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  • br Introduction Biocatalysis can be used to synthesize

    2019-12-03


    Introduction Biocatalysis can be used to synthesize chiral building blocks, such as monomers for polymer materials, and precursors for pharmaceuticals [[1], [2], [3], [4]]. Enzymes are very suitable for catalyzing reactions with high enantioselectivity to obtain chiral Apocynin products. For instance, alcohol dehydrogenases (ADHs, EC 1.1.1.X) – also known as carbonyl reductases or ketoreductases – depend on NAD(P)H to catalyze the asymmetric reduction of ketones to either (R)- or (S)-alcohols in excellent enantiomeric excess (ee) [5]. These enzymes, among others, have been applied for syntheses of pharmaceutical precursors [2,4]. Another group of redox Apocynin that depend on NAD(P)H are the Baeyer-Villiger monooxygenases (BVMOs) (EC 1.14.13.X). These FAD-containing enzymes can catalyze regio- and enantioselective transformations of ketones to esters or lactones, using dioxygen and NADPH. Interest in the application of BVMOs has grown, in particular for the transformation of substituted cyclic ketones to chiral lactones, for branched polyesters [[6], [7], [8], [9]]. Specifically, the recent discovery of robust BVMOs, such as the thermostable cyclohexanone monooxygenase from Thermocrispum municipale (TmCHMO), has led to great interest for exploring these monooxygenases for industrial applications, as most of the previously reported BVMOs were quite unstable [10]. Both ADHs and BVMOs rely on the cofactor NAD(P)H for catalysis, which is too expensive to apply in stoichiometric amounts, and therefore should be regenerated. There are a number of different approaches to recycle NAD(P)H [5,11,12]. One approach is to apply another enzyme which can use the oxidized NAD(P)+ and a sacrificial cheap cosubstrate to regenerate the reduced nicotinamide cofactor: the so-called “coupled-enzyme” approach. Three commonly used coenzyme regenerating enzymes are formate dehydrogenase (FDH), glucose dehydrogenase (GDH), and phosphite dehydrogenase (PTDH), all using relatively cheap substrates [12]. Instead of producing and adding these enzymes separately, the recycling enzyme can also be covalently fused to the NADPH-dependent enzyme through enzyme engineering (Scheme 1). In this way, a bifunctional and self-sufficient fusion biocatalyst is produced enabling conversion using merely one biocatalyst. Moreover, some studies on enzyme fusions provided evidence that tethering of two enzymes can improve the productivity of a multi-enzyme system [13]. In 2008 Torres Pazmiño et al. developed a platform for expressing BVMOs fused to PTDH for efficient cofactor regeneration [14,15]. Later, a few other studies reported enzyme fusions with FDH [16], GDH [17], or PTDH [[18], [19], [20]], though no study yet has compared a single biocatalyst with different regenerating enzymes. The aim of this work is to explore different recycling enzymes as fusion partner for two oxidoreductase enzymes (BVMO and ADH), and to compare their strengths and weaknesses for biocatalytic applications. We fused three different regenerating enzymes to a BVMO (TmCHMO, from Thermocrispum municipale) [10] and an (R)-selective alcohol dehydrogenase (LbADH, from Lactobacillus brevis) [[21], [22], [23]], to generate a panel of self-sufficient biocatalysts. The three recycling enzymes are: FDH from Burkholderia stabilis (BsFDH) [24], GDH from Sulfolobus tokodaii (StGDH) [25], and an engineered PTDH from Pseudomonas stutzeri (PsPTDH) [26,27]. The fused enzymes were produced and purified, and applied for biotransformations. Both enzymes act on ketone substrates to form interesting products, though many of such organic substrates have low solubility in water. Therefore, the enzyme fusions were tested with a wide range of solvents. In particular, the tolerance of these biocatalysts to deep-eutectic solvents (DES) was evaluated, which at present has not been studied to a great extent.
    Results and discussion
    Conclusions