Screening Of A Fibrinolytic Enzyme And Amylase Inhibitor Producing Strain Streptomyces Sp.CC5,and The Study Of Its Products

Actinomycete is a group of spore-forming, chiefly filamentous, aerobic or anaerobic gram-positive bacteria. They have various shapes including spheres, rods, branching and so forth. In general, it can develop aerial mycelium, substrate mycelium and different shape of sporophyte. Most of actinomycetes are saprophytic, rare of them are parasitic or symbiotic with other creatures.Actinomycete resources were isolated and collected from soil sampled all over China.Ten genera, 406 strains of actinomycetes were isolated including Streptomyces,Nonomuraea,Micromonospora,Microbispora,Nocardia,Pseudonocardia,Actinomadura,Planomonospora, Mycobacterium and Catellatospora. Based on natural products screening,10 a-amylase inhibitor productive actinomycete strains and 18 strains which can secrete fibrinolytic enzyme were obtained. Prominent one is Streptomyces sp. CC5, and significant a-amylase inhibition and fibrinolytic activity can be detected in its fermentation supernatant.In addition, plasminogen activation activity and antibacterial effect were also observed in its fermentation supernatant, indicating Streptomyces sp. CC5 can secrete various active natural products.Fibrinolytic proteases, which can directly degrade fibrin and dissolve thrombi rapidly and completely, have potential applications in cardiovascular disease therapy. A novel fibrinolytic protease, AfeE, with strong thrombolytic activity was purified from Streptomyces sp. CC5 using macroporous resin, 201×7 ion-exchange resin, ammonium sulfate precipitation and Superdex 200 gel filtration. AfeE displayed maximum activity at 40? in the pH range of 7.0-12.0. It was strongly inhibited by phenylmethanesulfonylfluoride (PMSF), soybean trypsin inhibitor (SBTI), tosyl-L-lysine chloromethyl ketone (TLCK) and tosyl-L-phenylalanine chloromethyl ketone (TPCK),indicating it is a serine protease inhibitor. The activity of the enzyme was partially inhibited by Cu2+, Co2+ and Zn2+. AfeE exhibited higher substrate specificity for fibrin than fibrinogen, hemoglobin, bovine serum albumin, and albumin chicken egg. The chains of fibrinogen for degrading ratio was A? chain>B? chain>y chain. The degrading ratio for fibrin is faster than fibrinogen degradation, which has rarely been reported in fibrinolytic enzymes. Fibrin plate assays revealed that it was able to hydrolyze fibrin clot directly, and can perhaps transform plasminogen into plasmin, with assistant effect in the fibrinolysis reaction as well. The afeE gene was cloned from the genome of Streptomyces sp. CC5.Full-length AFE-CC5E contained 434 amino acids and was processed into a mature form consisting 284 amino acids by posttranslational modification, as revealed by high-resolution mass spectrometry analysis.AfeE performs high thrombolytic activity in a carrageenan-induced mouse tail thrombosis model and the dose of 0.5 mg/kg AfeE antithrombotic effect was greater than that induced by 20 mg/kg urokinase. AfeE significantly prolonged prothrombin time,activated partial thromboplastin time, and thrombin time in rat blood at dose of 8 ?g, 4?g and 2?g respectively. A bleeding time assay revealed that AfeE did not prolong bleeding time in mice at a dose of 1 mg/kg. However, urokinase can significantly prolong bleeding time in mice at a dose of 20 mg/kg, and 40% of urokinase treated mice bleeding time was more than 1800 s. This results suggested AfeE may have low bleeding risk than urokinase.No acute cytotoxicity was observed for AfeE at 320 ?g/well in human umbilical vein endothelial cells. These results indicate that AfeE is a prospective candidate for antithrombotic drug development.a-Amylase inhibitor was widely used in agricultural and medical industries, its application ranges from crop protection to the treatment of diabetes and obesity. A proteinaceous a-amylase inhibitor, AAI-CC5, was purified from Streptomyces sp. CC5 secretion by macroporous resin, DEAE anion exchange, ammonium sulfate precipitation and Superdex 75 gel filtration. AAI-CC5 specifically inhibited mammalian a-amylases, and no obvious inhibition activity was detected against ?-amylases from Bacillus amyloliquefaciens, Myxobacteria, Streptomyces and Galleria mellonella. The molecular weight of the inhibitor was determined to be 8,212 Da by MALDI-TOF Mass Spectrum.The N-terminal 15 amino acid residues of the purified AAI-CC5 were DTGSPAPECVEYFQS, which is dissimilar to other reported proteinaceous a-amylase inhibitors. AAI-CC5 is a pH insensitive and heat-stable protein and the activity shows no detectable decrease after treatment in pH 2.0-10.0 for 24 h or 80? for 1 h. AAI-CC5 was cloned and expressed in Escherichia coli BL21 (DE3) with a hexa-histidine tag on the C terminal. AAI-CC5 shared 82 % identity with Parvulustat. The recombinant ?-amylase inhibitor was purified to homogeneity by one-step affinity chromatography using Ni2+-NTA resin with molecular mass of 9,404 Da. Steady state kinetics studies of ?-amylase and the inhibitor revealed an irreversible, non-competitive inhibition mechanism with IC50 and Ki value of 6.43×10-11 and 4.45×10-11 M respectively. These results suggest this novel a-amylase inhibitor possessed powerful inhibitory activity for a-amylase, and it may be a candidate in research of diabetes therapy and obesity treatment.