| Nattokinase is an alkaline serine protease.Compared with other fibrinolytic agents,nattokinase is more efficient and has no side effects.It is a highly effective treatment for cardiovascular diseases.The low yield and activity of nattokinase hinder its application and popularization.In this study,ultraviolet light and ion beam were used to mutate Bacillus subtilis respectively,and stable strains with high nattokinase production activity were screened out,and the fermentation conditions of this strain were optimized.Finally,the enzyme-producing mechanism of mutagenic strain was studied from the aspects of morphological characteristics,fermentation performance,genome and transcriptome.The main research results are as follows:(1)Using Bacillus subtilis as the initial strain,the initial strain was mutagenized by ultraviolet and ion beam respectively.Bacillus subtilis UV-1,Bacillus subtilis UV-2,Bacillus subtilis IB-1 and Bacillus subtilis IB-2 with high enzyme activity were screened by casein preliminary screening and fibrin recombination.The genetic stability analysis of the four strains showed that strain B subtilis IB-2 with the highest enzyme activity and strong stability was obtained.The enzyme activity of B subtilis IB-2 reached 320 FU/m L,which was 19.4% higher than that of the original strain.(2)The fermentation performance of the initial strain B subtilis and the ion beam mutagenic strain B subtilis IB-2 was analyzed,and five single factors in the fermentation conditions of B subtilis IB-2 were optimized to determine the optimal fermentation environment: The inoculum dosage was 7.5%,the fermentation time was 33.5 h,the seed age was 9 h,the temperature was 37 ℃and the rotation speed was 200 r/min.Under these conditions,the enzyme activity of nattokinase reached 355.07 FU/m L,which increased by 10.2%compared with that before optimization.(3)The whole genome sequencing of the initial strain B subtilis and the ion beam mutated strain B subtilis IB-2 showed that the initial strain B subtilis contained 4524 protein-coding genes,and the total length of the coding genes was 3599220 bp.The mutant strain B subtilis IB-2 contained 4524protein-coding genes,and the total length of the genes was 3602,148 bp.After functional annotation of protein-coding genes,it was found that the differential genes of the two strains were mainly distributed in carbohydrate transport and metabolism,cell wall/membrane/envelope biogenesis and migration group(prophage,transposon).(4)Through transcriptomic analysis of initial strain B subtilis and mutagenic strain B subtilis IB-2,a total of 191 significantly different expression genes were detected,among which 183 were up-regulated and 8were down-regulated.GO annotation showed that the genes with different expression in mutant B subtilis IB-2 mainly focused on macromolecular catabolic process,organic acid biosynthesis process,carboxylic acid biosynthesis process and peptidoglycan metabolism process.Enrichment of differentially expressed genes in KEGG metabolic pathway was found.The results of KEGG were consistent with the results of GO functional enrichment.In the key pathways related to nattokinase synthesis,O antigen nucleotide sugar biosynthesis,nucleotide sugar biosynthesis,lysine biosynthesis,amino sugar and nucleotide sugar metabolism,histidine metabolism and five pathways related to protein synthesis are all up-regulated,which is conducive to protein synthesis.The downregulation of the secondary metabolite biosynthesis gene was beneficial to the growth of the strain itself and laid the foundation for the later protein synthesis.The up-regulation of genes related to glycerophospholipid metabolism is beneficial for energy supply during protein synthesis. |
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