Magnetic combined cross-linked enzyme aggregates of ketoreductase and alcohol dehydrogenase: An efficient and stable biocatalyst for asymmetric synthesis of (R)-3-Quinuclidinol with regeneration of coenzymes in situ

Abstract

Enzymes are biocatalysts. In this study, a novel biocatalyst consisting of magnetic combined cross-linked enzyme aggregates (combi-CLEAs) of 3-quinuclidinone reductase (QNR) and glucose dehydrogenase (GDH) for enantioselective synthesis of (R)-3-quinuclidinolwith regeneration of cofactors in situ was developed. The magnetic combi-CLEAs were fabricated with the use of ammonium sulfate as a precipitant and glutaraldehyde as a cross-linker for direct immobilization of QNR and GDH from E. coli BL(21) cell lysates onto amino-functionalized Fe3O4 nanoparticles. The physicochemical properties of the magnetic combi-CLEAs were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and magnetic measurements. Field emission scanning electron microscope (FE-SEM) images revealed a spherical structure with numerous pores which facilitate the movement of the substrates and coenzymes. Moreover, the magnetic combi-CLEAs exhibited improved operational and thermal stability, enhanced catalytic performance for transformation of 3-quinuclidinone (33 g/L) into (R)-3-quinuclidinol in 100% conversion yield and 100% enantiomeric excess (ee) after 3 h of reaction. The activity of the biocatalysts was preserved about 80% after 70 days storage and retained more than 40% of its initial activity after ten cycles. These results demonstrated that the magnetic combi-CLEAs, as cost-effective and environmentally friendly biocatalysts, were suitable for application in synthesis of (R)-3-quinuclidinol essential for the production of solifenacin and aclidinium with better performance than those currently available.