Studies On The Identification Of A Marine Sulfitobacter Sp. M44 And Its Metabolites

In the present study, M44 was isolated from 30 m-deep seawater from the East China Sea (122.8o E,30.9o N). This testing strain is a halophilic microbe and its optimum salt concentration is 2.5%. The evidence from MTT model screening demonstrated that the fermentation broth of this strain powerfully inhibited the growth of HL-60, HepG2 and SW1990. The influence of different temperatures and pH values on the cytotoxic activity of fermentation broth was also tested. The fermentation broth was found to be stable to heating because its cytotoxic activity was not marked changed after treatment at 90℃for 30 min, while the cytotoxic activity of fermentation broth varied with pH value and decreased rapidly at pH 3.0 and pH 11.0 particularly.Based on the genomic DNA extraction, PCR, sequence analysis of the 16S rDNA and neighbor-joining tree construction by blasting in GenBank, strain M44 was found to have the most similarity to Sulfitobacter dubius strain with a homology of 99%. The subsequent analysis of the Gram stain, cultural characteristics, morphology, physiological and biochemical characteristics, fatty acid content and G+C% of genome also indicated that it belongs to the genus of Sulfitobacter.The fermentation conditions have been further examined. The optimum fermentation conditions were as follow: Temperature, 25℃; Initial pH, 7.0; Inoculation dose, 4 %; Containing dose, 50/250 mL; Fermentation time, 7 d.The fermentation was extracted by petroleum, acetic ether and n-butyl alcohol by turns. Guided by MTT test, we found the activity substances were in acetic ether phase. A total of 60 L fermentation broth was centrifuged at 4000 r/min for 10 min, and then the supernatant was completely extracted by equal volume acetic ether for three times. The acetic ether extract was subjected to chromatography on Sephadex LH-20, ODS CB18B reversed-phase column and HPLC for constituents. 25 compounds were isolated, and structure of 6 compounds was elucidated by physicochemical properties and spectral analysis (¹H-NMR,DEPT,¹³C-NMR,ESI-MS,¹H-¹HCOSY,etc.). These structure were identified as follow: Cyclo(Leu-val), Cyclo(Phe-val), Cyclo(Phe-Leu), Cyclo(Leu-Ile), Cyclo(Phe-Ile) and Cyclo(Trp-Pro) . Cyclo(Leu-Ile) showed significant inhibitory activities against HepG2 and Hep2 with the IC₅₀ value of 118μg/mL and 135μg/mL, respectively. But no compound showed antibacterial activity against Escherichia coli, Bacillus subtilis, Staphylococcus aureus and secondary metabolites production of M44. The rest compounds isolated from the fermentation broth of strain M44 showed little activity in these screenings.In summary, a marine strain M44 was identified and six cyclodipeptides was isolated. The present study provided a basis for further exploiting microbial resource of the East China Sea.