Coloning,Expression And Enzymatic Characterization Of Perhydrolases From Bacillus Sp.

Peracetic acid is an important chemical material.The traditional chemical synthesis method has many defects,such as explosive and unstable product quality.So the development of enzymatic catalytic synthesis is one of the important ways to achieve safe production.The perhydrolysis activity is generally considered as the catalytic activity that is kept by the enzyme proteins of the?/?hydrolytic enzyme fold family in the evolutionary process,and it belongs to promiscuous activity of enzyme proteins of this family.Therefore,the discovery and excavation of enzymatic proteins with perhydrolysis activity are of positive significance for the development of green synthesis of peracetic acid.Through investigation of existing patents and literature reports,it was found that a variety of?/?hydrolase folding family enzyme proteins produced from Bacillus sp.strains showed good perhydrolysis activity,inclouding Bacillus subtilis subtilisin Carlsberg(SCP),Bacillus pumilus acetyl xylan esterase(AXE)and Bacillus subtilis cephalosporin c acetyl hydrolase(CAH).On the basis,this paper mainly carried out the following three aspects of work:(1)Screening Bacillus sp.strains were from a specific habitat;(2)Gene of scp,gene of cah and gene of axe were cloned from B.subtilis NSYT-3 and B.pumilus OSLJ-13,and were Heterologously expressed;(3)The above recombinant proteins were separated and puried,and the enzymatic constant of perhydrolysis catalytic activity were determined,and cascade catalytic reactions with glucose oxidase were attempted to set up.The specific results were as follows:1.Twenty-four samples were collected from 19 plants species rhizosol soil of 8areas of china(Yongtai of Fuzhou,Hongdong of Linfen,Laixi of Qingdao,Yanlin of Xuchang,Gangu of Tianshui,Changyuan of Xinxiang,Lianjiang of Fuzhou and Pinghe of Zhangzhou)and five kinds of nato products were purchased from market.These samples were preliminarily screened.A total of eighty-five protein-producing Bacillus sp.strains were obtained.The 16S rDNA gene fragments of the above Bacillus sp.strains were amplified by PCR,and restriction fragment length polymorphism(RFLP)was analyzed using three restriction enzymes Hha?,Msp?and Rsa?.The results of RFLP analysis showed that the 85 strains of Bacillus were divided into 12 groups.,one strain or two strains with high protease activity were selected from each of the above groups.Their16S rDNA gene fragment sequences were determined,and cluster analysis was performed.The results of cluster analysis showed the four groups that were Bacillus subtilis,Bacillus cereus,Bacillus pumilus and Bacillus megaterium.2.The CAH protein coding gene sequence cloned from B.subtilis NSYT-3 strain had a total length of 960 bp and encoded 318 amino acids;The AXE protein coding gene sequence cloned from B.pumilus OSLJ-13 strain had a total length 963 bp in length and encoded 320 amino acids;SCP protein coding gene cloned from B.subtilis NSYT-3 strain had a total length of 1146 bp and encodes 381 amino acids.The polypeptide chain of SCP recombinant protein was composed of three parts:29 amino acids of signal peptide,77amino acids of leader peptide and 275 amino acids of mature peptide.The simulated 3D structures of the above three proteins all conformed to the protein structure of the?/?hydrolase folding family enzymes.The above three enzyme proteins of?/?hydrolase folding family coding genes were cloned into the expression vector pET28a,and then introduced into E.coli BL21(DE3)strain for heterologous expression,purification and SDS-PAGE analysis.The results of experiment showed that the relative molecular weights of recombinant AXE,CAH and SCP were 35.90 kDa,35.60 kDa and 29 kDa,respectively;the purification folds were 1.30 times,7.60 times and 6 times,respectively;The enzyme activity recovery rate were 57.30%,46.50%and 31%,respectively.Recombinant AXE and CAH showed perhydrolase activity and their specific activities were 0.90 U/mg and 2.00 U/mg,respectively.3.The enzymatic kinetic parameters K_m,V_max,and k_cat/K_mof the activity of perhydrolysis of recombinant protein AXE were 112.60 mmol·L^-1,10.40 mol·mg^-1·min^-1and 3.50×10³min^-1,respectively;The enzymatic kinetic parameters K_m,V_max,and k_cat/K_mof the activity of perhydrolysis of recombinant protein CAH were 76.80 mmol·L^-1,22.20?mol·mg^-1·min^-1and 1.60×10³min^-1,respectively.4.The four perhydrolases which were Mycobacterium smegmatis Acyl transferase(Ms Ac T),AXE,CAH and Tmermotoga maritima Acetyl xylan esterase(Tm AXE)were respectively coupled with glucose oxidase and Ms Ac T and Tm AXE were purified and prepared by other students in the research group.The kinetic analysis results showed that:It required respectively 2.50 h,4.70 h,4.30 h and 4.70 h for the coversion of 0.047?mol MCD in cascade catalytic system of GOD-MsAc T,GOD-AXE,GOD-CAH and GOD-Tm AXE under dynamic monitoring.Then,the MCD conversion rate was analyzed every 20 min(or every 8 min),and it was found that the reaction rate increased with the accumulation of hydrogen peroxide.In the coupling systems of GOD-MsAc T,GOD-AXE,GOD-CAH and GOD-Tm AXE,the maximum conversion rate for MCD was 34.70?mol/L/h,15.30?mol/L/h,18.00?mol/L/h and 17.00?mol/L/h,respectively.