1. Investigation of a New Type I Baeyer–Villiger Monooxygenase fromAmycolatopsis thermoflavaRevealed High Thermodynamic but Limited Kinetic Stability
- Author
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Mansouri, Hamid R., Mihovilovic, Marko D., and Rudroff, Florian
- Subjects
biocatalysis ,Stereochemistry ,monooxygenases ,Protein Engineering ,Kinetic energy ,medicine.disease_cause ,enzyme catalysis ,Biochemistry ,Mixed Function Oxygenases ,Substrate Specificity ,Enzyme catalysis ,Bacterial Proteins ,Enzyme Stability ,in silico analysis ,Escherichia coli ,medicine ,Amycolatopsis ,Molecular Biology ,Phylogeny ,chemistry.chemical_classification ,Chemistry ,Communication ,Organic Chemistry ,Substrate (chemistry) ,Hydrogen-Ion Concentration ,Monooxygenase ,Communications ,Recombinant Proteins ,Actinobacteria ,Kinetics ,Enzyme ,Biocatalysis ,Thermodynamics ,Molecular Medicine ,Chemical stability ,Half-Life - 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., In silico screening: A Baeyer–Villiger monooxygenase (BVMO) from a thermophilic origin is identified through an in silico approach. There is a strong deviation between the thermodynamic and kinetic stabilities of the new BVMO, which might have a major impact for future enzyme discovery of BVMOs and their synthetic applications.
- Published
- 2020