ARTP Mutagenesis Of Protease-producing Strain And Analysis Of Its Degrading Protein Metabolic Pathway

In this study,a strain with high protease production activity was screened from the soil near Dalian Zhongjia Food company limited,Liaoning Province.Then,Atmospheric and Room Temperature Plasma(ARTP)was applied to the strain.The activity of protease production was significantly improved.On the basis of optimizing the optimum enzyme production conditions of this strain,this paper used genomics combined with transcriptomics technology to analyze and compare the differences of genes,major metabolic pathways and key enzymes related to protein degradation of this strain before and after ARTP mutagenesis breeding,so as to explore the metabolic pathways of protein degradation of this strain.The specific research results are as follows:1.Ten protease-producing strains were isolated from protein-rich soil,and a strain Y-6with high protease-producing activity was screened by using transparent circle method and folinol method to measure the protease-producing activity of the strain.After ARTP mutagenesis,a strain Y-6-NEW with high protease yield was obtained.The results showed that the protease-producing activity of strain Y-6-NEW increased from 252.16 U/mL to658.15U/mL after 72 h culture in casein medium,and the enzyme producing activity increased by 161%.The strain was identified as Bacillus cereus.2.Single factor test and Box-Behnken response surface test were used to optimize the enzyme-producing conditions of mutant strain Y-6-NEW.The results showed that the optimum enzyme-producing conditions of strain Y-6-NEW were: Under the conditions of 2%casein content,4% NaCl concentration,2% liquid loading,7 initial pH,3 d fermentation time,4% inoculation amount and 28 ℃,the protease production activity of strain Y-6-NEW could reach 1288.27 U/mL,which was 48.91% higher than that of strain before optimization.3.The whole genome of strain Y-6 was determined and analyzed by Illumina.The results showed that the genome size of strain Y-6 was 5,694,983 bp,GC content was 34.95%,and it contained 5856 protein-coding genes.KEGG annotation results showed that there were463 genes related to protein degradation in strain Y-6 genome,and these genes mainly encoded enzymes related to protein degradation,such as serine protease gene deg P/htrA,zinc protease gene ppqL,periplasmic zinc protease gene ppqL2,ATP-dependent protease gene Clp B,alkaline serine protease gene apr A,peptidase(S54 family member)aar A,aminopeptidase pep P,dipeptidase pep D,et al.The genes associated with protein degradation described above indicted the strain Y-6 displayed potent protein degrading ability.4.The ability of protein degradation of strain Y-6 was greatly improved through ARTP mutation breeding.High-throughput sequencing platform was used to determine the transcriptome of strain Y-6 after treated with ARTP.The results showed that there were 1095 significantly different genes in strain Y-6 before and after ARTP mutagenesis,including 692up-regulated genes and 403 down-regulated genes.The genes involved in protein degradation,such as ppqL2,ppqL,aprA,aarA,pepP and pepD were upregulated,and the fold change values were 4.15,1.59,1.78,2.61,1.59 and 1.52,respectively.The mutagenesis of Y-6-NEW strain might enhance its ability to degrade protein by enhancing the expression level of these genes.Differential genes were performed by GO and KEGG enrichment analysis.GO annotation of differential genes showed that 279 significantly different genes were enriched in strain Y-6 during the biological process,among which 41 genes were related to protein metabolism.KEGG annotated differential genes showed that the differential genes were mainly concentrated in phenylalanine,glutamate and histidine metabolic pathway.In the phenylalanine metabolism pathway of strain Y-6 after ARTP treatment,the gene dad A encoding D-amino acid dehydrogenase was up-regulated 2.41 times,and the gene paa G encoding 2-acetyl-CoA isomase was up-regulated 2.73 times.In the metabolism pathway of alanine and aspartic glutamate,the gene put A encoding 1-pyrrolin-5-carboxylate dehydrogenase was up-regulated 2.7 times.In the histidine metabolism pathway,the gene hut H encoding histidine deaminase was upregulated 3.87 times,and hut I encoding imidazolinone propionate was upregulated 3.93 times.In conclusion,the Y-6-NEW strain genes involved in protein degradation,such as ppqL(zinc protease gene),apr A(alkaline serine protease gene)and Clp B(ATP-dependent protease gene)were upregulated.In Y-6-NEW strain,protein is hydrolyzed by proteases like ppqL to form polypeptide.Secondly,it is further hydrolyzed by peptidases like aarA to produce different kinds of amino acids such as phenylalanine alanine histidine.Finally,different kinds of amino acids are further degraded by amino acid catabolic pathways to complete the protein degradation process.