Isolation,Identification And Antibacterial Activity Of Strain Bacillus Sp.GFP-2 Isolated From Shark Intestine

The abuse of antibiotics and following rapidly increasing of antibiotic-resistant pathogens is the serious threat to human being and natural products from microorganism were regarded as the important substitutes for antibiotics.Most of natural products currently used are derived from terrestrial sources.However,marine-derived active substances can also provide valuable resources for the discovery of new drugs.The comparative analysis shows that the natural products obtained from marine sources are more chemically diverse than land-based natural products.In this subject,an active strain was isolated from the intestine of streaked bamboo shark and named as GFP-2.The antibacterial activity of this strain was preliminary investigated.At the same time,the efficacy of the corresponding GFP-2 bacterial preparation in the oncorhynchus mykiss breeding was studied.The results of antibacterial experiments showed that the GFP-2strain had a good inhibitory effect on Gram-positive bacteria and Gram-negative bacteria.The 16S rRNA gene of Bacillus sp.GFP-2 was detected and compared in NCBI database,indicating that Bacillus sp.GFP-2 is a new strain of B.velezensis.Bacillus sp.GFP-2 has been deposited with the CGMCC.The number is CGMCC NO.13337.The State Intellectual Property Office also accepted the isolation method of the strain and its application,the patent application number is 201710044718.4.The crude protein in the extracellular metabolite of Bacillus sp.GFP-2 was extracted by ammonium sulfate precipitation.When the ammonium sulfate saturation reached 20%,a large amount of precipitate was found.Resuspend the pellet with 1 mmol/L Na₃PO₄?pH 7.0?buffer to obtain a clear yellow liquid,which is the crude protein in the extracellular metabolites.The results of antibacterial experiment showed that the crude extract had obvious inhibition zone on Bacillus subtilis WB800N and Escherichia coli BL21,indicating that Bacillus sp.GFP-2 inhibits the growth of Gram-positive and Gram-negative bacteria by secreting proteins.Crude protein was separated by ultrafiltration tube with molecular weight of 10 k Da to obtain the small molecule and the macromolecule crude protein samples.The antibacterial experiments were carried out respectively.The results of antibacterial experiment showed that no matter the macromolecules or small crude protein samples both had obvious inhibition zone on Bacillus subtilis WB800N and Escherichia coli BL21.It was speculated that Bacillus sp.GFP-2 might express different sizes of proteins on the indicator strains to the bacteriostatic effect.The genome of Bacillus sp.GFP-2 contains 3711 protein cording sequences and an average GC content of 46.4%on one circular chromosome?3975220 bp?.The genes encode LCI,YFGAP and hGAPDH-like antimicrobial peptides?AMPs?and?-1,3-1,4-glucanase?bgl?.Secondary metabolites such as Difficidin,Bacillibactin,Bacilysin,Surfactin,Butirosin,Macrolactin,Bacillaene,Fengycin and Lanthipeptides can be predicted in the genome.Bacteriocin clusters such as Lanthipeptides and LCI are also predicted.Therefore,we speculated that Bacillus sp.GFP-2 may cooperate with bgl,antimicrobial peptides,secondary metabolites and bacteriocins to complete the antibacterial mechanism.Through bgl identification experiment found that,Crude protein,a metabolite of Bacillus sp.GFP-2,showed obvious hydrolysis circle on the identification medium,indicating that bgl substances are indeed present in this metabolite crude protein.In order to verify the antibacterial activity of bgl,bgl was first cloned,expressed and purified by using prokaryotic expression system to obtain high purity bgl,and then identified by antibacterial experiment.The experimental results showed that bgl had a significant inhibition circle on Bacillus subtilis WB800N and Escherichia coli BL21,indicating that bgl does have inhibitory effects on both Gram-positive and Gram-negative bacteria.In the oncorhynchus mykiss animal experiment with active strain GFP-2,the total weight of oncorhynchus mykiss fed with GFP-2 bacterium preparation was 811 g heavier than that of the control group after 30 experimental cycles,and the feed conversion rate was also 0.9 lower than that of the control group.Compared with the control group,feeding GFP-2 bacterium during the test period can increase the survival rate of oncorhynchus mykiss by 49.2%,which also provides some experimental data for other fish or other animals,which lays a good foundation for the subsequent research and development of new drugs.Therefore,the Bacillus sp.GFP-2 isolated from shark intestine maybe provide a useful antimicrobial tools to replace abused antibiotics in prevention and cure of pathogenic bacteria.