Biocatalyst Stability

An important issue concerning the usage of monooxygenase for biocatalytic purposes is enzyme stability. It is well known that CHMO is not a very stable enzyme. Based on literature data it seems that the instability is partly caused by cysteine oxidation. Reducing agents have been used to prolong the lifetime of the biocatalyst. Having a model structure of CHMO at hand (see above), it should be possible to engineer a more stable variant. We have also observed that binding of an NADPH coenzyme analog can drastically prolong the lifetime of BVMOs [33]. This feature can be exploited when storing these biocatalysts. Furthermore, the stability of the monooxygenase can be increased by addition of a specific salt in the medium or by immobilization on a solid carrier [56]. The co-immobilization of CHMO with a dehydrogenase that recycles the NADPH coenzyme has been shown to be very effective [56, 57]. The latter approach yields a formulation that enables facile reuse of the biocatalyst.

Instability of CHMO has not only been observed for the isolated enzyme. During conversion of cyclohexanone by non-growing CHMO-expressing E. coli cells the amount of intercellular biocatalyst rapidly decreased [58]. A 24 h incubation of recombinant cells with cyclohexanone and glucose resulted in almost full degradation of the recombinant protein, prohibiting effective bioconversion. The active degradation of the monooxygenase could not be prevented by changing the medium components (e.g. addition of riboflavin, IPTG, or chloramphenicol) and limited the efficiency of a whole cell bioconversion. The intracellular instability of CHMO can be circumvented by employing a BVMO variant with increased stability. We have observed that HAPMO (ti/2 = 80 min at 36 °C [33]) is a more stable biocatalyst when compared with CHMO (ti/2 = 24 h at 25 °C [56]). The superior stability may be partly explained by the fact that HAPMO contains an additional domain which is important for the dimeric structure of this enzyme [41]. To assess the stabilizing effect of this domain we have attempted to express CHMO fused to this N-terminal domain. Unfortunately, the hybrid protein was poorly expressed, which prevented further studies.

The recently discovered PAMO represents a quite thermostable biocatalyst that only tends to become inactive at temperatures above 50 °C (ti/2 = 24 h at 52 °C) [35]. In addition to being thermostable, it is also tolerant towards a number of solvents [59]. Therefore, this BVMO is ideally suited for use in its isolated form. Interestingly, it has been observed that the enantioselectivity of this monooxygenase can be tuned by medium engineering. Several solvents were found to strongly influence the outcome of the enantioselective oxidations of aromatic sulfides. For example, the oxidation of thioanisole by PAMO in the absence of a cosolvent yielded the (R)-sulfoxide in 43% enantiomeric excess while with 30% methanol the enantiomeric excess increased to 89%.

Methanol is also able to cause a reversal of enantio preference in the case of a few other sulfide substrates. The first engineered PAMO mutants with altered substrate specificity have already been reported and were shown to be equally stable when compared with the wild-type enzyme [37].

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