Co-expression Of(S)-carbonyl Reductase Ⅱ And Glucose Dehydrogenase In Saccharomyces Cerevisiae Spore-encapsulations For Chiral Catalysis

Carbonyl reductase(EC 1.1.1.184), one of the most promising biocatalysts with high chemo-, region- and stereo-selectivity, is always used for preparation of chiral compounds.(S)-carbonyl reductase II(SCRII) from Candida parapsilosis CCTCC M203011 can catalyze asymmetric transformation of 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol efficiently. By site-directed mutagenesis in substrate catalytic region of SCRII, the mutant E228 S efficiently catalyzed acetophenone to(R)-1-phenylethanol. In this work, using genetic engineering technology, we obtained four kinds of excellent microencapsulated enzymes by heterogenously expressing SCRII, E228 S, the coupled SCRII and glucose dehydrogenase(SCRII-GDH), and the coupled E228 S and glucose dehydrogenase(E228S-GDH) in Saccharomyces cerevisiae osw2△, and embedding them in yeast spores. These microencapsulated enzymes catalyzed highly efficient bioconversion of(S)-1-phenyl-1,2-ethanediol and(R)-1-phenylethanol. The main results are as follows:(1) The recombinant yeasts S. cerevisiae osw2△/p RS424-TEFpr-scr II、S. cerevisiae osw2△/p RS424-TEFpr-E228 S 、S. cerevisiae osw2△/p RS424-TEFpr-scr II-SD-AS-gdh 、S. cerevisiae osw2△/p RS424-TEFpr-E228S-SD-AS-gdh were constructed. When cultured with potassium acetate as the sole energy source, yeast spores were produced and embedded the recombinant proteins to form yeast-microencapsulated enzymes: SCRII, E228 S, SCRII-GDH and E228S-GDH. All the four target proteins were proved in microencapsulated system by Western blotting technique. Meanwhile, the recombinant yeast was constructed in which the target enzyme and green fluorescent protein(GFP) were fused. By co-focus microscopy, the target proteins were further proved to be enbeded in yeast spore-encapsulations successfully.(2) Using four microencapsulated enzymes as biocatalysts, SCRII biotransformed 6 g?L-1 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol with an optical purity of 99.3% and a yield of 99.0% at 4h; E228 S biocatalyzed 6 g?L-1 acetophenone to(R)-1-phenylethanol with an optical purity of 99.1% in a yield of 95.1% at 5 h; SCRII-GDH bioconversed 20 g?L-1 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol with an optical purity of 99.7% and a yield of 92.1% at 3 h; E228S-GDH catalyzed 20 g?L-1 acetophenone to(R)-1-phenylethanol with an optical purity of 99.8% a yield of 89.7% at 4.5 h. Furthermore, all the four microencapsulated enzymes catalyzed the above biotransformations with good resistance to high temperature and wide p H ranges.(3) During the biotransformation, the reusability, organic solvent tolerance and storage stability of microencapsulated SCRII-GDH and E228S-GDH were investigated. After being reused for 10 times, the microencapsulated SCRII-GDH biotransformed 20 g?L-1 2-hydroxyacetophenone to(S)-1-phenyl-1,2-ethanediol with a stable optical purity of 99% and a yield of 63%; the microencapsulated E228S-GDH biotransformed 20 g?L-1 acetophenone to(R)-1-phenylethanol with an optical purity of 99% maintaining a yield over 55%. Both the two microencapsulated enzymes were highly resistant to 15% organic solvents, such as alcohol, ester and alkane. Significantly, microencapsulated SCRII-GDH biotransformed 20 g?L-1 2-hydroxyacetophenone with a high yield about 85.4% under 15% DMSO. After being air-dried at 30oC for 24 h, both two microencapsulated enzymes catalyzed the biotransformation in about 50% yield; After being stored in –20oC for 70 days and repeated freezing and thawing for 10 times, both two microencapsulated enzymes catalyzed the biotransformation with a stable optical purity and only about 5% decrease of yield compared with untreated microencapsulated enzymes.(4) The addition of glucose for coenzyme-regeneration possibly leads to spore germination in coenzyme-coupled systems(SCRII-GDH and E228S-GDH). Cycloheximide was added in each reaction to inhibit spore germination. After the morphologic changes of ascospores as a novel enzyme-immobilization carrier to embed the target proteins was monitored. the enzyme-encapsulation spores were confirmed to be dormant during asymmetric synthesis.