Study On Reductions Of Carbonyl Compounds With Microbial Whole Cells In Organic Media

On the background of whole-cell biocatalysis for asymmetric synthesis in organic media, the solvent tolerance of baker's yeast was examined and a model for calculating activity retention of microbe in the presence of organic compounds was established. On the other hand, the asymmetric reductions of ethyl 4-chloroacetoacetate, l-phenyl-1,2-propanedione and benzaldehyde with baker's yeast in organic media were investigated in detail. The research work contributes to the application of whole-cell biocatalysis in the field of chiral synthesis in theory and practice.By determining the saccharide metabolic activity retention of baker's yeast after 24 h exposure to various organic solvents, the solvent tolerances of immobilized baker's yeast were studied. The results showed that the tolerance of immobilized baker's yeast was higher than that of free cells and increased with the increase of logP value of the organic solvent. The effects of temperature and pre-incubation time on the tolerance of immobilized baker's yeast were examined in detail. The tolerance of immobilized baker's yeast reached maximum at 30癈 and the activity retention of cells having been pre-incubated for 10 h decreased markedly.Using the method of Quantitative Structure-Activity Relationship of organic compounds, a new model for calculating activity retention of microbe in the presence of organic compounds was established on the basis of "target theory" and the relationship between the activity retention and relevant parameters was gained for the same compound or the homologous compounds. The new model successfully correlated the data of 11 a -hydroxylase activity retention of Rhizopus nigricans cells incubated in the presence of eight primary alcohols and photosynthetic activity retention of Monoraphidium braunii cells incubated in the presence of several alcohols. In addition, the saccharide metabolic activity retentions of baker's yeast incubated in the presence of butyl acetate, sec-butanol, benzaldehyde and ethyl 4-chloroacetoacetate were determined. It showed that these compounds were toxic to baker's yeast and the toxicity increased with the increase of the compound concentration in aqueous phase. The calculated data of the saccharide metabolic activity retention of baker's yeast using the new model accorded with the experimental data.The effects of some factors were examined in detail on the asymmetric carbonyl reduction of ethyl 4-chloroacetoacetate with baker's yeast(Type II ,Sigma) in organic media, including the selection of organic solvents, the immobilization conditions, the reaction temperature and the substrate concentration. Ethyl 4-chloroacetoacetate was reduced with baker's yeast to obtain ethyl (R)-4-chloro-3-hydroxybutyrate (ee=58%) when dibutyl phthalate was used as reaction media.Meishan yeast which showed higher stereoselectivity was used to catalyze the reduction of ethyl 4-chloroacetoacetate in dibutyl phthalate. As a result, (R)-4-chloro-3-hydroxybutyrate with higher ee value(ee=76%) was produced. The effects of the buffer used and its pH and the reaction temperature on the bioreductions with Meishan yeast or baker's yeast(Type II) were different.When Na3PO4, NaF and malonic acid was added to the reaction system respectively, the reduction of ethyl 4-chloroacetoacetat with baker's yeast(Type II) was inhibited and the yield decreased notably. This reflected that BMP and HMP pathways and the tricarboxylic acid cycle(TCA cycle) were very important to the bioreduction.Finally, the reductions of l-phenyl-l,2-propanedione and benzaldehyde with baker's yeast in organic media were investigated, too. The yield and chemical selectivity were influenced by organic solvents, temperature and pH of the buffer used for cell immobilization.