| Because of the existence of spores,food will cause corruption and some safety problems.However,spores are difficult to be killed by various bactericidal methods.Therefore,it is urgent to find a way to kill spores.The key to spore death lies in the hydrolysis of spore cortical peptidoglycan.Due to spore germination,core hydration and activation of cortical lyase,spore resistance disappears,so it is particularly important to isolate and purify the cortical lyase.The extraction of cortical lyase from natural strains is labor-intensive,difficult to isolate and purify,and the yield is still low.Therefore,genetic engineering engineering techniques have been used to construct genetically engineered bacteria of cortical lyase CwlJ to obtain a large number of cortical lyases.The raw materials were provided for the subsequent experiments,which laid a foundation for promoting the hydrolysis of peptidoglycan by CwlJ to kill spores and applied to food sterilization.In this paper,the cloning and bioinformatics analysis of subtilis subtilis cortical lyase CwlJ gene,the induced expression and purification of cortical lyase CwU gene,and the analysis of cortical lyase CwlJ hydrolyzed peptidoglycan were studied.The main research contents and conclusions are as follows:(1)In order to construct a clone containing the cortical lyase CwU gene,a specific primer was designed based on the gene sequence of CwlJ,and then the CwlJ gene fragment was synthesized,and ligated with the pCZN 1 vector,and the recombinant transformed into a Top 10 competent cell,and Positive clones were sequenced.Afterwards,the information analysis tools of NCBI and ExPASy were used for the sequencing results,and the bioinformatics analysis of the CwU gene sequence was carried out in combination with DNAMAN software.The results showed that CwlJ protein was a basic,unstable,hydrophilic and non-secretory protein;its secondary structure accounted for 26.06%of a-helix,P-extension chain accounted for 21.83%,and ?-turn angle accounted for 4.93%.The rule curl accounted for 47.18%;the possibility of CwlJ gene expressed enzyme protein forming inclusion bodies was 68.11%;Bacillus subtilis cortex lyase CwlJ and Bacillus thuringiensis cortical lyase CwlJ,Bacillus thuringiensi Bt18247 cortical lyase CwlJ in phylogenetic analysis Relative kinship.The above results provide reference information for the heterologous expression,purification and functional studies of the subsequent CwlJ gene.(2)In order to obtain large quantities of cortical lyase CwlJ,recombinant bacteria were induced to express the target protein CwlJ by IPTG,and the expression of recombinant cortical lyase was examined by sds-page and western-blot,and the recombinant protein CwlJ was purified by affinity chromatography.The study found that the 0.5 mM IPTG can realize CwU gene expression of the restructuring of the cortex lytic enzymes purified CwU molecular weight was 17.9 KD,optimum temperature of 40?,the optimum pH value of 5,enzyme activity than 146.67 U/mg,extracted from natural spores in the cortex than lyase,restructuring cortex lytic enzymes increased the activity of 2 times,to ensure the cortex lyase CwlJ further hydrolysis of peptidoglycan experiment.(3)In order to further understand the product characteristics of peptidoglycan hydrolyzed by enzyme,the spore cortex peptidoglycan was extracted first,and then the product hydrolyzed by peptidoglycan with enzyme CwlJ was analyzed.The results showed that there were 7 protein bands in the SDS-PAGE diagram of the hydrolysate,and the mass spectrometry data showed that there were 1300 kinds of proteins in the hydrolysate,with the majority of molecular weight ranging from 20 KD to 40 KD,and 2609 peptides.Amino acid analysis showed that there were four kinds of free amino acids in the hydrolysate,which were lysine,arginine,cysteine and methionine.The content of protein and NAG in the hydrolysate was 0.1600 mg/mL and 0.1625 mg/mL respectively.The elemental analysis showed that the C and N ratios were 2.95%and the C and H ratios were 5.88%.Relevant studies have shown that ultrahigh pressure can promote the activity of enzymes,so the peptidoglycan hydrolyzed by the cortex lyase CwlJ was treated with high pressure.In this study,a genetically engineered strain of cortical lyase CwlJ was constructed,which was successfully induced to express and purified active CwlJ,providing research materials for subsequent experiments.There are more than 1300 kinds of protein species in the analysis of peptidoglycan hydrolysate.The reason for the impure extraction of peptidoglycan is not excluded.The hydrolysis product is analyzed.The results provide reference information for further study on the hydrolysis of peptidoglycan and other bactericidal techniques to kill spores. |
PDF Full Text Request