Abstract
Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family have been thoroughly investigated for the stereospecific reduction of activated prochiral C=C double bonds. In this work, OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by affinity-based adsorption on EziGTM particles (OYE3-EziG). The immobilized OYE3-GA was demonstrated to be active (activity recovery = 52%) and to retain almost 100% of its activity under the enzymatic assay conditions (50 mM phosphate buffer pH 7, 28 °C) for six days, whereas the activity of the non-immobilized enzyme dropped to 50% after two days. In the case of EziGTM, the highest activity recovery (54%) was achieved by using the most hydrophilic carrier (EziGTM Opal) that was selected for the full characterization of this type of enzyme preparation (stability, recycling, re-use, enzyme leakage). OYE3-EziG was slightly less stable than OYE3-GA under the same experimental conditions. OYE3-GA could be recycled and re-used for up to 12 reaction cycles in the bioreduction of α-methyl-trans-cinnamaldehyde; after 12 runs, the highest conversion achieved was 40%. In the case of the co-immobilized OYE3/GDH-EziG, the conversion dropped to 56% after two reaction cycles. No enzyme leakage was detected over 48 h for both OYE3-GA and OYE3/GDH-EziG (50 mM phosphate buffer pH 7, 28 °C). These seed results pave the way for a true optimization of the immobilization of OYE3, as well as for the use of immobilized OYE3 for preparative applications both in batch and continuous flow conditions.
Highlights
In the case of the co-immobilized OYE3/glucose dehydrogenase (GDH)-EziG, the conversion dropped to 56% after two reaction cycles
Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family (EC 1.6.99.1) are flavin mononucleotide (FMN)-containing oxidoreductases, which are able to catalyze the stereoselective reduction of C=C double bonds activated by the presence of a suitable electron-withdrawing group (EWG) as a substituent of the alkene moiety [1,2]
OYE3 was immobilized both by covalent binding on glyoxyl-agarose (OYE3-GA), and by metal affinity interaction on EziGTM particles (OYE3-EziG)
Summary
Ene-reductases (ERs) belonging to the old yellow enzyme (OYE) family (EC 1.6.99.1) are flavin mononucleotide (FMN)-containing oxidoreductases, which are able to catalyze the stereoselective reduction of C=C double bonds activated by the presence of a suitable electron-withdrawing group (EWG) as a substituent of the alkene moiety [1,2]. The enzyme-bound flavin (reduced by NAD(P)H cofactor to FMNH2 ) transfers a hydride to the olefin carbon atom in β position with respect to the EWG. The OYE-mediated bioreduction has been widely investigated in the last decade to establish substrate scope and stereoselectivity of these enzymes [4,5,6], and to study the combination of this reaction with other chemo- and/or biocatalyzed transformations, both in concomitant and subsequent cascade procedures [7,8]. Several works showed the advantages of using OYE-catalyzed hydrogenation to prepare chiral building blocks for the synthesis of APIs (Active Pharmaceutical Ingredients) [9,10,11,12], flavors and fragrances [13,14,15]
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