Screening Of Active Marine Myxobacteria And Characterization Of Strain HW-1 Secondary Metabolites

Myxobacteria are unique gram-negative bacteria and are common in many types of terrestrialhabitats including soil, vegetarian dung, decayed plant materials, and bark of living or dead trees.Myxobacteria, one of the most important nature product producers, can produce lots of novel natural products with potent biological activities because of their potential to produce a variety of metabolites which have novel structures. So myxobacteria are recognized as the wonderful resource in research and development of microorganism medicament.In the present study, firstly, we evaluated the antibacterial and antifungul activities of 15 myxobacteria strains sending by the Laboratory of Myxo and Pharm Microbio Technology using Pyriularia oryzae , Escherichia coli, Bacillus subtilis, Cladosporium cucumerinum, Saccharomyces cerevisia. The results showed that 12 strains can inhibit the growth of Pyriularia oryzae.The MAD(Maxium activity dilution) of 8#, 12# and 15# is 256. Combining the results of antibacterial, the 12# was decided to be the object of our next study.The strain 12# was isolated from the seawater of the marine coast in the Huang China Sea, Qingdao, Shandong province, which was salt-tolerant myxobacteria, can grow under the condition of seawater (about 3.8% salt concentration) or distilled seawater. It was recognized as Mx.fulvus HW-1 according to the identification of the strain.A total of 20 compounds were isolated from the strain HW-1 by thin layer chromatography, column chromatography on silica gel, ODS-C₁₈ and Sephardex LH-20, followed by reverse-phase high performance liquid chromatography. The structures of 9 compounds were mainly elucidated by using spectroscopic methods (¹H-NMR, ¹³C-NMR, EI-MS/ESI).They are compound 1:cyclo(L-Ala-L-Phe),2:cyclo (L-Ala-L-Val),3:cyclo(L-Ala-L-Leu),4:cyclo-8-hydroxy-(Pro-Phe),5:4-(3-aminohexan -2-ylamino)benzamide,6:uridine,7:3-(pentan-2-yl)piperazine-2,5-dione,8:3-(hexan-2- yl)piperazine-2,5-dione,9:3-(4-methylpentyl)piperazine-2,5-dione。 Among the 9 compounds,7 compounds are cyclodipeptides. But compounds 7, 8 and 9 are not composed of natural amine acid and firstly reported of natual products. And compounds 4, 7, 8 and 9 are firstly reported as the secondary metabolites of Myxobacteria. Fouthermore, there are several active compounds, and their structures are under elucidation at present, beacause of large molecular weight, small sample weight,unclearly signals,etc.By using Pyricularia oryzae model and paper diffusing method, we found that compound 4, 5, 7, 8 and 9 could strongly inhibit the growth of Pyriularia oryzae. Especially, compound 4 could not only inhibit the hyphal growth of Pyriularia oryzae, but could make the mycelia distortion and the MAD was 64. Besides the effect to Pyriularia oryzae, compounds 4, 5, 7, 8 and 9 also had antibacterial activity to Bacillus subtilis, the inhibition zone diameter on plate were 20 mm,15 mm,19 mm,18 mm,16 mm.