Study On The Production Of Methionine From 2-amino-4-(methylthio)butylnitrile By Biotransformation

Methionine as an only skeleton amino acid containing a sulfur is extensively used in the fields of feed additives,food additives,pharmaceutical,cosMetics and so on.But in most time,it is used as the feed additives.In prsent,the main Methods for Methionine production is the chemical synthesis.In this experiment,Methionine was prepared from 2-amino-4-(Methylthio)butylnitrile by biotransformation.The analysis Methods of HPLC for substrate and product were developed.Through enrichment and directional screening technology,eight strains capable of converting 2-amino-4-(Methylthio)butylnitrile to Methionine were screened.Based on morphology,physiological,biochemical experiment and 16S rDNA sequence,the strain ZJB-12001 was identified as Pseudomonas fluorescens and the phylogenetic tree analysis was constructed.The strain was deposited in the China Center for Type Culture Collection(CCTCC M 12449,Wuhan,China).To improve the enzyme production of ZJB-12001,the medium components and cultivation conditions of ZJB-12001 were optimized.By single-factor and orthogonal Method,the medium compositions were optimized as follows(g/L):glycerol 15.80,tri-sodium citrate 14.70,yeast 32.0,sodium glutamate 5.0,ε-caprolactam 0.5,MgS04-7H20 0.15,KH2PO4 0.75,K2HPO4-3H20 0.75.The optimal cultivation conditions were initial pH 7.5,temperature 28℃,inoculum volume 4%(v/v),medium volumn 16%(v/v).Under the above conditions,the optimum culture time was 48 h.The enzyme activity and biomass reached 152.30 U/L and 7.43 gDCW/L,5.59 times and 2.09 times than the initial culture conditions,respectively.The influences of reaction conditions on the hydrolysis of 2-amino-4-(Methylthio)butylnitrile by nitrilase from ZJB-12001 were also investigated.Results indicated that the optimal pH was 8.0 with Tris-HCl buffer,and the optimal temperature was 45℃.When the reaction temperature was in the range of 25℃ to 45 ℃,the reaction keeped to the Arrhenius equation and the apparent activation energy Ea was 21.88 kJ/mol;The half lives of nitrilase at 40,45 and 50℃ were 97.76,54.92 and 5.94 h,respectively;In addition,nitrilase did not require Metal ions in the biocatalytic process;The reusability of the resting cells was not ideal,and only 11.46%of activity was remained in reaction after seven batches.