Enhancement Of The Synthetic Efficiency Of Chiral Alcohols By Yeast Cell-mediated Asymmetric Oxidoreduction

Optically active secondary alcohols are important intermediates of pharmaceuticals and fine chemicals, and enantiomerically pure phenyl-1,2-ethanediol (PED) is an important representative for optically active secondary alcohols, which is a versatile chiral building block for the synthesis of pharmaceuticals, agrochemicals, pheromones, and liquid crystals. Compared with chemical synthetic methods, biocatalysts offer distinct advantages in terms of of regio-, chemo- and enantioselectivity under benign conditions. Amonies, microbial deracemization and asymmetric reduction have been the front and topic research of present biocatalysis thanks to the maximum theroretical yield of 100% and easy-to-handle conditions. Nevertheless, some weaknesses frequently met in both microbial deracemization and asymmetric reduction are small substrate concentrations, the inefficient regeneration of cofactors and the poor stability of biocatalysts. In order to achieve highly effective synthesis of chiral PED by yeast-mediated asymmetric oxidoreduction, the bottleneck of Candida parapsilosis-catalyzed deracemization of PED to (S)-PED and S. cerevisiae-mediated asymmetric reduction of 2-hydroxyacetophenone to (R)-PED were investigated. The efficiency of above-mentioned reactions was significantly enhanced by process engineering and media engineering. Furthermore, the mechanism of coupling involving D-xylose metabolism and C. parapsilosis-catalyzed deracemization of PED was also uncovered. Those findings not only further enriched the theory of oxidoreductase-catalyzed biocatalysis, but also helped to systhesis of other fine chemicals.The main results were shown as follows:(1) C. parapsilosis catalyzing deracemization of PED was used as a model reaction to systemically investigate the factors that might limit the reactivity of such cells. It was found that there was a marked inhibition of reaction when the substrate and product concentration exceeded 25 g/1 and 17.5 g/1, respectively. Besides, excessive substrate or product concentration (such as>30 g/1) led to a high cell mortality rate of more thanl8.3%. Furthermore, NADPH regeneration was one of the factors limiting the reaction efficiency under higher substrate concentration (such as 30 g/1).(2) A highly efficient process for C. parapsilosis-mediated deracemization of PED was described, based on a "two-in-one" resin-based in situ substrate feeding and product removal methodology. The macroporous resin H103 was selected as the adsorbent material thanks to its high adsorbent capacity and greatest promoting effect on the reaction. The optimal conditions were selected as follows:pH 8,30℃,120 g/1 cells,72 g/1 resin H103 and 150 r/min. Under the optimal conditions,30 g/1 of racemic substrate was converted to (5)-PED with 99.2% enantiomeric excess (e.e) in 93.6% yield after 48 h. Based on the characteristic of biocatalytic deracemization by stereoinversion, a more rational method was proposed to make use of a "two-in-one" resin-based in situ substrate feeding and product removal strategy. Using this new method, when substrate concentration was 50 g/1, a reaction yield of 92.0% and e.e of 99.3% were obtained for (S)-PED within 90 h.(3) D-xylose added to multi-batch reactions had no influence on the activity of (S)-carbonyl reductase catalyzing the key step in deracemization, but performed a promoting effect on the recovery of the metabolic activity of the cells in each batch. Based on the fact that the activities of xylose reductase and xylitol dehydrogenase from cell-free extract of C. parapsilosis were detected, the depression of the pentose phosphate pathway (PPP) and the investigation of the cofactor pool were performed to uncover the mechanism of coupling between D-xylose metabolism and the deracemization of PED. The proposed mechanism was as follows:D-xylose was converted to xylulose via a two-step reduction and oxidation mediated by xylose reductase and xylitol dehydrogenase, respectively. Xylulose was phosphorylated to xylulose 5-phosphate, which then enters into PPP. When glucose-6-phosphate and 6-phpspho-gluconate, the intermediate metabolites of PPP, were catalyzed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, respectively, they can afford more NADPH for deracemization of PED to enhance the reaction efficiency and the stability of biocatalytic system. Furthermore, a strategy involving extractive biocatalysis (resin/buffer system) coupled with cofactor regeneration (adding D-xylose) not only alleviated the substrate/product inhibition, but also enhanced the sustainability of the biocatalyst.(4) S. cerevisiae JUC15 was successfully obtained by target reaction-oriented screening, which asymmetrically reduced 2-hydroxyacetophenone to (R)-PED of excellent e.e (>99.9%). There was no significant decrease in the yield and optical purity of (R)-PED when the free cells were reused for 40 repeated cycles at 2 g/1 substrate concentration. The biocatalyst used cheap sucrose for cofactor regeneration and worked over a considerably wider range of pH (4-9). The product e.e. kept above 99.9% in all examined conditions. When 2-hydroxyacetophenone concentration was below solubility limit (11.4 g/1), product inhibition was the primary bottleneck of the asymmetric reduction. However, when the substrate concentration exceeded its solubility, especially more than 20 g/1, the observed decrease in the yield of (R)-PED can be mainly attributed to a combination of product inhibition and the inactivation of the biocatalyst. Moreover, the catalytic decomposition of partial product by S. cerevisiae occurred. Biphasic systems consisting of buffer and a water-immiscible organic solvent were applied to circumvent those limitations. Dibutyl phthalate was selected as the suitable organic solvent thanks to its higher organic phase-buffer partition coefficent for the substrate and product, and the largest promoting effect on the reduction. Use the appropriate volume ratio could make the product concentration reach 20.7 g/1, which was remarked higher than that of other bio-methods reported so far.