Preparation Of (R)-(-)-Mandelic Acid By Two-step Biotransformation

(R)-(-)-mandelic acid is an important intermediate for synthesis of penicillin,cephalosporin,antineoplastic drugs goniothalamus styryllactones and(+)-crassalactone A.Therefore,(R)-(-)-mandelic acid become an important chiral chemical intermediate.Nowadays,the resolution of racemic mandelic acid ethyl ester was regarded as common technology to prepare R-(-)-mandelic acid.The highest conversion of racemic resolution is 50%theoretically.(R)-(-)-mandelic acid was synthesized by two-step biotransformation in this thesis and the conversion can get to 100%theoretically.Saccharomyces cerevisiae CGMCC No.3361 preserved in our laboratory was used in asymmetric reduction of ethyl benzoylformate in the first step.Microorganism used in hydrolysis of(R)-(-)-mandelic acid ethyl ester was screened from the soil samples in the second step.The DNA sequence of 16S ribosomal DNA(rDNA)identification showed that the isolated strain from the soil belong to the Genus of Bacillus,and it was named as Bacillus cereus CGMCC No.12336 preserved in China General Microbiological Culture Collection Center.The optimum culture conditions for lipase production were as follows:30 ?,pH 7.5,36 h,shaker speed 180 r/min.The optimum medium for B.cereus CGMCC No.12336 was composed of 9.23 g/L glucose,5.00 g/L yeast extract,2.12 g/L peptone,2.00 g/L NaCl,2.00 g/L K2HPO4·3H20,2.00 g/L KH2PO4 and 0.51 g/L MgS04.The predicted value of maximum hydrolase activity was 755.29 U/L by response surface methodology.The kinetics models for lipase batch fermentation by B.cereus CGMCC No.12336 were studied.Based on the Logistic and Luedeking-Piret equation,the kinetics models of cell growth,lipase formation and substrate consumption were established by nonlinear least-squares fitting method and kinetic parameters were also obtained.Cell growth:(dX)/dt()=0.304X(1-X/5.135)dt 5.135Lipase formation:(dP)/(dt)= 104.156((dX)/(dt)+ 1.857XSubstrate consumption:-(dS)/(dt)= 1.577((dX)/(dt)+ 0.008XThe kinetics models were in good agreement with the experimental data.The kinetics models provide a reasonable description for the fermentation process.The optimum hydrolysis conditions were as follows:80 g/L cell concentration,0.06 mol/L substrate concentration,5%methanol as cosolvent,48 h,shaker speed 180 r/min,pH 8.0,30 0C.The conversion of R-(-)-mandelic acid ethyl ester got to 97.2%.The kinetics models of B.cereus CGMCC No.12336 hydrolysis of(R)-(-)-mandelic acid ethyl ester can be explained by the Michaelis-Menten equation.Based on the Michaelis-Menten equation,the kinetics models were established and kinetic parameters were also obtained.The value of Vmax and Km were 1.56 mmol·L-1·h-1 and 6.78 mmol·L-1.The experimental data was in good agreement with the simulated value.