Studies On Asymmetric Biosynthesis Of (R)- (-)-epinephrine Using Acinetobacter Sp.UN-16 Cells

(R)-epinephrine, a typical representative of catecholamine and adrenaline drugs, plays an important role in clinical applications and the treatment of other potential diseases. At present, (R)-epinephrine is produced traditionally by chiral resolution of racemic epinephrine achieved with organic synthesis. So, it is of great significance to find a green and effective biosynthetic pathway that produces (R)-epinephrine, which could potentially represent chemical method.This pathway can also provide a reference to green synthesis of catecholamine and adrenaline drugs whose core structure are similar to (R)-epinephrine.A bacteria strain G92, which can produce (R)-epinephrine by asymmetric reduction of adrenalone with high stereoselectivity (enantiomeric excess,>99%) and high product yield (31.2%), was isolated from wet land of min river. The 16S rDNA sequence of G92 showed that the strain belongs to the Acinetobacter and it was named Acinetobacter sp. G92. To achieve higher product yield, UV mutagenesis, nitrosoguanidine(NTG) mutagenesis and UV-NTG combined mutagenesis were employed in strain screening. A mutant, named Acinetobacter sp.UN-16, exhibiting a high product yield of 54.9% was got from UV-NTG combined mutagenesis and used for the next study.Types of carbon, nitrogen source and inorganic salts in the fermentation medium of Acinetobacter sp. UN-16 cells were determinated by using single factor method. The fermentation medium composition ratio was optimized by response surface methodology, and the optimum medium contained:glycerol(9.12 g/L)、beef extract(7.82 g/L)、NH4H2PO4(5.65 g/L)、K2HPO4(0.1 g/L)、MgSO4(0.1 g/L)、 MnSO4(0.01 g/L) and ZnSO4(0.01 g/L). Under the condition of the optimal culture medium, the shaking flask fermentation conditions were optimized sequentially, and the optimized conditions were:inoculating amount (1%,V/V), pH (7.4), fermentation volume (20 mL), adrenalone concentrations (8mM), addition time of adrenalone(18h), fermentation termination time (22h),culture temperature (34℃), rotation speed (200rpm). The yield of (R)-epinephrine was promoted to 68.2% with optimized medium and fermentation conditons.In order to break through the restrictions of solubility and enzyme inhibition of the substrate and product on cellular asymmetric reduction reaction in water phase, this study examined the asymmetric synthesis of (R)-epinephrine with Acinetobacter sp.UN-16 cells in 14 different hydrophilic ionic liquid medium, The result of this study showed that product yield in the [HOOCEMIM] NO3-medium reached 75.4%, significantly more than 68.2% in the water phase; Ultraviolet absorption value and flow cytometry results showed that, in different ionic liquids medium, the cell membrane permeability and integrity changed with different degrees; and the cell membrane integrity in the [HOOCEMIM] NO3-medium was the best of all. Through process optimization of bioconversion, product yied in [HOOCEMIM] NO3-medium was enhanced up to 79.5%, and the substrate content, by contrast with water phase, increased from 8mM to 12mM.