Isolation,Purification And Biological Activity Of A Kind Of Antitumor Protein From Antarctic Marine Bacillus

With the development of society,cancer has already seriously threatened human health.The biodiversity of the marine environment constitutes a practically unlimited resource of new antitumor drugs in the field of the development of marine bioactive substances.In this study,the strain N6-2 was isolated from a marine surface water sample collected from Antarctic sea by MTT methods.The 16 S rDNA sequences indicated that strain N6-2 belonged to the genus Bacillus,which was further proved by the following characteristics.According to the tracking of antitumor activity,we developed a purification protocol involving ultrafiltration,ammonium sulfate precipitation anion-exchange and cation-exchange chromatography.A protein with native cytotoxicity activity was isolated and named NCBG(a cytotoxicity ?-glucanase from Bacillus sp.N6-2).The moleculer weight was about 24.26 kDa according to ESI-TOF analysis.The gene of NCBG was cloned.Bioinformatic analysis showed that NCBG gene encoded 242 amino acids,the signal peptide were 1-28 amino acids,and had three potential glycosylation sites NCTW,NVSM,NRSV.The calculated molecular weight was 24.28 kDa.Theoretical pI of the NCBG was 6.21.The N-terminal sequences of the NCBG was QTGGS FYEPF NNYNT.The Mascot search results and the N-terminal amino acid sequences analysis showed that protein NCBG was a ?-glucanase.The optimal pH and temperature of the NCBG were 6.0,40?,respectively.NCBG maintained considerable ?-glucanse activities and stabilities at wide temperature and pH ranges.Further,according to the result of substrate specificity and the cDNA sequences,it could be inferred that theenzyme was ?-1,3-1,4-glucanase.N6-2 also exhibited the highest cytotoxicity to a variety of cancer cells.The IC50 values of the NCBG were 33.93,34.53,91.14,20.68,8.93,158.45 ?g/mL for BEL-7402,HepG2,panc-28,NCI-H460,A549 and L-02 cells,respectively.Also,NCBG could inhibit the migration and invasion of A549 cells.Overall,NCBG has the potential for new anticancer drugs development.