Asymmetric Reduction Of β-Carbonyl Ester With Baker's Yeast

The research and development of chiral compounds has become the hot field and prosperous direction of new drug internationally.β-hydroxy ester with optical activity, whose enantiomer was often used as chiral block in the organic synthesis and intermediate for drug production,was one of the important compounds.β-hydroxy ester having optical activity could be synthesized in asymmetric reduction by baker's yeast or other microorganisms under mild reaction condition.The main purpose of this work was to study the characteristics of asymmetric reduction catalyzed by baker's yeast,the method to improve stereo-selectivity of the reaction,the tolerance of the yeast to organic solvent,and the possibility to apply immobilized yeast in calcium alginate beads in industrial process. Respiratory-deficient yeast was also used to study the effect of enzyme in respiratory chain on the reaction.The(S)-methylβ-hydroxybutanoate(MHB)was synthesized from methyl acetoacetate(MAA)by different yeasts in aqueous phase.Effects of reaction time, yeast concentration,glucose concentration,buffer pH,reaction temperature and substrate concentration on both the yield and the stereo-selectivity of(S)-MHB were investigated.The results showed that Baker's yeast effectively catalyzed the reaction. The optimal condition was reaction time 4 h,Baker's yeas 75 g/L,glucose 0.6 mol/L, substrate MAA 0.05 mol/L,pH7,35℃.Under the condition,methylβ-hydroxybutanoate yield and enantiomeric excess(e.e.)57%and 92%,respectively.Methyl 3-oxobutanoate was chosen as the model substrate,and the condition of immobilization and reaction in flask were investigated in details.A new fixed-bed process,which combined reaction function and cell-activity regeneration,was designed for the asymmetric reduction.The result showed that after immobilized in calcium alginate beads,yeast has high stereo-selectivity:e.e.of the product could achieve 99%under proper condition.Two fixed-bed reactors were able to work steadily for at least 16 days for reaction and regeneration alternately in 2 fixed-beds. The yield could achieve 80%and e.e.could keep above 95%in the fixed beds.In order to achieve high stereo-selectivity with yeast,ethyl acetoacetate was used as the model substrate,and a novel approach was proposed to selectively inhibit R-enzymes in yeast before reaction.In this method,Baker's yeast was pretreated with acrylic acid,ether,dimethylsulfoxide,acryl amide and hexane,respectively.After the pretreatment with organic solvents,the enantiomeric excess of S-product was raised from 70%to 85%-98%.Among these solvents,dimethylsulfoxide can improve the stereo-selectivity with the original yield while the other 4 solvents decreased the yield to different degrees.The mechanism on alteration of enantioselectivity by allyl compounds added in the reaction system was studied.The results showed that inhibition on enzymes system of yeast by allyl compounds was reversible,and the extent of inhibition was determined by the group linked to the allyl.The activities of intracellular hydrolases and mitochondrial complexes were decreased at different degree,respectively.Succinic acid dehydrogenase or its subunit in the mitochondrial might be the S-enzyme which catalyzes the asymmetric reduction.By gradually adding low-content of methyl acetoacetate into the solid medium,a new yeast strain,called MAA yeast,was separated from the commercial baker's yeast. The new yeast was used for catalyzing asymmetric reduction of methyl acetoacetate in aqueous phase,and results showed that it was more efficient than the regular baker's yeast in asymmetric reduction of methyl acetoacetate.The influence of external environment,pH and temperature on MAA yeast was as same as baker's yeast.When MAA yeast was used to catalyze other systems,such as reducing ethyl 4-chloro-3-oxobutanoate to ethyl(S)-4-chloro-3- hydroxybutanoate and ethyl acetoacetate to ethyl(S)-3-hydroxybutyrate,the yield also improved at various degrees.In addition,the main product was methyl(S)-β-hydroxybutanoate at low substrate concentration,however,methyl(R)-β-hydroxybutanoate would be produced when the concentration of substrate exceeded 0.4 M.The morphology of the new strain was as same as that of baker's yeast. By mutagenesis,respiratory-deficient(RD)yeast was prepared to study the effect of enzyme in respiratory chain on the reaction.The e.e.of product catalyzed with RD yeast was improved.The activity of complexesⅠ-Ⅳin mitochondrial were decreased,which means the improvement of e.e.was not caused by change of enzyme activity in respiratory chain.When extraneous electron acceptor was added,the biomass of RD yeast would increase,but no obvious improvement on the effect of asymmetric catalyzation was observed.