Genome Sequence And Fermentation Of Douchi Fibrinolytic Enzyme Analysis With Quantitative Proteomics Of Bacillus Amyloliquefaciens

Douchi fibrinolytic enzyme（DFE） has been confirmed as a kind of serine protease,which not only has thrombolytic activity, but also can activate plasminogen to increase thebody’s inner courtyard of plasmin activity and have lower blood pressure, anti-oxidation,anti-tumor and thrombolytic and other health effects. fermented soya beans as a traditionalChinese flavor of food, it’s records having appetizers by food, digestion of stagnation,diaphoresis, Chufan asthma and other effects as early as in the "Compendium of MateriaMedica". Modern research shows that fermented soy contains isoflavones, brown pigment,thrombolytic enzymes and antibiotics, which are substances very beneficial to the humanbody. And brown pigment and soy isoflavones have antioxidant effects, then brown pigmentand antibiotics have nutritionally balanced role in regulating the body, soy isoflavones andfibrinolytic enzyme also has high medicinal value.In this paper, a strain of Bacillus amyloliquefaciens which can secrete Douchifibrinolytic enzyme as the research object, extract its genome and genome sequencing, thensystem analysis the serine proteases enzyme species in genome to get the types of serineprotease. And the initial point to producing fibrinolytic enzyme and the total fibrinolyticenzyme activity of the highest point in time obtain by growth curve and fibrinolytic enzymefermentation accumulation curve. Extracting the extracellular proteins, cell wall proteins,membrane proteins and cytoplasmic proteins of Bacillus amyloliquefaciens at this two timepoints, and proteomic analysis by iTRAQ to obtain the impact of related proteins plasmin.Which will provides a research base for for the subsequent research of fibrinolytic enzymemechanism in Bacillus amyloliquefaciens. In this paper, the main research results are asfollows:(1) Collection the Bacillus amyloliquefaciens cultivate8-12h in LB medium, extractedgenome and sequencing by Beijing Genomics Institute (BGI). Obtain the unknow sequenceby polymerase chain reaction(PCR) and upload the genome sequence to NCBI by sequin.Then comparison the genome with Bacillus amyloliquefaciens FZB42and building thephylogenetic trees and some others bioinformatic analysis, there may be three kinds offibrinolytic enzyme and seven kinds of serine protease in Bacillus amyloliquefaciens DC-12.(2) Bulk fermentation of Bacillus amyloliquefaciens DC-12, choose the logarithmicphase which8.5h as seed fermentation time, transfer to the fermentation medium, thefibrinolytic activity of different fermentation supernatant was measured using fibrin plate method, fomation plasmin accumulation curve. That point in time in Bacillusamyloliquefaciens plasmin generated at6h, the highest fibrinolytic enzyme activity timepoint is54h obtained by the accumulation curve..(3) Were extracted extracellular proteins, cell wall proteins, membrane proteins andcytoplasmic proteins from Bacillus amyloliquefaciens which fermented at6h and54h bydifferent methods. Protein integrity and uniformity are identified by SDS-PAGE, and proteinconcentration measured using bradford Kit. Then sent those sample to Shanghai AppliedProtein Technology Co.Ltd for iTRAQ protein quantitative determination. Analysis thedifferential proteomics by the results of protein quantitative determination.