Asymmetric Synthesis Of Chiral Alcohols Catalyzed By Acetobacter Sp. CCTCC M209061and Characterization Of The Anti-prelog Carbonyl Reductase

Optically pure chiral alcohols and their derivatives are important intermediates for thesynthesis of chiral pharmaceuticals, pesticides and functional materials. For examples,enantiopure aromatic alcohols are chiral building blocks for the synthesis of pharmaceuticalswhich could be used for the treatment of diseases of the cardiovascular and nervous systems,as well as allergic response and inflammation. Optically pure aliphatic alcohols such as(S)-2-octanol and (R)-2-octanol are key chiral synthons for the synthesis of chiral liquidmaterials, steroid, insect sex pheromones. Currently, among the studies on the production ofchiral alcohols, biocatalysis using enzymes or microbial cells as catalysts has attracted muchattention.Our research group has successfully isolated a novel strain Acetobacter sp. CCTCCM209061from Chinese kefir grains, which showed exclusive anti-Prelog stereoselectivity forthe reduction of4-(trimethylsilyl)-3-butyn-2-one (TMSBO) to(R)-4-(trimethylsilyl)-3-butyn-2-ol [(R)-TMSBL]. In the previous works, it was found thatthis new strain exhibited low biomass when cultivated in tomato juice medium withdisappointingly low activity and stability, which limited its industrial application. Therefore,in this dissertation, the effects of medium composition and culture conditions on growth andcatalytic activity of Acetobacter sp. CCTCC M209061were explored to improve the biomassand the reduction activity of this strain; and immobilized Acetobacter sp. CCTCC M209061cells with good stability, reusability and activity were prepared by immobilizing the activecells. The effects of different reaction media on the asymmetric synthesis of (R)-2-octanolwith immobilized Acetobacter sp. CCTCC M209061cells were also examined and thebiocatalytic reaction systems used for highly efficient production of enantiopure alcohols havebeen well established. Moreover, purification and characterization of the carbonyl reductasecatalyzing the asymmetric reduction of ketones to enantiopure alcohols with anti-Prelogstereoselectivity from Acetobacter sp. CCTCC M209061were carried out. The enzymologicalproperties of AcCR were systematically investigated for further studies and practicalapplications.The effects of medium components and culture conditions on the strain’s growth andreduction activity were explored using a one-at-a-time method and a central compositerotatable design (CCRD). The optimal medium and culture conditions were found to be asfollows: glucose8.3g/L, fructose2.5g/L, soy peptone83.9g/L, MnSO4·H2O0.088g/L, pH5.7,30°C,80r/min and10%(v/v) inoculum. Under the above-mentioned conditions, the biomass after30h cultivation reached1.10g/L, which is9.5-fold higher than that obtainedwith basic medium. Also, the reduction activity towards4’-chloroacetophenone was markedlyenhanced to39.49μmol/min/g from29.34μmol/min/g, with the product e.e. being above99%.Comparable improvements were also seen with the enantioselective bioreduction of TMSBOto the key pharmaceutical precursor (R)-TMSBL.Although Acetobacter sp. CCTCC M209061cells cultivated in optimized medium hadpromising catalytic properties, its stability and reusability were relatively poor compared toother biocatalysts. Hence, the effects of various immobilizing methods on the operationalstability of the cells were examined here. It was found that Ca-alginate gave the bestimmobilized biocatalyst, which was then coated with chitosan to further improve itsmechanical strength and swelling-resistance properties. Conditions were optimized forformation of reusable immobilized beads which can keep high activity and be used forrepeated batch asymmetric reduction of4’-chloroacetophenone. The optimal immobilizationconditions were found to be as follows: sodium alginate2.5%(w/v), CaCl20.2mol/L,chitosan0.9%(w/v), pH of chitosan solution5.0, coating time20min, cell loading17.2mg-dw cell/g-ww catalyst. The resulting immobilized biocatalyst was very promising, with aspecific activity of85%that of the free-cell biocatalyst. The immobilized cells showed betterthermal stability, pH stability, solvent tolerance and storability compared with free cells. After25cycles reaction, the immobilized beads still retained>50%catalytic activity, which was3.5times higher than that of free cells (7cycles). The immobilized cells could be recultured infresh medium to regain full activity and perform a further25cycles of the reduction reaction.The external mass transfer resistances were negligible as deduced from Damkohler modulusDa <<1, and internal mass transfer restriction affected the reaction but was not therate-controlling step according to effectiveness factors i<1and Thiele modulus0.3<<1. Ca-alginate coated with chitosan is a highly effective material for immobilization ofAcetobacter sp. CCTCC M209061cells for repeated use.The asymmetric reduction of2-octanone catalyzed by immobilized Acetobacter sp.CCTCC M209061cells in various media was explored to broaden its application inproduction of chiral alcohols. In the aqueous monophasic system, the optimal pH value,reaction temperature, co-substrate and its concentration, substrate concentration and shakingrate were5.5,35°C, glucose,100mmol/L,6mmol/L,200r/min, respectively, under which,the initial reaction rate, the yield and the product e.e. with0.3g/mL immobilized cells were0.393mmol/L/min,99.0%and97.3%, respectively. However, the poor solubility of substrates and the pronounced inhibition of the reactants and the products were observed in aqueousmonophasic system, resulting in dissatisfactory reaction efficiency. Therefore, water-miscibleionic liquids were firstly added into aqueous buffer system for evaluating the effects ofvarious ionic liquids on asymmetric reduction of2-octanone to (R)-2-octanol catalyzed byimmobilized Acetobacter sp. CCTCC M209061cells. It was found that the addition of[C4MIM]·Ac could effectively make the cell membrane more permeable, and lower theproduct concentration in the cells, thus enhancing the efficiency of the reaction. The optimal[C4MIM]·Ac content was3%(w/v), and the concentration of2-octanone, the initial reactionrate and the product e.e. were10mmol/L,0.558mmol/L/min and99.3%, respectively, whichwere higher than the corresponding values in the aqueous buffer system. To further improvethe reaction efficiency, n-tetradecane as a second phase was introduced into the[C4MIM]·Ac-containing buffer system, which could effectively extract2-octanone and2-octanol to keep substrate and product concentrations at low level in aqueous phase, thusalleviating the inhibition of the substrate and the product. The optimal volume ratio ofaqueous phase to organic phase and2-octanone concentration were3:1and500mmol/L,respectively, under which the obtained yield, product concentration and product e.e. for thebioreduction were53.4%,267mmol/L and>99%, respectively. Obviously, the[C4MIM]·Ac-containing buffer/n-tetradecane biphasic system significantly enhanced thereaction efficiency.The anti-Prelog carbonyl reductase，the key enzyme in cytoplasm of Acetobacter sp.CCTCC M209061(AcCR) for the asymmetric reduction of carbonyl compounds with highstereoselectivity, was isolated and purified via four steps. The enzyme was enriched27.5-foldafter purification with an overall yield of0.4%and specific activity of3.85U/mg. AcCR hada homotetrameric structure with an apparent molecular mass of104kDa and each subunit of27kDa.AcCR can catalyze the reduction of carbonyl groups and the oxidation of correspondingalcohols utilizing either NAD(H) or NADP(H) as coenzyme. For the reduction of4’-chloroacetophenone using NADH as coenzyme, its optimum activity was at pH5.0and25°C, which were similar with the optimum conditions of whole cell-catalyzed reduction. WhenNADPH acted as the cofactor, the optimum pH and the reaction temperature were7.5and25°C. For isopropanol oxidation, similar catalytic properties of AcCR were found using eitherNAD+or NADP+as cofactor. The optimum pH and reaction temperature were8.0and35°C.Metal ions such as Mn2+, Ni2+and Fe2+remarkably activated the enzyme, followed by Zn2+and Ca2+. Co2+and Mg2+showed weak activation on the enzyme. Hg2+and Ag+completely inhibited the activity of the enzyme, while Cu2+showed relatively weaker inhibition on theenzyme. Iodoacetamide and β-mercaptoethanol showed little effect on enzymatic activity.5mmol/L EDTA completely inhibited the activity of AcCR. Furthermore, a broad range ofcarbonyl compounds such as aryl ketones, α-ketoesters and aliphatic ketones could beenantioselectively reduced by this enzyme with high activity.The reaction kinetics of4’-chloroacetophenone reduction catalyzed by AcCR was explored.It was found that the Kmvalue of AcCR for NADH was over25-fold greater than that forNADPH (0.66mmol/L vs0.026mmol/L), showing that the enzyme had a preference forNADPH over NADH. When NADH was used as cofactor, the response of carbonyl reductaseactivity to increasing concentration of4’-chloroacetophenone was clearly sigmoidal with aHill coefficient of3.1, suggesting that the enzyme might possess four substrate-binding sitescooperated with each other when NADH was present. The Vmaxvalue for NADH-basedreduction of4’-chloroacetophenone was slightly higher than that for NADPH-based reduction(0.21mmol/L/min vs0.17mmol/L/min). The reaction kenitics of isopropanol oxidationcatalyzed by AcCR was also explored. When NAD+acted as cofactor, the Kmand Vmaxvalueswere1.33mmol/L (NAD+),48.32mmol/L (isopropanol) and0.069mmol/L/min. WhenNADP+acted as cofactor, the Kmvalues for NADP+and isopropanol were1.12mmol/L and67.82mmol/L, respectively, and the Vmaxvalue was0.074mmol/L/min.This study provides not only a better understanding of biocatalysis and biotransformations,but also a novel route to the preparation of enantiopure chiral alcohols.