Regulation Of Marine Stachybotrys Longispora Of Fibrinolytic Compounds Based On Secondary Metabolic Synthesis

Marine microorganisms live in the extreme environment of high pressure,high salinity,low dissolved oxygen,low nutrition and low light.This situation can also lead to the uniqueness of growth,reproduction and secondary metabolism.They can produce secondary metabolites with novel structure and diversity of biological activities.They have anti-tumor,neuroprotection,anti-fungal,anti-oxidation,anti-atherosclerosis,anti-virus and anti-inflammatory activities.It is believed that the regulation of microbial secondary metabolism can significantly increase the yield of target compounds and may produce new compounds.The discovery of novel small molecular fibrinolytic active compounds may make a breakthrough in the development of new drugs for thrombosis.It also can guide the exploration of novel compounds.In this experiment,the secondary metabolites were affected by regulating the secondary metabolic pathway of Stachybotrys longispora.The fermentation conditions and methods were optimized.The secondary metabolites were analyzed by liquid chromatography,and the suitable secondary metabolic regulators were determined.The goal products were separated and purified by liquid chromatography,and the molecular structure and fibrinolytic activity of the compounds were analyzed.The work to optimize of culture conditions of Sphaeromyces longsporum FG216.Stachybotrys longispora FG216,a marine Stachybotrys longispora strain preserved in a refrigerator at – 80?,was extracted and fermented in Chapffer liquid medium after being activated by seed medium.The enrichment of secondary metabolites was taken as the main index and the quality of reference bacteria was optimized by changing fermentation temperature,adding carbon source and adding precursors.The mass of the strain reached a stable stage on the 3rd day of fermentation,and declined on the10 th day.The richness of secondary metabolites reached its maximum on the 6th day.By adding sucrose in the fermentation process and analyzing the production of secondary metabolites,it was concluded that FG216 did not need to be supplemented with carbon sources in the fermentation process.In the fermentation medium,supplementation of L-ornithine hydrochloride,a precursor,which can increase the secondary metabolites by 1.90 times.The large-scale fermentation conditions of Stachybotrys longispora FG216 were determined as Chapman liquid medium with inoculation amount of 5%,28 ?,180 r/min,activation culture for 3 days andfermentation culture for 7 days.The culture and fermentation conditions of Spiromyces longsporum FG216 were optimized.The discovery of secondary metabolic regulators.Stachybotrys longispora FG216 was fermented in Cha's liquid medium for 7 days.Methanol was extracted from 5 mL fermentation broth and dried in vacuum to obtain crude methanol extract.Secondary metabolites were analyzed by liquid chromatography with centrifugal filtration membrane after 2 mL methanol resolving.Compared with the control group added ornithine,new retention peaks were detected when the secondary metabolic regulator was tryptophan or phenylalanine,which determined that tryptophan or phenylalanine regulated the secondary metabolic pathway of Stachybotrys longispora,thus affecting the production of secondary metabolites.The work of isolation,purification and structure analysis of secondary metabolites from Spirulina longspora FG216 is done.FG216 strains added ornithine,tryptophan and phenylalanine were cultured in large-scale fermentation to obtain 1 L fermentation broth.After methanol extraction,the crude samples were 70 mg,880 mg,18 mgand 60 mg,respectively.The concentration of resolving and methanol was 24mg/mL,44 mg/mL,3 mg/mLand 20 mg/mL.The methanol extract was separated and purified by liquid chromatography,and the structure of the compounds was analyzed by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.Fibrinolytic activity of new small molecule compounds was studyed.Fibrinolytic activity of fibrolytic small molecule compound FGFC1 was evaluated in vitro by simulating human thrombolytic system pro-uPA/Plg.The results showed that compound FGFCD could promote fibrinolysis in pro-uPA/Plg system.The fibrinolytic activities of separated compounds,such as FGFCA,FGFCB and FGFCC,were lower than those of control FGFC1.ADP,trisodium citrate,lysine,histidine and asparagine were added to the metabolic process which compared with ornithine.It is concluded that there is no new retention peaks were found,i.e.no new compounds were formed.Among the four new compounds,FGFCB and FGFCD showed good fibrinolytic activity,while FGFCA,FGFCC and FGFCE did not show good fibrinolytic activity.According to the retention time,the polarity of the compounds was sorted from large to small:FGFCA > FGFCC > FGFCB > FGFCD > FGFC1.