Asymmetric Bioreduction Of 4-hydroxy-2-butanone To (R)-1, 3-butanediol By Yeast

Chiral compounds have been widely applied in pharmaceuticals, food, pesticide and chemical industry due to their special optical activity. With the progress in pharmacology of chiral pharmaceuticals and the strict regulation on the application of new racemic drug in recent years, the research and development of chiral pharmaceuticals have become the new focus and direction. Enantiomerically pure alcohols are important intermediates for synthesis of pharmaceuticals. Asymmetric reduction pro-chiral ketones catalyzed by microorganism or the oxidoreductase in the cell has been an important method of synthesis enantiomerically pure alcohols.(R)-1,3-butanediol((R)-1,3-BDO) is an important material for synthesizing various optically active compounds such as azetidinone derivatives,which are intermediate material forantibioti-cs, pheromones,fragrances and insecticides. It has reported that microbials can be used to reduce carbonyl compounds into the chiral alcohol. In this work,the reduction of reactions of 4-hydroxy-2-butanone(4H2B) into chlorine 1,3-butanediol catalyzed by yeast cells will be studied. The maincontents of this work are as follows:(1) HBY61 was screened from 80 yeast strains preserved in our laboratory,which showed a high bioreduction activity and enantioselectivity.The product of asymmetric reduction was enantiomerically pure (R)-1,3-BDO. The identities of the strains were established based on their 18S rRNA sequences and physiological characteristics. Results indicated the strain which designated as HBY61was a variety of Pichia jadinii with similarities of 99.7%.(2) The sample of substrate and product were analyzed qualitatively comparing with gas chromatographic chart of standard,the external standard method was used to determine their quantity of sample.(3) The experiment was used to optimize parameters of the bioreduction condition of yeast. Composition of culture media was discussed,the analytical results indicated that there are three factors including glcose, trypone and K2HPO4 which have remarkable effect on biomass of yeast.Based on the experiment,the maximal yield of the biomass (15.1g/L) could be obtained when the concentrations of glcose, yeast extract, tryptone, KH2PO4 and MgSO4 were set at 120 g/L, 20 g/L, 20 g/L, 5 g/L and 0.75 g/L, respectively at the optimal cultivation natural pH, rotate speed 200 r/min, inoculation size 2%. In this condition the biomass is 1.9 times of the initialization. The (R)-1,3-BDO(5.8 g/L) and the biomass(15.2 g/L) were achieved in standard reaction system using the optimized medium. The growth curve of yeast was detected,which showed the high-activity yeast could be obtained for 26-28 hr.(4) The factors having effect on asymmetric bioreduction 4H2B by yeast HBY61 had been investigated in standard reaction system.The technical conditions of bioreduction by yeast HBY61 were obtained,including appropriate temperature(30℃), PH(7.0), initial sub concentration(1%),carbon resource,and glcose(6%) as so on.These investigation of technical conditions helped to improve yield and enantiomeric excess(e.e) of (R)-1,3-BDO in bioreduetion by yeast HBY61.