Abstract
Baeyer–Villiger monooxygenases (BVMOs) are remarkable biocatalysts, but, due to their low stability, their application in industry is hampered. Thus, there is a high demand to expand on the diversity and increase the stability of this class of enzyme. Starting from a known thermostable BVMO sequence from Thermocrispum municipale (TmCHMO), a novel BVMO from Amycolaptosis thermoflava (BVMOFlava), which was successfully expressed in Escherichia coli BL21(DE3), was identified. The activity and stability of the purified enzyme was investigated and the substrate profile for structurally different cyclohexanones and cyclobutanones was assigned. The enzyme showed a lower activity than that of cyclohexanone monooxygenase (CHMOAcineto) from Acinetobacter sp., as the prototype BVMO, but indicated higher kinetic stability by showing a twofold longer half‐life at 30 °C. The thermodynamic stability, as represented by the melting temperature, resulted in a T m value of 53.1 °C for BVMOFlava, which was comparable to the T m of TmCHMO (ΔT m=1 °C) and significantly higher than the T m value for CHMOAcineto ((ΔT m=14.6 °C)). A strong deviation between the thermodynamic and kinetic stabilities of BVMOFlava was observed; this might have a major impact on future enzyme discovery for BVMOs and their synthetic applications.
Highlights
Baeyer–Villiger monooxygenases (BVMOs) were identified, isolated, and characterized in the late 1960s and since have become highly versatile biocatalysts for the oxidation of ketones and aldehydes into the corresponding esters or lactones (Baeyer–Villiger reaction).[1]
The phylogenetic tree was constructed by PhyML and visualized by TreeDYN to find the position of BVMOFlava between different groups of BVMOs (Figure 1)
The midpoint rooted maximum likelihood phylogram shows the diversity of different BVMOs from groups 1 to 7.[20]. As observed in the maximum likelihood phylogram (Figure 1), the sequence of BVMOFlava is close to the sequence TmCHMO, with a strong bootstrap statistical support of 100 %
Summary
Intrigued by the work of Romero et al.,[14] we aimed to find a novel BVMO with altered thermodynamic stability, but that maintained high activity and substrate acceptance, based on a sequence similarity in silico approach. We envisaged exploiting the sequence space of thermophilic bacteria by using the TmCHMO sequence as a starting point. Among sequences found in the NCBI databank, a new putative BVMO sequence from the thermophilic organism Amycolatopsis thermoflava, which was isolated from heat-treated soil,[15] was selected. Multiple sequence alignment with different BVMOs[16,17,18,19] was performed to investigate the Type I BVMO family motifs. Ty and stability (kinetic and thermodynamic) were measured, and the substrate profile of this novel BVMO was investigated
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