Mutagenesis Breeding Of Protein Glutaminase High-producing Strains By Atmospheric Room Temperature Plasma(ARTP)

Protein-glutaminase can hydrolyse glutamine residue in the macromolecular protein specificily,while asparagine residues and free glutamine will not be influenced.There is an excellent application prospect in protein modification.Microbiology laboratory of East China Normal University has worked on PG for several years and 33 PG-producing strains,Chryseobacterium proteolyticum have been isolated,which are proved to be edible safe strains.However,the enzyme activity produced by present strains is too low to satisfy the industrial requires,mutagenesis breeding will be further carried on.In order to improve the protein-glutaminase production,iterative mutagenesis breeding with atmospheric room temperature plasma has been conducted on PF-Y0360in this study,which was screened by protoplast fusion previously.During the research,compound mutagenesis has been applied with ARTP followed by ultraviolet.Resequencing and transcriptome sequencing of the high-producing strain AU-J0789has been performed by the second-generation sequencing after the mutagenesis screening,and the molecular mechcnism of high yield has been preliminary inquired from from the perspective of genomics.1.Optimization of atmospheric room temperature plasma mutagenesisWith the fatality rate and the positive mutation rate as the criteria,the optimal value of bacteria concentration,processing time and gas flow have been determined through the single element experiment.The appropriate OD₆₀₀ of bacteria solution is 1.0,and the processing time and gas flow are set to 80 s and 10 splm respectively.2.Five rounds of iterative mutagenesis breeding with ARTPPF-Y0360 is used as the original strain in the first round of mutagenesis,and the high-yielding strain is used as the original strain in next round and so on.12,720mutants have been screened all over the five rounds and the increasment of enzyme activity in each round is 14.0%?17.3%?16.5%?10.4%?8.1%.3.Two rounds of compound mutagenesis with ARTP followed by UVCompound mutagenesis with ARTP followed by UV has been conducted to deal with fatigue effect after iterative mutagenesis.A total of 3,348 mutant strains have been screened and the superposition of different mutagenesis methods shows favourable synergistic effect.Finally,the enzyme activity of the high-yielding strain AU-J0789 is2.04±0.02 U/m L,which has been increased by 137%comparing to the original strain PF-Y0360.4.Study on the properties of high-yielding strainsThe logarithmic phase of AU-J0789 is 6-12 h according to the prowth curve.The enzyme activity can be detected at the 6^th h and the maximum shows near the 14^th h,which can maintain for 3 hours between the 12^th and 15^th.The high-yielding strain owns good genetic stability.There is no mutation in the PG coding sequence of AU-J0789,while the expression levels of PG are significantly higher than that of PF-Y0360at the 10^th,12^th and 14^th h of fermentation in the fluorescence quantitative PCR experiment.5.Preliminary study on the molecular mechanism of high yieldResequencing and transcriptome sequencing have been carried out for the high-yield strains with the whole genome of L11 used as reference genome,which is wild type chryseobacterium proteolyticum.52 SNP and 6 Indel sites have been selected in resequencing.SNPs site function annotation mainly focuse on carbohydrate metabolism,nucleotide metabolism and amino acid metabolism.Indels site functional comments are associated with transmembrane transport and protein modification.The analysis of the GO annotation of the differentially expressed genes shows that the genes of high-yielding strains are up-regulated in the biological process in the transcriptome sequencing.KEGG pathway analysis indicates that the histidine metabolism is significantly down-regulated in the high-yielding strain.It is speculated that there is less ribulose-5-phosphate in the pentose phosphate pathway flowing into histidine biosynthesis,but more transformed into fructose or glyceraldehyde 3-phosphate,which return to glycolytic pathway.As a result,it is strengthened of the growth of the high-yielding strain.