Coupled(s)-carbonyl Reductase â…¡ And Glucose Dehydrogenase In Mesoporous ZnO/C Composite Nanomaterials For Efficient Chiral Catalysis

Carbonyl reductase(EC 1.1.1.X) is one of the most promising biocatalyst in preparation of chiral compounds because of its high chemo-, region- and stereo-selectivity.(S)-carbonyl reductase II(SCRII) from Candida parapsilosis CCTCC M203011 catalyzes asymmetric transformation of 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol. To realize efficient biosynthesis of(S)-1-phenyl-1,2-ethanediol, we designed a co-expression system containing SCRII and Bacillus sp.YX-1 glucose dehydrogenase(GDH) in Escherichia coli BL21(DE3), basing on the optimal ratio between the specific activities of two enzymes. In addition, we successfully synthesized a new type of mesoporous ZnO/C composite nanomaterials, and then immobilized the fusion enzyme SCRII-GDH in ZnO/C nanomaterials. The immobilized SCRII-GDH showed excellent performance to produce(S)-1-phenyl-1,2-ethanediol. The main results are as follows:(1) SCRII and GDH exhibited specific activities of 1.3 U?mg-1 and 13.5 U?mg-1. When the total enzyme activity was 1 U, the optimal ratio of their activities is between 1:1 and 5:1, and the optimal temperature and pH are 30 oC and 7.0, respectively. Basing on these results, we designed a co-expression system E. coli BL21/S-SD-AS-G, in which the ratio of the SCRII and GDH genes is 1:1. The specific activities of SCRII and GDH are 0.76 U?mg-1 and 0.73 U?mg-1 in the cell-free extracts of E. coli BL21(DE3)/S-SD-AS-G, respectively. The ratio between SCRII and GDH activity is about 1:1. Under the optimal conditions, the recombinant showed excellent performance to produce(S)-1-phenyl-1,2-ethanediol with an optical purity and a yield both over 99% during the reduction of 2-hydroxyacetophenone. With respect to the recombinant E. coli BL21(DE3)/pET-SCRII, the co-expression system obviously improved the yield of(S)-1-phenyl-1,2-ethanediol and reduced biotransformation time from 24 h to 13 h.(2) A facile synthesis of mesoporous ZnO/C composites was achieved through an EISA method by using a cheap zinc acetate and resol as the Zn and carbon source, respectively, and an amphiphilic triblock copolymer Pluronic F127(PEO-PPO-PEO) as a template, followed by carbonization, and with it as a support for immobilization of fusion enzyme SCRII-GDH. X-ray diffraction(XRD) indicated the ZnO was successfully embedded in carbon layer with a good crystal shape. Transmission electron microscopy(TEM) showed a clear 2-D structure of the mesoporous ZnO/C composites, in which the uniform and good dispersion pores were circlewise dispersed in the carbon matrix. The formation of nanocrystalline ZnO with a hexagonal structure is further confirmed by the diffusive selected area electron diffraction(SAED). Fourier transform infrared spectroscopy(FTIR) further proves the SCRII-GDH was successfully immobilized on the mesoporous ZnO/C composites.(3) The amount of immobilized SCRII-GDH was found to be 110 mg?g-1 of support. The immobilized SCRII-GDH catalyzed the asymmetric transformation of 10 g?L-1 2-hydroxyacetophenone to(S)-1-phenyl-1, 2-ethanediol within 1 hour at 30 oC and pH 6.0, while the free enzyme SCRII-GDH required about 8 h to finishe the biotransformation process. In addation, the optimal temperature of the immobilized SCRII-GDH and free enzyme are both 35 oC, while thermal stability of the immobilized SCRII-GDH is better than free enzyme. SCRII-GDH shows a broader optimal catalytic pH range when it is attached on the ZnO/C composite nanomaterials.(4) The reusability, conversion efficiency of high-concentrated substrate, organic solvent tolerance and storage stability of the immobilized SCRII-GDH were investigated. After being reused for 12 times, the immobilized SCRII-GDH biotransformed 10 g?L-1 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol with a stable optical purity over 99% and a yield of 64.3%; when the substrate was at an impressive concentration of 50 g?L-1, the yield of(S)-1-phenyl-1,2-ethanediol also reached 57.7%. Immobilized SCRII-GDH shows a high resistance to organic solvent of alkane with a yield all over 90%. After being stored at-20 oC for 100 days and repeated freezing and thawing for 10 times, immobilized SCRII-GDH catalyzed the biotransformation with a stable optical purity over 99% and a yield over 75%.