Studies On The Stereoselective Conversion And Preparation Of (R)-2-Octanol By Microorganism

Enantiomerically pure 2-octanol is important intermediates for the synthesis of pharmaceuticals, agrochemicals, pheromones, steroid, liquid crystals and as chiral auxiliaries in asymmetric synthesis. Therefore, the studies on the preparation of Enantiomerically pure 2-octanol and screening of biocatalysts for producing chiral alcohols are very important both for the knowledge on biocatalytic mechanism and its application.In this research, Candida cylindracea ATCC 14830 selected for the asymmetric conversion of racemic 2-octanol among various microorganisms including bacteria, yeast and molds. (R)-2-octanol was obtained with the optical purity of 95.21%e.e. and the yield of 45.38% catalyzed by C. cylindracea ATCC 14830. By investigating the reaction process, the intermediate was identified as 2-octanone analyzed by GC-MS. (S)-2-octanol was oxidized to the intermediate of 2-octanone, and (R)-2-octanol was reserved.After optimizing reaction conditions, it was observed that the enantiomerically pure 2-octanol was increased from 95.21%e.e. to 99.20%e.e. This was obtained by the whole cells of C. cylindracea ATCC 14830 under the follow conditions: n-octane as the organic solvent , PE as the buffer, pH6.5, aqueous phase/organic phase=1:1, cell concentration 10%(w/v), tempreture 30℃, rotate speed 150rpm/min. Reaction finished in 10 hours with substrate concentration of 40g/L, 25 hours with substrate concentration of 80g/L.To develop a practical process for (R)-2-octanol production from racemic 2-octanol, a two step microbial reaction was investigated. The reaction employed the eantioselective oxidation of (S)-2-octanol by C. cylindracea ATCC 14830 and the asymmeric reduction of oxidated 2-octanone by Oenococcus oeni CECT 4730. O. oeni CECT 4730 was a strain preserved which can reduce 2-octanone to (R)-2-octanol. After redox-coupling ,the yield and optical purity was over 92% and 99%. The substrate concentration was 40g/L.After optimizing culture medium and conditions, it was observed that the cell mass of O. oeni CECT 4730 increased from 4.32g/L to 6.87g/L. The optimized medium compositions were under the follow conditions: Yeast extract 0.503%, Glucose 1%, Tryptone 1%, D-Fructose 0.5%, L-Cysteine hydrochloride monohydrate 0.05%, MgSO4·7H2O 0.02%, MnSO4·4H2O 0.005%, Ammonium citrate dibasic 0.458%, Tween 80 0.1%, Tomato juice 14.395%. The optimized culture conditions were as following: inoculation percentage 10%, pH5.5, tempreture 28℃, culture time 42h.