Mutation Breeding And Omic Analysis Of Bacillus Subtilis With High Yield Nattokinase

Nattokinase(NK)is a serine protease produced by Bacillus Subtilis.It has the advantages of strong thrombolytic activity and long action time.As a new type of thrombolytic agent and health food,it is popular extensive attention.Nattokinase is generally produced by fermentation of Bacillus subtilis,but it still has problems such as low enzyme activity and low yield.This thesis intends to use the Bacillus subtilis JNFE0126 strain selected in the laboratory as the starting strain to obtain a high-yielding nattokinase strain through combined mutagenesis of ultraviolet and ⁶⁰Co-?rays,and to optimize the fermentation conditions of the mutagenized strain to further improve the enzyme activity.Finally,the second-generation sequencing technology and the third-generation sequencing platform were used to sequence the genome and transcriptome of the original original strain and the mutagenized strain,and explore the molecular mechanism of the mutagenized strain for high-yielding nattokinase from the perspective of omics.The main findings are as follows:(1)The original strain Bacillus subtilis JNFE0126 was used as the research object.The UV-4 mutant strain has the highest enzyme activity under the conditions of UV lamp power of15 W/m² and treatment time of 120 s,which can reach 1282.47 IU/m L under 24 h fermentation conditions.Taking the UV-4 mutant strain as the starting strain for ⁶⁰Co-?-ray mutagenesis,under 400 Gy treatment dosage mutagenesis conditions.A mutant strain with stable genetic traits and high-yield nattokinase was obtained,and it was named Bacillus subtilis JNFE1126.After 60 h of fermentation in the shake flask,its enzyme activity reached 12656.46 IU/m L,which was 1.27 times higher than that of the original strain.(2)The fermentation conditions of the Bacillus Subtilis JNFE1126 mutant strain were optimized from the aspects of carbon source,nitrogen source and surfactant,and the optimal formula of fermentation medium was determined to be 10 g/L glucose,30 g/L soybean meal powder,2 g/L disodium hydrogen phosphate,1 g/L sodium dihydrogen phosphate,0.2 g/L calcium chloride,0.5 g/L magnesium chloride,1 m L/L Tween 80.After fermentation for 60 h,the enzyme activity of nattokinase was 15030.31 IU/m L.The enzymatic properties of the mutagenized strain nattokinase were studied.When the temperature was lower than 20?,the enzyme activity was relatively stable.The enzyme activity is relatively stable in the range of p H 8-p H 12.(3)The whole genomes of the original strain Bacillus subtilis JNFE0126 and the mutant strain Bacillus subtilis JNFE1126 were sequenced through the Illumina Hi Seq second-generation sequencing system and the Pac Bio third-generation sequencing system.Combined with genome-wide data,GO,COG,KEGG metabolic pathways,homologous genes and SNP analysis were performed.It was found that the original original strain had 6 more CDS than the mutagenized strain.13 insertions,2 deletions and 28 SNP were detected in the JNFE1126genome.The functions of mutant genes mainly focus on amino acid transport and metabolism,carbohydrate transport and metabolism,transcription,energy production and conversion.(4)Transcriptome sequencing was performed on the original strain JNFE0126 and the mutant strain JNFE1126 at the 24 h fermentation time point,and 1,425 differentially expressed genes were screened.The differential genes were analyzed by GO,KEGG function annotation and metabolic pathway enrichment analysis,and the differentially expressed genes were searched for and induced The possible mechanism of high-yield nattokinase by the mutant strain The results showed that the mutagenic bacteria involved in amino acid metabolism and energy production related genes such as Nad B(L-aspartate oxidase),Ndh F(NADH dehydrogenase)and other genes were up-regulated,which promoted energy production and increased biomass;In addition,the expression of Com P(histidine kinase)genes related to the regulation of nattokinase transcription level is up-regulated,Cod Y(transcription factor)and other genes are down-regulated,and the secretion-related Sec Y(internal membrane protein transposase component)and other genes are up-regulated to promote nattokinase.The production and secretion of enzymes increase the activity of enzymes.Real-time fluorescent quantitative PCR was used to verify the differential expression of genes,and the results were consistent with the results of transcriptome sequencing.