Purification,Cloning And Expression Of Bacillus Cereus Esterase And Its Application In Resolution Of Indoxacarb Intermediate

Microbial esterase?EC 3.1.1.1?have been considered as high-performance biocatalysts due to their commercial availability,high stereoselectivity,and mild reaction,and have been widely used in fine chemicals,agriculture,food,and pharmaceutical industry.In this study,Bacillus cereus WZZ006,an esterase-producing strain deposited in the laboratory,was used as the research object.Bacillus cereus esterase was isolated and purified by chromatography,and its enzymatic properties were explored.The esterase gene was cloned and expressed,and explored its application in the resolution of the indoxacarb chiral intermediate.Bacillus cereus WZZ006 esterase was isolated and purified by ultrasonic disruption,DEAE ion exchange chromatography,and Octyl hydrophobic interaction chromatography.The esterase was purified to homogeneity,with an 89.50-fold purification,specific activity of 1.79 U/mg,and the recovery was 26.70%.SDS-PAGE and MALDI-TOF-MS mass spectrometry analysis showed that the apparent molecular weight of the esterase was 96 k Da.Activity staining showed that the esterase possessed new carboxyesterase properties.Using p NP-hexanoic acid?C6?as a substrate to investigate its biochemical characteristics in detail,it showed moderate thermal stability?50??and alkali resistance?p H 8.5?.Kinetic parameters such as K_m,V_max,k_cat and respectively.Using the genomic DNA of Bacillus cereus WZZ006 as a template,the primers were designed to perform PCR amplification to obtain the esterase gene.The sequencing revealed that the esterase gene was 2433 bp in length and was responsible for encoding 810 amino acids.It was successfully ligated with p ET-28a?+?vector by one-step cloning,and finally expressed heterologously in E.coli BL21?DE3?.The recombinant esterase was purified by nickel column affinity chromatography,and it was found that the target esterase eluted with 150 m M imidazole.After analysis by SDS-PAGE electrophoresis,a single band was found,and the band size was about 100k Da,which was in line with the expected size.The three-dimensional structure of Bacillus cereus WZZ006 esterase was analyzed by homology modeling method,and the process of resolution substrate?R,S?-5-chloro-2,3-dihydro-2-Hydroxy-1-oxo-1H-indene-carboxylic acid methyl ester was simulated by molecular docking method.The purified BL21/p ET-28a?+?-BCE recombinant esterase was used as a catalyst to selectively resolve the racemic substrate.The effects of different catalytic conditions on the resolution reaction were investigated and the resolution conditions were optimized.The optimal p H of the reaction was 8.0,the optimal temperature was 35°C,the optimal speed was 210 rpm,and 85 m M was the maximum saturation concentration of the recombinant esterase.Compared with the wild strain Bacillus cereus WZZ006,the recombinant esterase proved to be a natural biocatalyst with greater application value in the preparation of a key chiral intermediate of indoxacarb.