Exploring the Temperature Effect on Enantioselectivity of a Baeyer-Villiger Biooxidation by the 2,5-DKCMO Module: The SLM Approach.

Temperature is a crucial parameter for biological and chemical processes. Its effect on enzymatically catalysed reactions has been known for decades, and stereo- and enantiopreference are often temperature-dependent. For the first time, we present the temperature effect on the Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one by the type II Bayer-Villiger monooxygenase, 2,5-DKCMO. In the absence of a reductase and driven by the hydride-donation of a synthetic nicotinamide analogue, the clear trend for a decreasing enantioselectivity at higher temperatures was observed. "Traditional" approaches such as the determination of the enantiomeric ratio (E) appeared unsuitable due to the complexity of the system. To quantify the trend, we chose to use the 'Shape Language Modelling' (SLM), a tool that allows the reaction to be described at all points in a shape prescriptive manner. Thus, without knowing the equation of the reaction, the substrate ee can be estimated that at any conversion.

Divorce in the two-component BVMO family: the single oxygenase for enantioselective chemo-enzymatic Baeyer-Villiger oxidations.

Two-component flavoprotein monooxygenases consist of a reductase and an oxygenase enzyme. The proof of functionality of the latter without its counterpart as well as the mechanism of flavin transfer remains unanswered beyond doubt. To tackle this question, we utilized a reductase-free reaction system applying purified 2,5-diketocamphane-monooxygenase I (2,5-DKCMO), a FMN-dependent type II Baeyer-Villiger monooxygenase, and synthetic nicotinamide analogues (NCBs) as dihydropyridine derivatives for FMN reduction. This system demonstrated the stand-alone quality of the oxygenase, as well as the mechanism of FMNH2 transport by free diffusion. The efficiency of this reductase-free system strongly relies on the balance of FMN reduction and enzymatic (re)oxidation, since reduced FMN in solution causes undesired side reactions, such as hydrogen peroxide formation. Design of experiments allowed us to (i) investigate the effect of various reaction parameters, underlining the importance to balance the FMN/FMNH2 cycle, (ii) optimize the reaction system for the enzymatic Baeyer-Villiger oxidation of rac-bicyclo[3.2.0]hept-2-en-6-one, rac-camphor, and rac-norcamphor. Finally, this study not only demonstrates the reductase-independence of 2,5-DKCMO, but also revisits the terminology of two-component flavoprotein monooxygenases for this specific case.

Efficient and Selective Carboligation with Whole-Cell Biocatalysts in Pickering Emulsion.

Pickering emulsions (PEs) are particle-stabilized multiphase systems with promising features for synthetic applications. Described here is a novel, simplified set-up employing catalytically active whole cells for simultaneous emulsion stabilization and synthetic reaction. In the stereoselective carboligation of benzaldehyde to (R)-benzoin catalyzed by a benzaldehyde lyase in E. coli, the set-up yielded maximum substrate conversion within very short time, while economizing material demand and waste. Formation and activity of freshly produced PEs were enhanced when the catalytic whole cells were covered with hydrophobic silicone prior to PE formation. Benchmarked against other easy-to-handle whole-cell biocatalysts in pure organic solvent, neat substrate, an aqueous emulsion in substrate, and a micro-aquatic system, respectively, the cell-stabilized PE outperformed all other systems by far.

Convergent Cascade Catalyzed by Monooxygenase-Alcohol Dehydrogenase Fusion Applied in Organic Media.

With the aim of applying redox-neutral cascade reactions in organic media, fusions of a type II flavin-containing monooxygenase (FMO-E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell-free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane-1,4-diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert-butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ-butyrolactone, resulting in about 27 % analytical yield.