Study On Microbial Enzymatic Synthesis Of The Chiral Intermediate Of Mefenoxam

Methy(R)-N-(2,6-dimethylphenyl)alaninate((R)-DMPM)is key intermediate for the synthsis of metalaxyl-M,which is typically consisits of the R-isomer of metalaxyl.It is effective to soil fungi,including Rhizoctonia which can cause blight and root rot,especially to Basidiomycetes.Currently,Chemical routes are the main method for preparation of(R)-DMPM with strict operation condition,serious environmental pollution and low purity.The use of biocatalysts for the industrial synthesis of chemicals has been attracting much attention as an environment-friendly synthetic method.Compared with chemical catalysts,whole-cell biocatalysts can be much more readily and inexpensively prepared on an industrial scale with better stability and adaptability.A strain which could asymmetric hydrolysis(R,S)-DMPM well was isolated from soil.The strain was identified as Pseudochrobactrum asaccharolyticum by 16S rDNA sequence analysis,named as Pseudochrobactrum asaccharolyticum WZZ003,and preserved in China Center for Type Culture Collection.The condition for enzyme production of Pseudochrobactrum asaccharolyticum WZZ003 had been studied and determined the appropriate medium composition:yeast extract 10 g/L,peptone 5 g/L,MgSO40.01 g/L and NaCl 0.5 g/L.The initial pH was 7.0.Cultivated with this condition for 24 h the enzymatic activity of Pseudochrobactrum asaccharolyticum WZZ003 was increased from 34.07 U/L to 118.57 U/L,the bio-mass was increased from 1.30 g/L to 6.18 g/L compared with the data before optimization.Lyophilized mycetome of Pseudochrobactrum asaccharolyticum WZZ003 was successfully developed to enantioselective hydrolysis of(R,S)-DMPM for production of(R)-MAP-acid in this paper.The key biocatalytic process parameters(temperature,pH,rotation speed and substrate concentration)were optimized.The optimized reaction conditions were as follows:the concentration of substrate and Pseudochrobactrum asaccharolyticum WZZ003 were 0.256 mol/L and 50 g/L,respectively.The reaction mixture was shaken(250 rpm)in a water bath shaking bed at 30? for 2 h.The optical purity of(R)-MAP-acid was improved up to>98%when the conversion was approximately 50%.Finally,A 3 L scale reaction was done under optimized reaction conditions,in order to explore high substrate concentration(0.256 mol/L)reaction.(R)-MAP-acid and(R)-DMPM were separated and extracted by ethyl acetate,based on chemical properties between them.After that,the organic extract was dried over evaporated under vacuum with rotary to get 45 g of(R)-MAP-acid with 97.3%(e.e.).