Asymmetric Bioreduction Of Ethy1-4-chloroacetoacetate By Baker's Yeast

The asymmetric reduction of ketones by yeast was studied in this paper. For the purposes of the investigation, the asymmetric reduction of ethyl-4-chloroacetoacetate by baker's yeast in water media was investigated. The research work contributes to the application of whole cell biocatalysis in the field of chiral synthesis in theory and practice.The gas chromatogram (GC) with FID (Fire iron detetector) was applied to detect and analyz their appearance, in this bioreduction of ethyl-4-chloroacetoacetate by baker's yeast.The sample of substrate and product were analyzed qualitatively comparing with gas chromatographic chart of standard, the internal standard method was used to determine their quantity of sample, chloroform as internal standard sample.Fermenting baker's yeast, has important effect on bioreduction by baker's yeast. Using statistic software Minitab with model block such as Fraction Factor Design, Central Composite Design, Response Surface Method (RSM), Composition of culture media was discussed, the analytical results indicated that there are three factors including sucrose. (NH4)2SO4. K2HPO4 which have remarkable effect on biomass of yeast; The model equation with biomass of yeast was also obtained. Based on the equation, the most optimized media is determined. The growth curve of yeast was detected, which showed the high-activity yeast could be obtained for 16-18hr.After the chiral additives were screened from many compounds, additive M was proved to help asymmetric bioreduction of ethyl-4-chloroacetoacetate by baker's yeast remarkably .By virtue of additive M, the standard reaction system of bioreduction by yeast was established to determine influences of bioconversional conditions on yield and enantiomeric excess (E. e) of Ethyl- (S)-chloro-3-hydroxybutyrate.The factors having effect on asymmetric bioreduction of ethyl-4-chloroacetoacetate by baker's yeast had been investigated in standard reaction system .The technical conditions of bioreduction by baker's yeast were obtained, including appropriate temperature, pH, the sort of pH buffering system, initial sub.concentration, carbon resource, the adding mode of sub. and sucrose as so on. These investigation of technical conditions helped to improve yield and enantiomeric excess (E.e) of ethyl- (S)-chloro-3-hydroxybutyrate in bioreduction by baker's yeast.Based on investigations of factors and technical conditions which have effect on asymmetric bioreduction of ethyl-4-chloroacetoacetate by baker's yeast, the technical factors such as feeding mode of sucrose and substrate, addition of buffer and chrical additive were studied further and optimized, using Orthogonal Design [L9(34)] to perform, the results analyzed by statistic software showed that the concentration of substrate as high as 2.5ml/120ml can be reduced at 96%(yield) and 100% E. e (which was tested by chiral colomn )under optimized reaction condition such as addition of sucrose 15g, feeding-on mode of substrate, 0.5ml(3), chiral additive M 2g, addition of buffer [K2HPO4-Na2CO3(8/2)] 1.5g.