Preparation Of Optically Active 2-octanol By Microbial Asymmetric Reduction

(R)- and (S)-2-octanol are important optically active alcohols and used as synthons for the preparation of chiral natural products and related compounds such as many pharmaceuticals. Compared with chemical and biological method of preparing optical active 2-octanol, the way of bioreduction is of the most convenient and safety.Asymmetry reduction of 2-octanone by Yeast, Mold and Bacteria was investigated. The Candida boidinii CECT 10035 was selected, which reduce 2-octanone to (S)-2-octanol with higher optical purity. When the concentration of 2-octanone was 78 mmol/L, the conversion and enantiomeric excess were 32.6% and 95.6%. Meanwhile Oenococcus oeni CECT 4730 was selected, which reduce 2-octanone to (R)-2-octanol with higher activity and optical purity. When the concentration of 2-octanone was 78 mmol/L, the conversion and enantiomeric excess were 99.8% and 97.8%.Anti-Prelog reduction by Oenococcus oeni CECT 4730 with high efficient was reported for the first time and its reaction condition was optimized. The suitable buffer was Tris-Borate (TBE), the optimum co-substrate was glucose, the best organic solvent was n-nonane. When the concentration of 2-octanone was 0.5 M, with 300 mmol·L-1 TBE and 300 mmol·L-1 glucose (aqueous phase/organic phase=1:1), the conversion and enantiomeric excess were 99% and 99%.In order to solve the substrate inhibition, fed-batch reaction was applied for the reduction of 2-octanone by Oenococcus oeni CECT 4730. During the fed-batch course, the substrate concentration was kept with 10 g/L and 0.3 M Tris solution was used for pH titration. After 48-h incubation, the concentration of R-2octanol reached 120g/L with optical purity higher than 99%.It is discovered that the alcohol dehydrogenase of Oenococcus oeni CECT 4730 exhibits a broad substrate specificity in the course of reduction, especially those ketones which possess a long chain and a short chain beside the carbonyl carbon. It allows the anti-Prelog reduction of aliphatic and aromatic ketones to furnish (R)-configurated alcohols in high optical purity as well.At last, the co-enzyme recycling system of Oenococcus oeni CECT 4730 was studied. The NADPH was regenerated by the metabolism of glucose through the PK metabolic pathway. When 1 mol glucose was degraded, nearly 2 mol NADPH was regenerated and 1 mol lactic acid and 1 mol acetic acid was produced.By adding 2.5 mmol/L Mn2+ and Fe2+ or 10 mmol/L ethanol, the co-enzyme recycling system was enhanced. Under the optimized condition, the conversion could increase 50% than before.The conditions for reduction by Candida boidinii CECT 10035 were also investigated. The suitable pH was 8, the best biomass concentration was 200 g/L, the proper substrate concentration was 8 g/L and the temperature was 33℃. Under this optimized condition, the conversion and enantiomeric excess were 47.8% and 96.1%.