Expression Of Candida Parapsilosis(R)-carbonyl Reductase And Its Efficient Asymmetric Biotransformation Of L-phenyl-1,2-ethanedioi

Carbonyl reductases are excellent biocatalysts with high chemo-, regio-, andstereo-selectivity, so they are usually used for catalyzing asymmetric oxidation/reductionreaction to prepare chiral alcohol compounds with optical activity. In Candida parapsilosisCCTCC M203011,(R)-carbonyl reductase catalyzes a reversible reaction between(R)-1-phenyl-1,2-ethanediol and2-hydroxyacetophenone, and the (S)-carbonyl reductasebiotransforms2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol. In this work, to furtherimprove the catalytical performance of stereoselective reductase, and its efficiency of theasymmetric biotransformation, the secretory expression of (R)-carbonyl reductase wasperformed in Escherichia coli. The specific activity of extracellular (R)-carbonyl reductaseand its bioconversion efficiency were substantial increased by improving the permeability ofthe recombinant cells to accelerate the secretory rate and protein floding of enzyme.Furthermore, the in-situ expression system of Candida parapsilosis of (R)-carbonyl reductasewas first constructed. The enzyme activity and biotransformation function of the (R)-carbonylreductase were significantly increased through biotransformation condition optimization.Moreover, the enzymatic properties of the recombinant (R)-carbonyl reductase in in-situexpression system of C. parapsilosis were studied. The contents in details are as follows:(1) The secretory expression system of C. parapsilosis (R)-carbonyl reductase in E. coliwas constructed. The specific activity of the recombinant enzyme in the periplasmic spaceand extracellular were0.72U/mg and0.26U/mg, respectiveily. In order to accelerate thesecretory rate of enzyme from the periplasm to the outside of cells, a mild chemical penetrant,glycine was used. Then the extracellular recombinant enzyme activity was increased to1.10U/mg. When using the extracellular crude enzyme as catalyst,(R)-1-phenyl-1,2-ethanediolwas transformed from2-hydroxyacetophenone with an optical purity of93.9%and a yield of88.1%at8h.(2) The in-situ expression plasmid pCP in C. parapsilosis was successfully designed bycloning the promoter and terminator of the gene, anti-Nourseothricin gene and the righthomologous regions on the plasmid pUC57. When the plasmid pCP was integrated into thegenome of C. parapsilosis, the URA3gene was knockout making the host cell be a uracilauxotrophy. The in-situ expression system was not only resistant to Nourseothricin and5-fluoro-orotic acid as the positive filter, but also was a uracil auxotrophy as the negativefilter, thus significantly improving the probability of screening positive clones. The in-situexpression system can be widely used for a variety of enzymes from C. parapsilosis and bebeneficial for their correct protein-folding and post-translational modification.(3) The efficient in-situ expression of (R)-carbonyl reductase was realized by cloning itscoding gene RCR on the plasmid pCP to construct pCP-rcr and recombinant strain C.parapsilosis/pCP-rcr. The reductive activity and oxidative activity of the recombinantenzyme reached0.74U/mg and0.15U/mg, respectively. The recombinant enzyme showedthe highest reductive activity for2-hydroxyacetophenone at an optimum pH6.5and35℃.The enzyme was relatively stable at pH5.5-8.0, and over80%of the whole activity was remained within48h; and it showed a good stablility when the temperature was below40℃for1h. The recombinant enzymes showed the highest oxidative activity for(R)-1-phenyl-1,2-ethanediol with an optimum pH5.0and30℃. It was stable when pH wasfrom4.5to6.0since more than80%enzyme activity was remained within48h, and showed agood stablility when the temperature was lower than40℃for1h. When using therecombinant crude enzyme as catalyst,2g/L (R)-1-phenyl-1,2-ethanediol was transformedfrom2-hydroxyacetophenone with an optical purity of99.9%in a yield of99.6%within2h.Compared to the recombinant enzyme in E. coli, the reaction duration was shortened by4times by the in-situ expression system of C. parapilosis.(4) When using the recombinant C. parapsilosis cells as catalysts, the oxidative reactionof (R)-1-phenyl-1,2-ethanediol to (S)-1-phenyl-1,2-ethanediol was carried out at optimum pH5.0and30℃. The product (S)-1-phenyl-1,2-ethanediol was biotransformed with an opticalpurity of91.8%and a yield of92.2%. During the reductive reaction of2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol,(R)-1-phenyl-1,2-ethanediol wasyielded within the first12h at optimum pH6.5and30℃, then (R)-1-phenyl-1,2-ethanediolwas transformed to (S)-1-phenyl-1,2-ethanediol during12h-50h, with an optical purity of94.4%in a yield of95.9%.