Facile fabrication of 3D porous hybrid sphere by co-immobilization of multi-enzyme directly from cell lysates as an efficient and recyclable biocatalyst for asymmetric reduction with coenzyme regeneration in situ.

Ni-agarose bead-wrapped multi-enzyme/inorganic hybrid sphere composed of the immobilized enzymes as organic component and NaHPO and NaCl as inorganic component was developed by co-immobilizing extracellular His-tagged 3-quinuclidinone reductases and glucose dehydrogenase without pre-purification. The resulting biocatalysts has 3D porous architectures as confirmed by SEM and FESEM, and it enabled the continuous biotransformation of 3-quinuclidone to (R)-3-quinuclidinol with cofactor regeneration in situ. The 3D porous biocatalysts were formed via three steps: First, immobilization of the His-tagged enzymes directly from the cell lysates supernatant. Next, formation of enzyme aggregates, ribbons and gels. Finally, the enzymes, the formed aggregates/ribbons/gels and salt were incorporated to the foam and then covered the Ni-agarose bead. The technique made the immobilization of these enzymes effective such that specific enzyme loading of 60.8mg/g support and enzyme loading efficiency of 92.3% were achieved. As a direct consequence, the biocatalyst catalyzed the conversion of 3-quinuclidinone (204g/L) to (R)-3-quinuclidinol in 100% yield and 100% ee at 4.5h, and the recyclability of the biocatalyst was excellent, retaining>95% conversion yield and 100% ee even after the fifteenth runs. Overall, our strategy is demonstrated to be a promising method for developing efficient and robust biocatalyst for asymmetric synthesis.