Study On Biocatalytic Asymmetric Reduction Of2-octanone To (R)2-octanol With Acetobacter Pasteurianus Cells

Optically pure chiral alcohols are widely used as important building blocks in chemicalsynthesis. Among them,(R)-2-octanol is an important intermediate for synthesis of steroid,insect sex pheromones (biological insecticide), and other optically active pharmaceuticals andpesticides. Especially,(R)-2-octanol is necessary materials for preparation of highperformance liquid crystals and their subassemblies. Biocatalytic asymmetric synthesis of2-octanone to (R)-2-octanol by microbial cells has gained increasing attraction because ofunparalleled enantioselectivity, high theoretical yield (100%), being environmentally friendly,and no requirement of expensive coenzyme. To date, the biocatalytic asymmetric reduction2-octanone to (S)-octanol has been extensively studied, but the biocatalytic anti-Prelogasymmetric reduction of2-octanone to (R)-2-octanol with microbial cells has remainedunexplored largely, with only few accounts where the used microbial cells showed lowcatalytic activity, poor ability to tolerate high substrate concentration and unsatisfactoryenantioselectivity. In this dissertation, Acetobacter pasteurianus GIM1.158was screened outfrom various microorganisms and was able to catalyze the biocatalytic reduction of2-octanone to (R)-2-octanol with high efficiency and stereoselectivity; a comparative study wasmade of the biocatalytic asymmetric reduction of2-octanone to (R)2-octanol withAcetobacter pasteurianus GIM1.158cells in various reaction media [especially deep eutecticsolvents (DESs)-containing reaction systems], and the effects of diverse reaction conditionson the biocatalytic reduction of2-octanone were explored systematically. Additionally, theoperational stability of Acetobacter pasteurianus GIM1.158cells was examined. As a result,the biocatalytic reaction system for highly efficient and enantioselective synthesis ofenantiopure (R)-2-octanol was established.Among the tested nine microorganisms, Acetobacter pasteurianus GIM1.158showed tobe the best biocatalyst for the asymmetric reduction of2-octanone to enantiopure (R)-2-octanol. The optimal temperature, buffer pH, co-substrate and its concentration, substrateconcentration, cell concentration and shaking rate were35oC,5.0,500mmol/L isopropanol,40mmol/L,25mg/mL and120r/min, respectively. Under the optimized conditions, themaximum yield and the product e.e. were83.2%and99.1%, respectively.However, the maximum yield and the optimal substrate concentration for the bioreduction carried out inaqueous monophasic system were relatively low, possibly due to the pronounced inhibition ofthe reaction by the substrate and/or the product in this system.In order to solve this problem, a metabolic regulation way, namely addingdeep eutectic solvents to the aqueous buffer to make the cell membrane more permeable andthus lower the product concentration in the cells and enhance the reaction efficiency, wasattempted. Initially, the biocompatibility of various DESs with Acetobacter pasteurianusGIM1.158cells and the effects of various DESs on the permeability and integrity of the cellmembrane were examined. Also, the effects of various DESs on the bioreduction of2-octanone were explored and the catalytic performances of the cells in the DES-containingsystems were characterized. It was found that the catalytic performance of the biocatalystdepended on the hydrogen bond donors. Among all the seven different DESs, Acetobacterpasteurianus GIM1.158cells had the highest sugar metabolic activity and suitable cellmembrane integrity in the reaction system involving the DES ChCl/EG (1:2), showing thebetter biocompatibility of the DES with Acetobacter pasteurianus GIM1.158cells. Besides,the DES ChCl/EG (1:2) can properly increase the cell membrane permeability, which ishelpful for the rapid transport of the formed product out of cells and thus partly relievesinhibitory and toxic effects by the product. All these can well account for the highest catalyticefficiency of the cells in the ChCl/EG (1:2)-containing system. Under the optimized reactionconditions for the bioreduction in the ChCl/EG (1:2)-containing system, the substrateconcentration was60mmol/L, and the initial reaction rate and the maximum yield weres2.60μmol/min and95.7%, resepectively, which were higher than the corresponding values with theaqueous monophasic system. However, it should be noted that the optimal substrateconcentration is rather low (60mmol/L), and the maximum yield needs further improvement.To tackle this problem, phase transfer method was used in this work with the expectationthat the second phase can effectively extract the substrate and the product. Generally, organicsolvent and hydrophobic ionic liquid are adopted for this purpose. So the asymmetricreduction of2-octanone with Acetobacter pasteurianus GIM1.158cells was carried out invarious organic solvent/buffer and ILs/buffer biphasic systems. The effects of hydrophobic solvents on the bioreduction of2-octanone varied widely. Compared with other hydrophobicsolvents, C4MIM·PF6showed the best biocompatibility. And the best efficiency of thebioreduction was observed in the ChCl/EG (1:2)-buffer/C4MIM·PF6system. The optimalreaction conditions for the bioreduction of2-octanol with Acetobacter pasteurianusGIM1.158cells were as follow:1.5mol/L2-octanone,3mol/L2-propanol,35℃and shakingrate220r/min. After48hours, the initial reaction rate, the yield and the product e.e. were47.3μmol/h,90.7%and above99.9%, respectively. Among the above-mentioned three reactionsystems examined, Acetobacter pasteurianus GIM1.158cells showed not only the highestcatalytic efficiency of the bioreduction of2-octanone in the ChCl/EG(1:2)-buffer/C4MIM·PF6biphasic system, but also the best operational stability.This study not only enriches the theory of biocatalytic process in DES-contaningsystems, but also provides a new way of efficient asymmetric reduction of prochiral ketones.