Purification,gene Cloning,expression And Application Of An Esterase From Bacillus Aryabhattai

(S)-indoline-2-carboxylic acid is a key intermediate of perindopril for hypertension treatment.Its production efficiency and purity will directly affect the price and quality of downstream drugs.At present,the high optical purity?S?-indoline-2-carboxylic acid is mainly prepared by chemical method,but the chiral auxiliaries needed by this method are expensive and difficult to recover,which increases the cost of industrial production and also causes great pressure on the environment.There are few reports on the preparation of?S?-indoline-2-carboxylic acid by biological methods around the world.Bacillus aryabhattai strain which can selectively hydrolyze?S?-ethyl indoline-2-carboxylate to produce?S?-indoline-2-carboxylic acid was screened from the natural environment by our lab.On this basis,we isolated and purified an stereoselective esterase from the strain,and it was successfully expressed in the Escherichia coli.Then,the industrial catalysis conditions of recombinant strain were measured.The main research results are as follows:1.The esterase from Bacillus aryabhattai B8W22 was isolated by DEAE anion exchange chromatography and Octyl-Sepharose chromatography.The specific activity of the esterase was increased from 0.01 U/mg to 0.57 U/mg with 59-fold purification and 20%yield.The protein was identified by MALDI-TOF as a carboxylesterase?WP?013057000.1?with a molecular weight of 34705 Da.The study of enzymatic properties showed that the esterase had broad spectrum to p-nitrophenol ester of C2-C8 with high substrate specificity toward p NP butyrate?C4?and p NP hexanoate?C6?.The optimum temperature was 30?and the optimum p H was 8.5.The activity was increased in the presence of 5 m M Fe²⁺and K⁺.But the activity was not promoted by Ca²⁺,indicating the esterase was calcium-independent.The activity of esterase was promoted by 0.1%?w/v?concentration of NP 40 and Tween 80.The K_mand V_maxof Ba CE were 0.52 m M and 6.39?M/min,respectively.The lower K_mvalue of esterase indicates that esterase has a higher affinity for p NPB.k_catand k_cat/K_mwere 26.87 min^-1and 51.67 m M^-1min^-1,respectively.2.The target gene was amplified by PCR using the genomic DNA of Bacillus aryabhattai B8W22 as the template.The target gene with homologous fragment was constructed in p ET-28a?+?vector and the recombinant product was transferred into E.coli BL21?DE3?.The total length of the esterase gene was 930 bp,which was responsible for encoding 310 amino acids.And the gene sequences of Ba CE was94.84%homology compared to previously identified protein.The recombinant esterase was purified by Ni²⁺column affinity chromatography and eluted with 80 m M imidazole.SDS-PAGE electrophoresis showed a single protein band with a molecular weight of 35 k Da,which was consistent with the expectation.3.The freeze-dried strain powder was prepared by vacuum freeze-drying.The optimal catalytic process was analyzed using?R,S?-ethyl indoline-2-carboxylate as the substrate.The optimized results were as follows:In 1 m L system,0.02 g of recombinant freeze-dried strain powder,2%concentration of?R,S?-ethyl indoline-2-carboxylate and 10%concentration of n-hexane were added,under the condition of p H 6.5,30?and 180 rpm,?S?-indoline-2-carboxylic acid with e.e._pof97%could be prepared by reaction for 40 min,and the conversion rate can reach34%.At the same time,the enzyme activity of recombinant engineered strain reached13 U/g,which was 480-fold higher than 0.027 U/g of the wild strain.This study indicates that recombinant engineered strain has the value of large-scale production of?S?-indoline-2-carboxylic acid.