Department of Biotechnology, University of the Free State, 205 Nelson Mandela Drive, Bloemfontein, 9300, South Africa.
Institute of Technical Biocatalysis, Hamburg University of Technology, Denickestr. 15, Hamburg, 21073, Germany.
Enzyme Microb Technol. 2017 Nov;106:11-17. doi: 10.1016/j.enzmictec.2017.06.017. Epub 2017 Jul 3.
FAD-dependent Baeyer-Villiger monooxygenases (BVMOs) have proven to be useful biocatalysts in the selective and specific oxygenation of various ketones. Despite the cloning, heterologous expression and characterization of close to 80 members of this enzyme family, some sub-groups of BVMOs still remain underrepresented and their evolutionary relationship uncertain. Until recently, very few fungal BVMOs have been described. Our previous investigations into BVMOs from the fungus Aspergillus flavus, yielded very little activity on simple cyclic ketones. Here we report on another four BVMOs from A. flavus that are more closely related to cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. Evolutionary analysis with other characterized BVMOs show their closest relationship to be with either cycloalkanone monooxygenase (CAMO) or 2-oxo-Δ-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase (OTEMO). The OTEMO-related BVMO and BVMO were heterologously expressed in E. coli, purified and shown to be able to convert a range of cyclic and substituted cyclic ketones. Of the unsubstituted cyclic ketones, cyclohexanone showed the highest conversion with maximum turnover frequencies reaching 4.3s for BVMO. Unlike CHMO, and many of the closely related BVMOs, no substrate inhibition was observed with cyclohexanone to a concentration of up to 30mM, creating the possibility for applications requiring high substrate loading. Aliphatic ketones were also readily converted with excellent regioselectivity. Similar to CHMO, acetophenones were not converted and the oxidation of rac-cis-bicyclo[3.2.0]hept-2-en-6-one occurs enantiodivergently, with the (1R,5S) isomer converted to the "normal" lactone and the (1S,5R) isomer to the "abnormal" lactone.
依赖 FAD 的 Baeyer-Villiger 单加氧酶（BVMO）已被证明是各种酮类选择性和特异性氧化的有用生物催化剂。尽管该酶家族的近 80 个成员已经被克隆、异源表达和表征，但一些 BVMO 亚组仍然代表性不足，其进化关系也不确定。直到最近，才描述了很少的真菌 BVMO。我们之前对黄曲霉真菌 BVMO 的研究，对简单的环状酮几乎没有活性。在这里，我们报告了黄曲霉中的另外四个 BVMO，它们与来自不动杆菌 NCIMB 9871 的环己酮单加氧酶（CHMO）更为密切相关。与其他已鉴定的 BVMO 的进化分析表明，它们最密切的关系是与环烷酮单加氧酶（CAMO）或 2-氧代-Δ-4,5,5-三甲基环戊烯基乙酰辅酶 A 单加氧酶（OTEMO）。OTEMO 相关的 BVMO 和 BVMO 在大肠杆菌中异源表达、纯化，并证明能够转化一系列环状和取代环状酮。在未取代的环状酮中，环己酮的转化率最高，BVMO 的最大转换频率达到 4.3s。与 CHMO 不同，而且与许多密切相关的 BVMO 不同，环己酮在高达 30mM 的浓度下没有观察到底物抑制，为需要高底物负荷的应用创造了可能性。脂肪酮也很容易被转化，具有极好的区域选择性。类似于 CHMO，苯乙酮没有被转化，rac-顺式-双环[3.2.0]庚-2-烯-6-酮的氧化发生对映体转化，（1R,5S）异构体转化为“正常”内酯，（1S,5R）异构体转化为“异常”内酯。
