Asymmetric Synthesis Of The Chiral Alcohol With Oenococcus Oeni And Characterization Of The Anti-Prelog Alcohol Dehydrogenase

Optically pure chiral alcohols are widely used as important building blocks in chemosynthesis. Among them, (R)- and (S)-2-octanol are important intermediates for synthesis of steroid, insect sex pheromones (biological insecticide), and other optically active pharmaceuticals and pesticides. And especially, they are necessary materials for preparation of high performance liquid crystals and its subassemblies. Among the reports on chiral synthesis, asymmetric synthesis of pro-chiral ketones to the corresponding enantiopure alcohols by microbial alcohol dehydrogenase (ADH) is the focus because of the unparalleled enantioselectivity, high theoretical yield (100%) and environmentally benign conditions. In the previous work, we have successfully screened out a novel strain, Oenococcus oeni CECT4730, capable of reducing 2-octanone to (R)-2-octanol with high enantioselectivity. Aim to apply the approach to manufacturing, we conducted this study on whole cells asymmetric synthesis of (R)-2-octanol in a water/organic solvent two phase system constructed by in situ separation technique,and purification, characterization of the key (R)-ADH which followed anti-Prolog rule. The main work and results are as following: (1) ATB was sceened out as the optimum fermentation medium for the yield of (R)-ADH by O. oeni CECT4730 from six common media for O. oeni. Through Plackett-Burman design we sceened out 3 key factors: tomato juice, yeast abstract and diammonium citrate according to the effect on the yield of (R)-ADH, and found higher concentration of tomato juice in ATB could increase the yield of (R)-ADH markedly. Subsequently, response surface analysis was applied to optimize the 3 key factors. And finally, the optimized medium compositions of ATB were considered to be: glucose 1%, tryptone 1%, L-cysteine hydrochloride monohydrate 0.05%, MgSO₄·7H₂O 0.02%, MnSO₄·4H₂O 0.005%, Tween 80 0.1%, tomato juice 23.8%, yeast extract 0.52%, diammonium citrate 0.34%, and pH was adjusted to 4.9. After the optimization, the whole cell enzyme activity could be raised 133.4% and the cell mass could be raised 12.8%.(2) The Non-nutritive conditions for the yield of (R)-ADH by O. oeni CECT4730 were optimized through stationary fermentation in flasks. And the results were as following: seed age 36 h, inoculum level 10%, temperature 30 oC, initial pH 4.9, working volume 200 ml/250 ml. We employed a 7 L fermentor for scale up fermentation and found the anaerobic condition in fermentor by supplying CO₂ could be beneficial to cells growth and the yield of (R)-ADH. And this strain is sensitive to the shearing force and the optimum stiring speed was 50 r/min. Compared the fermentation in flasks, the fermentation time could be shortened about 4 h in fermentaor and the enzyme activity (12.91 U vs 12.84 U), the cell mass (OD600) (1.597 vs 1.574) could be raised.(3) We constructed a suitable water/organic solvent two phase system consisting of Tris-borate and n-nonane by in situ separation technique for whole-cells catalyzed reduction. After evaluating the limiting factors during the reaction in flasks, we found various influential variables, such as Va/Vo (v/v), pH, temperature, shaking speed, cosubstrates and the substrate concentration could influence the product yield and the initial reaction rate markedly, but only pH and temperature did influence the product e.e. Under the optimized operation conditions (Va/Vo 1:1 (v/v), pH 8.0, temperature 30 oC, shaking speed 150 r/min, the ratio of glucose to biomass 5.4:1 (m/m), the ratio of biomass to substrate 0.51:1 (g/mol)), a product yield of approximate 90% could be achieved at the substrate concentration of 0.5 mol/L, and a maximum space time yield of 24 mmol/L·h occurred even at the substrate concentration close to 1.0 mol/L with the product ee (R) > 98%.(4) From approximate 11 g wet cells separated from 2 L fermentation liquid, the enzyme was purified to homogeneity 25.6 fold by sequential purification procedures: ultrasonication, (NH₄)₂SO₄ salting out, ion exchange chromatography by Q-sepharose-FF and gel exclusive chromatography by Sephacryl S-200. The purified enzyme showed an activity of 254.45 U/mg with a yield of 2.4 %. It followed anti-Prelog rule in reduction and the product ee (R) of the reduction of 2-octanone could reach 98.9%.(5) Study on main characteristics of the purified enzyme showed that its molecular mass was 100kDa (±10 kDa), and the subunit size was 49 kDa (±2 kDa) indicating that the native enzyme was composed of two identical subunits. Its optimum activity with 2-octanone was at pH 8.0 and 30 oC, which were in correspondence with the optimum conditions of whole cells-catalyzed reduction. Metal ions such as Pb²⁺, Ag⁺, and Hg²⁺ inhibited the enzyme activity markedly, as did Fe³⁺, Cu²⁺ and Zn²⁺ slightly. The bulkiness of substitutes of methyl ketones had a marked effect on the enzyme activity, the size of the substitutes increased from C₄H₉– < C₅H₁₁– < C₆H₅– < C₆H₁₃–, the enzyme activity increased from 2-hexanone < 2-heptanone < acetophenone < 2-octanone. The enzyme was NADPH-dependent and its kinetic constants: Km, Vm for 2-octanone and NADPH were 0.2 mmol/L, 67.1μmol min^-1 mg^-1 and 0.5 mmol/L, 147.1μmol min^-1 mg^-1, respectively. Its main characteristics showed marked difference in many aspects, such as molecular mass, subunit structure, temperature and pH optima, and dependence on cation, compared with the reported (R)-ADHs in literature.