Production Of(R)-Mandelic Acid And Its Derivatives By Enantioselective Bio-oxidation And Reduction

(R)-mandelic acid and its derivatives are a class of chiral synthons for production of important pharmaceuticals.The asymmetric carbonyl compounds by oxidoreductase is an important alternative for the synthesis of the active optical chiral alcohols.At the same time,there is a way that could make the process of deracemization more simple,named one-pot oxidation-reduction reaction,which employing enantioselective a-hydroxyacid dehydrogenases(Pseudomonas aeruginosa ZJB-1125 in our lab)transform the(S)-isomer in racemate mandelic acid and its derivatives to prochiral ketones,and then transform to the opposite configuration by reduction.In this paper,a strain named ZJB-5074,which could transform phenylglyoxylic acid to(R)-mandelic acid,was isolated from more than 100 soil samples,and the enantiomeric excess of product was 99.9%.Based on its morphology,physiological characteristics,18S rDNA sequences and phylogenetic analysis,ZJB-5074 was confirmed as Saccharomyces cerevisiae.We further investigated the thermal stability of the bacteria,and substrate affinity.Several important parameters affect the asymmetric reduction process such as pH,temperature,co-substrate,concentration of substrate and concentration of cell etc.were studied.The optimal reduction condition was as follows:temperature 35 ?,pH 7.5 potassium phosphate buffer(100 mM),wet cells of 50 g/L,substrate 30 mM,co-substrate glycerol 10 g/L.The yield of S.cerevisiae ZJB-5074 revert phenylglyoxylic acid to(R)-mandelic acid was 96.67%at 48 h,and the enantiomeric excess has remained upper 99.9%,under the optimum reaction conditions,and there's a lag phase about the first 10 h.We investigated the batches stability of the bacteria,results indicated that there's no lag phase,starting from the second batch,and the enzyme activity of the second batch was much higher than the first batch.Studied preparation of(R)-mandelic acid and its derivatives by carbonyl reduction,in which the conversion of the 17 ketones was 79.28-99.92%,e.e.%>99%.We investigated the best way to permeabileze S.cerevisiae ZJB-5074 cell,which was add 5 mM mandelic acid in bacterial suspension,stand for 10 min at 4 ?.The yield of convert phenylglyoxylic acid to(R)-mandelic acid was 61.45%at 19 h by employing the permeabilized cells.Then,we convert racemic mandelic acid to(R)-mandelic acid using P.aeruginosa ZJB-1125 and S.cerevisiae ZJB-5074,both simultaneously and consecutively,which was divided according to the addition sequence of S.cerevisiae ZJB-5074.The optimum condition for consecutive reaction was:20 mM substrate,pH 8.0 phosphate buffer(100 mM),the proportion of Pseudomonas aeruginosa ZJB-1125 in oxidation and Saccharomyces cerevisiae ZJB-5074 cell in reduction was 2:1(50 g/L:25 g/L),glycerol 3.33 g/L,at 35 ?;the optimal condition of simultaneous reaction consist:20 mM substrate,pH 7.5 potassium phosphate buffer(100 mM),the proportion of Pseudomonas aeruginosa ZJB-1125 in oxidation and Saccharomyces cerevisiae ZJB-5074 cell in reduction was 2:1(50 g/L:25 g/L),glycerol 3.33 g/L,at 30?.Under the above optimal conditions,the yield of deracemization of mandelic acid were 89.52%for consecutive reaction and 92.67%for simultaneous reaction,and their enantiomeric excess both greater than 99.9%.Investigated the reaction time,yield and enantiomeric excess value of preparation 17 types of(R)-mandelic acid and its derivatives from their racemate.We also detected the changes of racemic substrate,intermediate ketone and product for several mandelic acid derivatives.For scale up reaction,the yield of deracemized 2-chloromandelic acid simultaneously,was 95.63%after 33 h,e.e.value greater than 99.9%.