Study On Asymmetric Reduction Of Ethyl Acetoacetate Atalyzed By Immobilized Acetobacter Sp. CCTCC M209061Cells

Chiral alcohols are important building blocks for the synthesis of chiral pharmaceuticals,agrochemicals, flavors and functional materials. Among them, enantiomerically pure ethyl3-hydroxybutyrate (EHB) is a versatile intermediate for the synthesis of various biologicallyand structurally interesting compounds and pharmaceuticals. Currently, enantiopure chiralalcohols EHB can be prepared by biocatalytic asymmetric reduction with isolated enzymes aswell as whole cells which are adopted as biocatalysts. Generally, whole-cell biocatalysts arepreferable to isolated enzymes because they are more convenient and stable sources ofenzymes, with no need for enzyme purification and coenzyme addition. Additionally, the useof immobilized cells as biocatalysts can not only facilitate product separation, but also makecells recyclable and reusable, thus greatly simplifying the process and lowering the cost ofproduction. To our best knowledge, several attempts have been made to perform thebiocatalytic reduction of ethyl acetoacetate (EAA) and the obtained product is mostly(S)-EHB with moderate product e.e. and yield. However, the biocatalytic anti-Prelogstereoselective reduction of EAA to (R)-EHB using whole microbial cells has remainedlargely unexplored, and the achieved product e.e. and yield of (R)-EHB were unsatisfactory.In this dissertation, a comparative study was made of the biocatalytic asymmetric reduction ofEAA to (R)-EHB with immobilized Acetobacter sp. CCTCC M209061cells in variousreaction media, and the effects of diverse reaction conditions on the biocatalytic reduction ofEAA were explored systematically. The effects of various ILs on the bioreduction of EAAwere examined. The efficient biocatalytic process for EAA reduction to (R)-EHB withimmobilized Acetobacter sp. CCTCC M209061was evaluated on a preparative scale.Additionally, the novel biocatalytic reaction system used for highly efficient andenantioselective synthesis of enantiopure (R)-EHB was established.Among the tested five microbial strains, Acetobacter sp. CCTCC M209061, which wasisolated from Chinese kefir grains by our research group, showed to be the best biocatalyst forthe asymmetric reduction of EAA to enantiopure (R)-EHB. Then, Acetobacter sp. CCTCCM209061cells were entrapped by Ca-alginate and then coated with chitosan. Compared withfree cells, the immobilized cells showed better thermal stability, storability and recyclability. Additionally, the immobilized cells afforded the maximum yield (around71%) which wasclose or comparable to that with the free cells, and more than99%of the product e.e.Consequently, the immobilized Acetobacter sp. CCTCC M209061cells were chosen forsubsequent investigations.The poor concentration of EAA and the pronounced inhibitory of the reactants wereobserved in the aqueous monophasic system. The optimal substrate concentration, buffer pH,glucose concentration, reaction temperature, biocatalyst concentration and shaking speed were35mmol/L,5.5,80mmol/L,35,0.45g/mL and200r/min, respectively. Under theoptimized conditions, the initial reaction rate, the yield and the product e.e. were1.28mol/min,82.6%and above99.0%, respectively.To solve the aforementioned problems, organic solvent/buffer biphasic system wasconducted in this work, which was expected to effectively extract reactants to lower theconcentration of reactants in aqueous phase by the organic solvents. The effects of variousorganic solvents (ethyl acetate, cyclohexane, n-hexane, methylbenzene, n-octanol, n-octaneand isooctane) on asymmetric reduction of EAA with immobilized Acetobacter sp. CCTCCM209061cells were studied here. Compared with other organic solvents, n-hexane showedthe best biocompatibility. And the best efficiency of the bioreduction was observed in then-hexane monophasic system. The optimal reaction condition for the bioreduction of EAAwith immobilized Acetobacter sp. CCTCC M209061cells were as follow: co-substrate2-propanol,2-propanol concentration60mmol/L, substrate concentration40mmol/L,reaction temperature35and shaking speed220r/min. Under these conditions, the initialreaction rate, the yield and the product e.e. were0.72mol/min,85.2%and above99.0%,respectively.Using water-immiscible ILs, instead of traditional organic solvents could improve thecatalytic activity of cells. So the effects of various biocompatible water-immiscible ILs wereexamined in the biphasic system for the asymmetric bioreduction of EAA. Of these tested ILs,C4MIM·PF6showed the best biocompatibility with immobilized cells and the excellentsolubility for the EAA and EHB. In the C4MIM·PF6/buffer biphasic system, the optimalvolume ratio of IL to buffer, reaction temperature, buffer pH, shaking rate and substrateconcentration for the reaction were found to be2:1,35,5.5,220r/min and55mmol/L, respectively, under which the initial reaction rate, the yield and the product e.e. were1.16mol/min,90.8%and above99.0%.To further improve the reaction efficiency, adding hydrophilic imidazolium ILs to theaqueous buffer was thought to be able to make the cell membrane more permeable and lowerthe reactants concentration in the cells. It was found that the effects of water-miscible ILsaffected markedly on the catalytic performances of the immobilized cells in the bioreductionof EAA. With pairing of C2OHMIM+with Cl-, the immobilized cells exhibited the highestcatalytic activity and thus the C2OHMIM·Cl was chosen to be the optimal IL for thebioreduction. The optimized substrate concentration, IL content, buffer pH, reactiontemperature and shaking rate for the reaction conducted in the C2OHMIM·Cl-containingsystem were found to be45mmol/L,0.5mol/L,5.5,35and220r/min, under which theinitial reaction rate, the yield, and the product e.e. were4.90mol/min,95.3%and above99.0%. The500-mL preparative scale under the optimized conditions was carried out, whichthe initial reaction rate, the yield and the product e.e. were4.78mol/min,94.9%and above99.0%, respectively. Hence the biocatalytic reduction of EAA to (R)-EHB with immobilizedAcetobacter sp. CCTCC M209061cells is promising and competitive.Among the above-mentioned four reaction systems examined, the best operationalstability of immobilized Acetobacter sp. CCTCC M209061cells was observed in theC4MIM·PF6/buffer biphasic system, and the poorest one was recorded in the n-hexane system.This study provides not only a deeper understanding of immobilized cells catalysis in ILs,but also a novel and efficient route to reduce-ketone ester.