Study On Asymmetric Reduction Of Prochiral Ketones Catalyzed By Acetobacter Sp. CCTCC M209061 Cells

Chiral alcohols are important building blocks for the synthesis of chiral pharmaceuticals and functional materials. For example, enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol {(R)-TMSBL} is a key chiral block for the synthesis of (R)-benzyl-4-hydroxyl-2-pentynoate with the potential therapeutical function for Alzheimer's disease.Compared with the chemical ways, the biocatalytic routes have gained more and more attention owing to its mild reaction conditions, high enantioselectivity, high efficiency, and low environmental concerns. Using whole cells can avoid the need for coenzyme addition, protect the related enzymes and coenzyme from inactivation by keeping them in natural environment of cells. As a result, it is of great significance to prepare enantiopure (R)-TMSBL throught microbial cell-mediated asymmetric reduction of prochiral 4-(trimethylsilyl)-3-butyn-2-one (TMSBO). In recent years, a number of investigations have showed that many enzymes and microbial cells have higher activity, selectivity and stability in IL-containing systems. In this dissertation, the microbial strains, which are highly effective and enantioselective in catalyzing asymmetric reduction of TMSBO to enantiopure (R)-TMSBL, were screened out from various possible microbial samples following anti-Prelog rule for stereoselective reduction reactions, and a comparative study was made of the biocatalytic asymmetric reduction of TMSBO catalyzed by the novel microbial cells in various reaction media. The effects of various ionic liquids (ILs) on the bioreduction of TMSBO were examined and the catalytic performances exhibited by the cells in IL-containing systems were characterized. Additionally, the novel biocatalytic reaction system used for highly efficient and enantioselective synthesis of enantiopure (R)-TMSBL was established.Several microbial strains, capable of catalyzing the asymmetric reduction of TMSBO to enantiopure (R)-TMSBL have been screened out from the microbial samples. Among them, the strain XZY003 isolated from China kefir grains has proven to be the best one for the asymmetric reduction of TMSBO. It has been identified to be a member of Acetobacter species by morphological, physiological and biochemical tests and further 16S rDNA gene sequence analysis, and named as Acetobacter sp. CCTCC M209061. In comparison with the reported microbial strains following anti-Prelog rule, Acetobacter sp. CCTCC M209061 cells exhibited higher efficiency and enantioselectivity in catalyzing the anti-Prelog reduction of TMSBO to enantiopure (R)-TMSBL in TEA-HCl buffer. The optimal reaction conditions for the bioreduction of TMSBO with Acetobacter sp. CCTCC M209061 cells were as follows: buffer pH 5.0, cosubstrate 2-propanol, 2-propanol concentration 130.6 mmol/L, reaction temperature 30 oC, substrate concentration 6.0 mmol/L and shaking rate 180 r/min. Under these conditions, the initial reaction rate, the maximum yield and the product e.e. were 0.50μmol/min, 71% and >99%, respectively. Additionally, the novel strain Acetobacter sp. CCTCC M209061 was capable of catalyzing the anti-Prelog asymmetric reduction of a series of other prochiral ketones to the corresponding enantiopure chiral alcohols in aqueous monophasic system. However, the maximum yield and the optimal substrate concentration for the bioreduction carried out in aqueous monophasic system were relatively low, possibly due to the pronounced inhibition of the reaction by the substrate and/or the product in this system.In order to solve this problem, a metabolic regulation way, namely adding water-miscible imidazolium ILs to the aqueous buffer to make the cell membrane more permeable and thus lower the product concentration in the cells and enhance the reaction efficiency, was attempted. Also, the effects of various hydrophilic ILs on the bioreduction of TMSBO were examined and the catalytic performances of the cells in the IL-containing systems were characterized. It was found that the catalytic performance of the biocatalyst depended not only on the types of anion and cation of the IL, but also on their combination. Acetobacter sp. CCTCC M209061 cells displayed drastically low catalytic activity in the co-solvent systems involving BF4-- or TfO--based ILs. In the case of C_nMIM⁺-based ILs, the catalytic activity of the cells decreased with the increasing n value. With the pairing of C2OHMIM+ with NO3-, the cells manifested the highest catalytic activity and therefore the IL 1-(2'-hydroxyl) ethyl-3-methylimidazolium nitrate (C₂OHMIM·NO₃) was considered as the optimal IL for the bioreduction reaction. In the C₂OHMIM·NO₃-containing system, several crucial influential variables were examined. Under the optimized reaction conditions for the bioreduction in the C₂OHMIM·NO₃-containing system, the substrate concentration was 9.0 mmol/L, and the initial reaction rate and the maximum yield weres 1.7μmol/min and 91%, resepectively, which were higher than the corresponding values with the aqueous monophasic system. Also, the product e.e. was not affected and remained above 99%. Furthermore, the biocatalytic anti-Prelog asymmetric reduction of a series of other prochiral ketones catalyzed by Acetobacter sp. CCTCC M209061 cells were successfully performed in the C₂OHMIM·NO₃-containing system. Of all the tested 15 hydrophilic ILs, Acetobacter sp. CCTCC M209061 cells had the highest sugar metabolic activity and suitable cell membrane integrity in the reaction system involving the IL C₂OHMIM·NO₃, showing the better biocompatibility of the IL with Acetobacter sp. CCTCC M209061 cells. Besides, the IL C₂OHMIM·NO₃ can properly increase the cell membrane permeability, which is helpful for the rapid transport of the formed product out of cells and thus partly relieves inhibitory and toxic effects by the product. All these can well account for the highest catalytic efficiency of the cells in the C₂OHMIM·NO₃-containing system. However, it should be noted that the optimal substrate concentration is rather low (9 mmol/L), and the maximum yield needs further improvement.To tackle this problem, phase transfer method was used in this work with the expectation that the second phase can effectively extract the substrate and the product. Generally, organic solvent/buffer biphasic system is adopted for this purpose. So the asymmetric reduction of TMSBO with Acetobacter sp. CCTCC M209061 cells was initially carried out in various organic solvent/buffer biphasic systems. The effects of organic solvents on the bioreduction of TMSBO varied widely. The initial reaction rate and the maximum yield both increased with increasing Log P values of organic solvents when Log P values was lower than 3.5 and the initial reaction rate and the maximum yield both reduced with increasing Log P values of organic solvents when Log P values was higher than 3.5. n-Hexane was found to be the most suitable organic solvent for the bioreduction among all the tested organic solvents. For the bioreduction performed in the n-hexane/buffer biphasic system, the initial reaction rate, the maximum yield and the product e.e. were1.8μmol/h, 90% and >99%, respectively, under the optimal reaction conditions. Obviously, using n-hexane/buffer biphasic system failed to enhance the efficiency of the reaction efficiently. As shown by our experiments, the organic solvent n-hexane was quite toxic to the cells, leading to a substantial drop in the catalytic activity of the cells.Using ILs, instead of traditional organic solvents, could improve the cell activity. So the effects of various hydrophobic ILs on the bioreduction of TMSBO were examined. Of all the tested hydrophobic ILs, 1-butyl-3-methylimidzolum hexafluorophosphate (C₄MIM·PF₆) showed to be the most suitable one for the bioreduction. In the buffer/C₄MIM·PF₆ biphasic system, the optimal volume ratio of buffer to C₄MIM·PF₆, substrate concentration, buffer pH, co-substrate concentration, reaction temperature and shaking rate were 4/1, 60.0 mmol/L, 5.0, 555.7 mmol/L, 30 oC and 200 r/min, respectively, under which the initial reaction rate, the maximum yield and the product e.e. were 9.4μmol/h, 93% and >99%, respectively. The optimal substrate concentration (60.0 mmol/L vs. 9.0 mmol/L) and the maximum yield (93% vs. 91%) in the C₄MIM·PF₆-based biphasic systems were much higher than those achieved in n-hexane/buffer biphasic system. Among the examined 7 hydrophobic ILs, Acetobacter sp. CCTCC M209061 cells showed the highest sugar metabolic activity and cell membrane integrity in buffer/C₄MIM·PF₆ biphasic system, indicating the IL's excellent biocompatibility with the cell. Additionally, the IL C₄MIM·PF₆ can extrac the substrate and the product efficiently. The above-described results contributes to the observation that the efficiency of the asymmetric bioreduction catalyzed by Acetobacter sp. CCTCC M209061 cells was relatively higher in the C₄MIM·PF₆-based biphasic system.On the other hand, the FT-IR analysis showed that the IL C₄MIM·PF₆ was able to enter Acetobacter sp. CCTCC M209061 cells and accumulate within cell membrane, suggesting that the IL was likely to interact with the enzymes associated with the bioreduction within the cells. Furthermore, the biocatalytic anti-Prelog asymmetric reduction of a series of other prochiral ketones using Acetobacter sp. CCTCC M209061 cells were successfully carried out in the C₄MIM·PF₆-based biphasic system.Among the above-mentioned four reaction systems examined, the best operational stability of Acetobacter sp. CCTCC M209061 cells was observed in the buffer/C₄MIM·PF₆ biphasic system, and the poorest one was recorded in the n-hexane/buffer biphasic system.The study will not only establish the underlying theories for applications of ILs in microbial whole-cell biocatalysis, but also provide a novel and efficient route to enantiopure chiral alcohols.