Screening And Mechanism Study On Mutagenesis Of Protease-Producing Bacillus By He-Ne Laser And UV

The excessive addition of antibiotics and other drugs in feed is one of the important factors affecting the safety of animal derived food consumption.The protein in oil cake can be converted into active peptides that enhance animal immunity through microbial fermentation,which can partially bear the role of antibiotics.Therefore,the fermentation of oil cake has been highly valued by the feed industry.The ability of bacterial strains to produce protease is a decisive factor in the amount of oil cake protein converted into peptides,and mutagenesis is an important mean to improve the proteaseproducing ability of bacteria.Physical mutagenesis has the advantages of high safety and high mutation stability,and composite physical mutagenesis with multi target characteristics can compensate for the mutation limitations or resistance saturation phenomenon of single mutagenesis,resulting in better mutation effects.Therefore,this study carried out the screening of solid-state fermentation mode and protease-producing strains,the combined mutagenesis of He Ne laser and UV for improving protease production ability,the mutation mechanism of He Ne laser and UV light sources based on Ames test and transcriptome,and the excavation of red light receptor protein in Bacillus subtilis.The main research contents and results are as follows:(1)Screening of solid-state fermentation of soybean meal and protease-producing strains.Comparing the high-temperature solid-state fermentation(Bacillus subtilis CICC 22983)and simulated natural variable temperature solid-state fermentation(Bacillus subtilis CICC 21927)of soybean meal,it was found that After 3 days of fermentation,the total number of molds decreased significantly,and the coliform group was <0.3 MPN/g,which meets the hygiene standards.The maximum peptide content in simulated-natural variable temperature fermentation group was 135.63±2.36 mg/g.SDS-PAGE,particle size distribution and Zeta potential analysis showed that under the premise of ensuring fermentation quality,the soybean meal fermentation was more thorough.Bacillus subtilis CICC 21927 was selected as the starting strain for subsequent mutagenesis based on the levels of protease production and polypeptide production by fermentation.(2)He-Ne laser and UV were used to screen for high-yield protease mutant strains.A high-throughput culture,detection and screening method for protease-producing Bacillus subtilis CICC 21927 was established using microplate and enzyme marker as media.Using protease activity as the evaluation index,the optimal plate culture conditions were as follows: 48 deep-well plates,1 m L of liquid loading,500 r/min of oscillating speed,5% of inoculum volume.Under these conditions,the protease activity of the starting strain was 58.67 U/m L.Bacillus subtilis CICC 21927 was mutated by five mutagenesis methods: UV alone mutagenesis(UV),laser alone mutagenesis(LA),UV and laser sequential mutagenesis(UL),laser and UV sequential mutagenesis(LU),and UV and laser synchronous mutagenesis(UV+LA).It was found that the laser and UV sequential mutagenesis(LU)had the highest positive mutation rate of 4.30%,while UV and laser sequential mutagenesis(UL)reduced the mortality of Bacillus subtilis.A stable mutant strain LU-1-11 was identified through primary screening,secondary screening,and secondary mutagenesis.The 16 S r DNA sequence comparison showed that the starting strain and the mutant strain were both Bacillus subtilis,and there was no contamination by other bacteria.The simulated natural variable temperature solidstate fermentation of soybean meal showed that the peptide content of the mutant strain reached 153.1 mg/g,which increased by 12.91% compared with the starting strain.SEM showed that many mutant cells had irregular shapes and wrinkled surfaces.Amplification of two neutral protease genes npr E and npr B in the two strains revealed no changes in gene sequences.Genome resequencing revealed that the mutant strain had 5 SNP mutation sites and 8 structural variation sites involving deg S,yqb D,yqa S,puc L,srf AA,and ync B genes.Mutations in deg S may activate genes of intracellular proteases or inhibit the formation of flagella and cell membranes.Mutations in srf AA may affect the formation of biofilms,making intracellular proteases more easily released.(3)Mutation types induced by He-Ne laser and UV radiation based on Ames test.Ames strains TA97 a,TA98,TA100,and TA102 were used as model strains,the study investigated the effects of laser and UV on the reversion mutation and mutation types,with relative reversion mutation rate as an indicator.The results showed that the effect of laser on inducing reversion mutations in TA98 and TA100 was significantly higher than that in TA97 a and TA102.After 30 min of laser irradiation,the reversion mutation rate of TA98 was 2.16 times higher than that of the control group.After 10 min of laser irradiation,the reversion mutation rate of TA100 was 3.88 times higher than that of the control group.After 2 min of UV irradiation,the reversion mutation rate of TA102 was6.68 times higher than that of the control group.Based on sequencing data of 1500laser-treated TA98 reversion mutants,it was found that laser treatment produced unique large fragments of DNA insertions and deletions,significantly increasing the mutant variety of the strains.Based on sequencing data of 760 laser-treated TA100 reversion mutants,it was found that laser radiation promoted the production of C→A/T in base substitution.Based on sequencing data of 192 UV-treated TA102 reversion mutants,it was found that UV irradiation was more conducive to inducing multi-directional mutations in strains.(4)Study on mechanism of He-Ne laser and UV mutagenesis of Bacillus subtilis based on Transcriptomics.Five groups of samples(CK,UV,LA,UL,and LU)were analyzed by transcriptome sequencing.The statistical results comparing the differential gene expression levels among four groups(CK-vs-UV,CK-vs-LA,CK-vs-LU,and CKvs-UL)revealed that compared with the CK group,the LU group had the most differentially expressed genes.Direct analysis of 22 differentially expressed genes in the CK-vs-UV group revealed that UV enhanced the synthesis of pyrimidine nucleotides by upregulating key enzymes involved in pyrimidine nucleotide synthesis.GO enrichment and KEGG enrichment analysis of 536 differentially expressed genes in the CK-vs-LA group showed that laser stimulated growth metabolism by regulating the activity of transmembrane transporters.The KEGG pathway enrichment analysis was performed on 1891 differentially expressed genes in the UV-vs-LU group.According to the structural characteristics of red-light receptors,89 differentially expressed genes significantly enriched in the two-component system were analyzed,and the genes of histidine kinase and its response regulation protein that may be related to the phytochrome were found.KEGG pathway enrichment analysis of 784 differentially expressed genes in the UV-vs-UL group revealed that the expression levels of genes related to translation,replication,and repair in the genetic information process were significantly upregulated,confirming that laser treatment induced the repair function of UV damage.Nine key genes(lia S、yvf T、yxd K、che A、yko W、uvr C、pcr A、rec A and ruv B)were selected for fluorescence quantitative PCR detection,and the change trend was basically consistent with the transcriptomic data.(5)Analysis and functional prediction of phytochrome genes in Bacillus subtilis.Using Tbtools software,alignment analysis was performed on the putative phytochrome protein sequences in the reference genome of Bacillus subtilis,combined with functional domain prediction.It was inferred that the diguanylate cyclase encoded by gene ytr P might be a phytochrome-like protein in Bacillus subtilis.Combined with transcriptome analysis,it was found that the expression of this gene in the UV-vs-LU sample relationship was 50.62 and 102.08,respectively,which was significantly upregulated.After receiving red light irradiation,the protein encoded by the ytr P gene of Bacillus subtilis receives red light wavelength signals through the GAF domain,thereby activating the activity of diguanylate cyclase containing the GGDEF domain and phosphodiesterase containing the EAL domain to regulate the concentration of c-diGMP,which acts on the effector protein and produces various physiological reactions.