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.

Ni2+-agarose bead-wrapped multi-enzyme/inorganic hybrid sphere composed of the immobilized enzymes as organic component and NaH2PO4 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 Ni2+-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.

Supramolecular assemblies constructed from ß-cyclodextrin-modified montmorillonite nanosheets as carriers for 5-fluorouracil.

Supramolecular assemblies generated from self-assembling ß-cyclodextrin-modified montmorillonite nanosheets were evaluated as anticancer drug carriers in vitro. The results showed that the assemblies had a high loading capacity of 5-fluorouracil (5-FU) under the optimized conditions that included a temperature of 80 °C and a pH level of 11. Scanning electron microscopy (SEM) images showed no morphological changes in the assemblies even after 20 days of storage at room temperature. Moreover, SEM and atomic force microscopy (AFM) observations revealed that the incorporation of 5-FU hardly affected the morphology of the assemblies. Furthermore, the assemblies showed sustained release behavior in vitro, and SEM and AFM analyses indicated that the kinetics of 5-FU release were closely associated with morphological changes in the surface of the assemblies during drug release. Cell viability assays showed that the blank assemblies had low cytotoxicity against A549 cells, while the inhibitory effects of 5-FU-loaded assemblies against A549 cells increased significantly with an increased concentration. More importantly, fluorescence microscopy imaging and transmission electron microscopy (TEM) demonstrated that both blank assemblies and 5-FU-loaded assemblies can easily penetrate cultured human ovarian cancer SKOV3 cells. These results suggest that the supramolecular assemblies may potentially be used as building blocks for the development of new anti-cancer drug delivery systems.