| R-mandelic acid is the important fine chemical intermediates and chiral precursor with broad uses in pharmaceutical and chemical industry.Synthetizing chiral drug with biological catalysis method has many advantages,such as mild conditions,high efficiency and high stereoselectivity,etc.In addition,it is non-pollution and low energy consumption,which is an environment friendly green synthesis method.As nitrilases are important industrial enzymes that convert mandelonitrile directly into the R-mandelic acid,they receive extensive attention.This dissertation is devoted to the comprehensive understanding of the fermentation fundamental of nitrilase in the recombinant E.coli Q196S,optimizing the preparation technics of recombinant nitrilase and developing a novel technology of continuous regulating pH and glycerol fed-batch fermentation.Subseguently,the optimum process was verified in 30-L fermenter,and the production of nitrilase was 24991 U/L.The paper investigated the bioconversion conditions of mandelonitrile to R-mandelic acid with resting cells and preparation of R-mandelic acid at high accumulative concentration with fed-batch.The optimal catalysis conditions are as follows:50 g/L resting cells,1 mM Ba2+,1%(v/v)isooctanol as co-solvent,substrate 64 mM,pH 7.5 and 45℃.After continuous reaction for 9.5 h,acquired 587 mM R-mandelic acid and the e.e.value was 99%.Afterwards,the separation and purification of R-mandelic acid from the reaction mixture were studied.Based on lab experiment,the process conditions were investigated by magnified test and design of industrial production.After the process revised,KLa was the scale-up principle and extraction was replaced by ion exchange chromatography.To obtain process workshop of 500 tons R-mandelic acid per year,several contents,such as process design,material calculation,energy calculation,workshop layout design,"three wastes" treatment and comprehensive utilization were systematically investigated and designed. |
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