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

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 Tm value of 53.1 °C for BVMOFlava, which was comparable to the Tm of TmCHMO (ΔTm=1 °C) and significantly higher than the Tm value for CHMOAcineto ((ΔTm=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.

Microbial Lipases and Their Potential in the Production of Pharmaceutical Building Blocks

“…The advantages of the use of this lipase is to allow a green and cost-effective process that does not require cofactors such as flavin and NADP + , necessary in the case of Baeyer–Villiger monooxygenases [ 222 ], and to avoid the use of catalysts based on heavy metals (Cu 2+ , Sn +4 ), such as with those based on MOFs [ 19 , 223 ]. Extending the use of these lipase-mediated reactions could favor the synthesis of building blocks of APIs, as is the case of intermediaries for the design of κ opioid receptor agonists ( Scheme 21 ), taking into account morphinan pharmacophore such as Narcan, which could be obtained by using this type of oxidation [ 224 ].…”

Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer–Villiger Monooxygenase

“…NCIMB 9871, CHMO Acineto ). 18 − 20 , 22 − 24 Thus, there is an unsolved problem in engineering highly stable and active BVMOs for industrial applications.…”

Enzymatic Kinetic Resolution by Addition of Oxygen

“…In a recent report, the group was able to use the genomic sequence of a recently published and thermostable BVMO, TmCHMO, for database screening and identified a new BVMO from Amycolaptosis thermoflava. [28] This new thermostable monooxygenase was able to process a range of cyclic ketones with excellent conversion and % ee.…”

“…However,b oth CAMO and OTEMO were shown to be excellent catalysts for the desymmetrisation of 3-vinylcyclobutanone and this led to the first chemoenzymatic synthesis of the Taniguchi lactone,akey intermediate in the synthesis of multiple natural products.I narecent report, the Rudroff group was able to use the genomic sequence of ar ecently published and thermostable BVMO,TmCHMO,for database screening and identified an ew BVMO from Amycolaptosis thermoflava. [28] This new thermostable monooxygenase was able to process ar ange of cyclic ketones with excellent conversion and enantioselectivity.…”