| The extreme living environment of marine microorganisms has led to the formation of a unique secondary metabolite synthesis pathway from them in the process of evolution and adaptation to the environment.It has been found that marine microorganisms have significant biological activities and many compounds of new skeleton structure.The unique metabolic pathway and genetic background provide the possibility for the discovery of new drugs.Here,with Euphausia superba and sea water as the isolated source,the plasminotic active strains were screened,the species characteristics of the active strains were identified,the fermentation conditions were optimized,the small molecule plasminotic active compounds and plasminotic enzymes were isolated and purified,the structure of the small molecule plasminotic active compounds and the enzyme characteristics of plasminotic enzyme were analyzed,and the plasminotic characteristics of the active compounds were studied.Discovery of active strain.In this study,864 strains of polar marine fungi were isolated from Euphausia superba and sea water by selective fibrinolytic medium,and 19 strains with high activity(No.A-O1 to A-19)were obtained by plate screening of fermentation broth and high-throughput screening of secondary metabolites.The fibrinolytic activity of A-10 was similar to that of the control sample FGFC1(Fungi Fibrinolytic Compound 1).Identification of active strain.The sequence ratio of rDNA ITS gene in the active and stable strain A-10 was similar to that of Penicillium expansum.The physiological and biochemical characteristics showed that the strain grew well when glucose and sucrose were used as carbon sources.The culture characteristics of strain A-10 in the experiments of starch hydrolysis,cellulose utilization,milk coagulation,peptonization and gelatin liquefaction were all positive.Nitric acid reduction was negative and didn't produce hydrogen sulfide.Therefore,A-10 was identified as Penicillium.A-10 can not only produce fibrinolytic active secondary metabolites,but also secrete cellular molecular fibrinolytic protease.For the first time,a marine Penicillium strain produced simultaneously fibrinolytic secondary metabolites and fibrinolytic enzymes was found.Optimization of fermentation conditions for fibrinolytic secondary metabolites.When A-10 was cultured at 25±1?,both the growth amount of the bacteria and the fibrinolytic activity of the fermentation broth were optimal,and the growth amount of the bacteria and the activity of the fermentation broth were inhibited in cultures above 30±1?.The initial pH of the medium was 6.0,and the growth rate of the bacteria was 21.00 g/L.When the inoculation amount was 1%,the growth of the bacteria was too slow,and the volume of the bacteria on the sixth day was 18.00 g/L.However,when the inoculation amount was 3%,the bacteria grew too fast,and the bacteria volume reached 15.00 g/L on the third day.The bacteria declined early and could not accumulate enough fermentation active substances.The fibrinolytic activity of the fermentation broth was 0.25(OD405)at 180 rpm/min.The 5-day growth of strain A-10 was the highest at 26.00 g/L.Therefore,the optimal fermentation temperature of A-10 was 25?,the optimal fermentation pH value was 6.0,the optimal fermentation inoculation amount was 2%,the optimal fermentation ventilation volume(rotation speed)was 180 rpm/min,and the optimal fermentation time was 5 days.Isolation and purification secondary metabolites of fibrinolytic compounds.A-10 fermentation liquid was centrifuged,and the bacteria were extracted with acetonitrile-0.1%TFA(trifluoroacetic acid)aqueous solution as mobile phase.Elution conditions by HPLC(High Performance Liquid Chromatography):100%TFA+0%acetonitrile linearly changed to 15%TFA+85%acetonitrile(0-50 min),wavelength:254 nm,flow rate:0.8 mL/min,column temperature:35?,sample loading volume:10 ?L.Seven compounds were isolated and purified by analytical HPLC and preparative Pre-HPLC.C-2 with a retention time of 8.65 min showed excellent fibrinolytic activity,and EC50 was 200.00 ?g/mL.Structure analysis of secondary metabolites of fibrinolytic compounds.The structure of compound C-2 was analyzed by high resolution mass spectrometry and nuclear magnetic resonance.Compound C-2 was resolved to phytosterols 5?,8?-epidioxyergosta-6,22-diene-3?-ol.Compound c-2 was a rare sterol compound with a dioxygen bond at C5-C8.Compound C-2 is a fibrinolytic active sterol compound isolated and purified from Penicillium A-10 for the first time.Characterization of fibrinolytic activity of compound C-2.FGFC1 was used as the positive control,and the EC50 of compound C-2 was 200.00 ?g/mL.The Caco-2 cell model showed that compound C-2 had low permeability.Intravenous administration of compound C-2 was distributed in the liver(65.00%),heart(0.11%),spleen(0.41%),lung(0.90%),kidney(1.88%),intestine(0.75%),brain(0.18%),and muscle(0.83%)of beagles.Compound C-2 could be rapidly distributed to all tissues and organs of the animal,mainly concentrated in liver and bile.T1/2 was 49.00±2.00 min.Isolation and purification of fibrinolytic protease and its enzymatic properties.The fermentation broth was purified by ammonium sulfate salination,Q-sepharose Fast Flow anion exchange chromatography,and SuperdexTM 200 10/300 GL gel filtration chromatography in three steps.The electrophoretic pure plasminozyme was obtained by SDS-PAGE detection.The molecular weight of the target active protein was 27 kDa,the purification factor was 2.08,and the specific activity was 736.44±1.51 U/mg,with a recovery rate of 25.21%.The enzymatic properties experiment showed that the optimum temperature and pH of the enzyme were 37?and 8.0?,respectively,and the activity could still maintain more than 92.0%at 30?Besides,the purified enzyme had good stability at pH of 7.0-10.0 and a temperature not higher than 55?.Na+,Ba2+,K+,Co2+,Mn2+,Al3+ and Cu2+promoted the activity of this enzyme.Fe3+,Mg2+and Zn2+showed inhibitory effects on the enzyme activity.Ca2+and Fe2+strongly inhibit the enzyme activity of this plasminogen.Here,we isolated and obtained a rare polar marine Penicillium A-10 strain,which could not only produce fibrinolytic active secondary metabolites,but also secrete extracellular fibrinolytic active macromolecule fibrinolytic protease.This type of polar marine penicillium was found for the first time.Compound C-2 is a fibrinolytic active sterol compound isolated from polar marine Penicillum,which can penetrate the blood-brain barrier and has the characteristics of lead compound for cerebral thrombosis.Fibrinolytic proteinase showed excellent molecular structure and active sites,which provided new possibilities for the treatment of thrombotic diseases and had a good application prospect. |
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