The Lyase Gene Diversity Analysis From Eryuan Hot Spring And A Gene Mmplysin Cloning And Expression

Bacteriophage is a virus that infects microorganisms such as bacteria,archaea and fungi.Since bacteriophage has been found,because of its small genome,relatively simple mechanism,easy gene manipulation and other advantages,which usually as a gene replication and expression regulation model.Bacteriophage lyase is a kind of cell wall hydrolase that the release of phage-infected host,cleaves the bacteria by hydrolyzing the cell wall peptidoglycan,and releases the progeny phage.Traditional antibiotic treatment can lead to the emergence of antibiotic resistant strains,and phage lysate can be efficient,specifically to crack the bacteria,it is expected to replace the traditional antibiotic treatment,especially for multiple drug-resistant pathogens.In this paper,the mixed sample ya from Dali Eryuan hot spring were used as material.Phage was collected after ultracentrifugation of phage mixture,and phage DNA was extracted and sequencing by Novogene company.The sequencing result was staged by FastQC sequence analysis and IDBA-UD,and the spliced sequence was compared with NCBI in the gene library,and 1006 sequences were obtained.The sequence number of the cleavage enzyme were 1006,which were amidase,endopeptidase,transglycosylase and glucosaminidase.The contig number of these four kinds of lyase were 422,306,250 and 28,respectively,with the number of complete ORFs being 94,65,66 and 8.The conserved domains of amidase,endopeptidase,transglycosylase and glucosaminidase were analyzed for the subsequent search of new and more efficient phage lysates and phage lysates.It's benefit to lay the foundation for the cracking mechanism.The experimental object of this paper is a known sequence named mmplysin,from the Meiothermus bacteriophage of Eryuan hot spring.The base sequence length is 633 bp and the conserved domain was shown from the M23 peptidase family.The recombinant plasmid pET28a-mmplysin was constructed with pET28a by PCR amplification,ligation,transformation and induction.After induction of expression,the recombinant protein mmplysin was obtained.The recombinant protein was expressed at 28 ? for 5 hours.The recombinant protein has a molecular weight of about 23 kDa.The optimal activity temperature of the recombinant protein mmplysin was 56-65?,and the activity was the highest at 37? to 75?,which was higher than most of the reported phage lysates.The results showed that the activities of Mn2+,Ca2+ and K+ were decreased,while Zn2+,Cu2+ and Mg2+ had an activating effect on the enzyme.The results showed that the recombinant protein mmplysin had significant inhibitory effect on the phage MMP17 host strain TG17,and had certain inhibitory effect on Salmonella,Staphylococcus aureus and E.coli.In the antimicrobial experiment of Klebsiella pneumoniae,it was found that some strains also had certain inhibitory effect.Enzyme microscopic observation of the inhibitory effect of the lysozyme on Klebsiella pneumoniae showed that the action of the enzyme solution on the surface of the cell wall resulted in perforation of the cytoplasm leading to cell death.In this paper,the diversity of phage lysates were studied,and the characteristics of the cleavage enzyme mmplysin were studied,which provided some experimental basis for the application of the high temperature lysozyme in real life.