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Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability

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...

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Detalles Bibliográficos
Autores principales: Mansouri, Hamid R., Mihovilovic, Marko D., Rudroff, Florian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7187199/
https://www.ncbi.nlm.nih.gov/pubmed/31608538
http://dx.doi.org/10.1002/cbic.201900501
Descripción
Sumario: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 (BVMO(Flava)), 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 (CHMO(Acineto)) 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 BVMO(Flava), which was comparable to the T (m) of TmCHMO (ΔT (m)=1 °C) and significantly higher than the T (m) value for CHMO(Acineto) ((ΔT (m)=14.6 °C)). A strong deviation between the thermodynamic and kinetic stabilities of BVMO(Flava) was observed; this might have a major impact on future enzyme discovery for BVMOs and their synthetic applications.