Cloning and characterization of the Type I Baeyer–Villiger monooxygenase from Leptospira biflexa

Abstract

Baeyer–Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer–Villiger monooxygenase from Leptospira biflexa. This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the Baeyer–Villiger monooxygenase from L. biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0390-5) contains supplementary material, which is available to authorized users.

Unique Features of a New Baeyer–Villiger Monooxygenase from a Halophilic Archaeon

“…However, the highly negative surface of HtBVMO prompted us to further investigate this feature in comparison to other known halophilic and non-halophilic BVMOs. These enzymes have been classified into seven groups (plus a further group Catalysts 2020, 10, 128 5 of 20 of non-classified outlayers) based on phylogenetic clustering [28]. We considered the fifty BVMOs from this article, representative of the seven classes, added HtBVMO and ranked all of them on the basis of protein net charge (see supplementary Table S1).…”

“…The evidence that phylogenetic-based grouping into the seven reported classes [28] is not in agreement with net charge distribution (as it varies in each class, with highly overlapping ranges), prompted us to perform an electrostatics-based, comparative analysis of a set of 86 BVMOs (including HtBVMO and other important enzymes not previously reported [28]). To this aim, we considered the distribution of the electric isopotential at the protein surfaces, which proved useful to identify putative fingerprints in a "functional" grouping and classification of proteins [29,30].…”

Recent Developments in Protein Engineering and Catalytic Oxidations of Baeyer-Villiger Monooxygenase

“…野生型相反的区域选择性(48∶47＝1∶3), 其中 PAMO A435Y 转化所得的异常产物 47 还有着优异的立体选择 性(ee 90%). 而 CHMO [51] 、PAMO 的 V54I 和 I67T 突变 株 [19] 、 CAMO [26] 以 及 BVMO Lepto ( 来 源 于 Leptospira biflex) [4] 等酶则是等量产生 2 种区域异构产物(1∶1). 此…”

“…BVMO [2] . Rial 等 [4] 壬酮和 3-壬酮等长链酮能实现完全转化 [5] . 己酮可可碱 1 是一种广泛使用的药物, hFMO5(来源于 Human)对这 种具有羰基的药物有很强的体外氧合活性, 可以将其氧 化成相应的乙酸酯, 反应具有高度的区域选择性(30∶ 1) [6] (Scheme 1).…”

Towards biobased ethyl acetate and beyond : Identification and application of the elusive Eat1 enzyme

“…BVMOs have been used extensively for the biotransformation of cyclic ketones into lactones (cyclic esters) (Leisch et al, 2011). Several BVMOs exist that are also active towards aliphatic ketones, and are interesting catalysts for the production of volatile esters (Ceccoli et al, 2017;Pereira et al, 2018;Rehdorf et al, 2009). However, most bioconversions via BVMOs were performed in cell free systems.…”