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Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability
Author(s) -
Mansouri Hamid R.,
Mihovilovic Marko D.,
Rudroff Florian
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900501
Subject(s) - monooxygenase , chemistry , enzyme kinetics , substrate (aquarium) , enzyme , stereochemistry , chemical stability , cyclohexanone , kinetics , kinetic energy , catalysis , active site , organic chemistry , biology , cytochrome p450 , ecology , physics , quantum mechanics
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 ( Tm CHMO), 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 Tm CHMO (Δ T m =1 °C) and significantly higher than the T m value for CHMO Acineto ((Δ T 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.