BVMO is attractive when relatively large compounds are targeted.22 The enzyme proved to be effective in enantioselective Baeyer-Villiger oxidations of a range of bulky cycloketones. By this, it complements the substrate range that is covered by other known BVMOs.

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