Screening Of N-benzoyl Hydrolase Producing Microorganism And Its Application In Preparation Of 2-amidomethyl-3-oxobutanoate

N-benzoyl hydrolase can catalyze the hydrolysis of N-benzoyl derivants.Compared with chemical hydrolysis,N-benzoyl hydrolase catalyzed deprotection is advantageous due to its mild reaction conditions,high steroselectivity and environmental benign.（25,3R）-2-amidomethyl-3-hydroxybutanoate is the important building block of 4-AA from methyl acetoacetate.Thes-monomer of product was often obtained by enzyme catalysis of 2-benzamidomethyl-3-oxobutanoate due to the large substituent group located at C2,which is unfavorable to direct synthesis of 4-AA.This study aims to find novel N-benzoyl hydrolase producing microorganism to hydrolyze amide of 2-benzamidomethyl-3-oxobutanoate,relieving the interference of substituent group which located at C2 position of the molecule.It layed the foundation for the preparation of（2S,,3R）-2-amidomethyl-3-hydroxybutanoate by carbonyl reductase further.The HPLC analysis of substrate and product was first established.Strain ZJB-12031,a bacterium capable of hydrolyze amide of 2-benzamidomethyl-3-oxobltanoate,was isolated from soil samples.Based on morphology,physiological and biochemical characteristics,16S rDNA analysis,the strain was designated as Burkholderia tropica.The substrate preference of the enzyme from B.tropica ZJB-12031 was investigated with differemt N-benzoyl derivants.The result suggested that the enzyme is an L-aminoacylase.To improve the enzyme production of B.tropica ZJB-12031,the medium components and cultivation conditions of the strain were optimized by single factor method.The optimal medium composition was as follows（g/L）:sorbitol 3,NaNO3 2,peptone 1,K2HPO4 3,MgSO₄·7H2O 0.4,FeSO₄·7H2O 0.01.The optimal cultivation conditions were as follows:medium loading 50 ml/250 ml,inoculum size 2.0%（v/v）,intial pH 8.0,30℃,150 rpm and 48 h.Under the optimal medium composition and cultivation conditions,the N-benzoyl hydrolase activity reached 21.17 U/L,which was 4.7 fold of that obtained under the original conditions.Furthermore,the influences of factors such as pH and temperature on the enzyme catalyzed synthesis of 2-amidomethyl-3-oxobutanoate was investigated.The results showed that the optimal reaction temperature and pH were 35oC and pH 8.0（100 mM phosphate buffer）,respectively.The optimal Co²⁺ and substrate concentration were 0.5 mM and 10 mM,respectively.Under the optimal reaction conditions,the timecourse of the reaction with 20 mM substrate was investigated.The substrate conversion increases from 27.6%to 39.7%with the addition of 0.5 mM Co²⁺.In the case of fed-batch bioconversion,the substrate conversion reached 60.7%after 48 hours.