Bio-resolution Of Mandelic Acid And Its Derivatives

Optically active mandelic acid(MA)and its derivatives are a class of chiral synthons for the production of important pharmaceuticals.For example,R-MA is used for the production of semi-synthetic cephalosporins,antitumor agents,antiobesity agent etc;(R)-2-chloromandelic acid is an important intermediate to synthsis anti-platelet aggregation drugs,clopidogrel.Enantioselective a-hydroxyacid dehydrogenases(a-HADHs)have been widely used for the production of optically active a-hydroxyacids from their racemic mixtures by asymmetric oxidation.To isolate a-HADHs,two high-throughput screening methods were established.Screening mode I was based on the analysis of ketoacids which was released during the oxidation of mandelic acid or its derivatives by mandelate dehydrogenase(MDH).The ketoacids can react with 2,4-dinitrophenylhydrazine(DNPH)to produce colored hydrazones,which are easily quantified by spectrophotometric measurement.The colorimetry method was proved to be correlate well with those from HPLC analysis,with this method,one strain,ZJB-1125,which displayed the highest(S)-enantioselective MDH activity was obtained.Screening mode II was based on the analysis of phenylpyruvic acid which was released during the oxidation of Phenyl lactic acid.Phenylpyruvic acid can react with Fe3+ to form a stable blue-green complex in the presence of acetate acid and DMSO.To prove the accuracy of the screening system,the conditions of the chromogenic system were optimized.With the methods,two strains(ZJB-1125 and ZJB-1101)displayed higher(S)-enantioselective MDH activity were obtained.Based on morphology,physiological tests,ATB system and the 16S rRNA sequence,ZJB-1125 and ZJB-1101 were identified as Sinorhizobium sp and Pseudomonas aeruginosa,respectively.We have compared the biochemical properties on thermostability and specific growth rate about the two strains.It was concluded that the type of coenzyme of the MDH was NAD(P)+-independent enzyme.Both of the two strains had high activity and good S enantioselectivity,the e.e.of all substrates were above 91%and the recoveries of all the R substrates were above 45%after reaction.The catalysis of the p-substituted mandelates by the two strains consistented with the hammett equation and p was 1.2679 and 0.9749 respectively.The apparent kinetics parameters of eight substrates were determined.The effects of medium composition and the culture conditions on the production of S-MDH in Pseudomonas aeruginosa ZJB-1125 were evaluated.By single-factor and orthogonal method,the medium composition was optimized as follows(g/L):2-chloromandelic acid 2.5,mannitol 10,yeast extraction peptone 10,K2HPO4·3H2O 2.5,KH2PO4 2.5,CuCl2 0.01,NaCl 1.The optimum conditions for cell growth and enzyme production were as follows:initial pH 7.5,inoculum volume 4%(v/v).The enzyme activity reached 26.48 U/L,which was 5.1 times higher than the initial number(5.20 U/L).The influences of reaction conditions on bio-resolution of 2-chloromandelic acid by Pseudomonas aeruginosa ZJB-1125 were also investigated.Results indicated that the optimal pH was 7.5.The suitable concentration of the substrate was 30 mM and the initial rate reached 6.79?molmin-1 gDCW-1.The S-MDH showed the highest activity at 50?,and apparent activation energy Ea was 26.81 kJ/mol.The half-life of S-MDH activity in the strain at 35? and 45? were 35.8 h and 26.4 h respectively.After 5 reuse cycles,11%of the activity was remained.High dissolved oxygen can promote the reaction.The apparent kinetics parameters were determined to be Km=6.47 mM and Vmax=0.103 mM/min.