Screening Of Antibacterial Marine Actinomycetes And Separation And Structural Analysis Of Their Secondary Metabolites

Actinomycetes are potential sources of bioactive natural products and play an important role in microbial drug development.Marine actinomycetes have different metabolic pathways from terrestrial actinomycetes due to their special growth environment.They can produce natural products with biological activities,and they are important producers of natural medicinal compounds.It is important to find bioactive substances from secondary metabolites of actinomycetes from different marine environments.This paper reveals the distribution,antibacterial potential and diversity of actinomycetes in the Rizhao coastline by screening actinomycetes in marine sediments of Rizhao coastline.Conclusion as below:(1)A total of 208 strains were obtained using five different media and five different pretreatment methods.The Oxford cup method was used to detect antimicrobial activity.Among them,91 isolates exhibited inhibitory activity against pathogen.35 strains showed inhibitory effect on Aerobacter aerogenes(CGMCC 1.183),25 strains showed inhibitory effect on Proteus(CGMCC 1.491),17 strains showed inhibitory effect on Staphylococcus aureus(ATCC25923),9 strains showed inhibitory effect on Escherichia coli(ATCC25922),2 strains showed inhibitory effect on Pseudomonas aeruginosa(CGMCC 1.15148),3 strains showed inhibitory effect on Aspergillus flavus(CGMCC 3.3950).Moreover,the 16 S rRNA gene of the 25 representative isolates with with high antibacterial activity was amplified by PCR and then sequenced.Results indicated that the 25 isolates could be sorted into 22 phylotypes belonging to three different genera as follows: Streptomyces(18),Nocardiopsis(4),Saccharomonospora(3).(2)The optimal fermentation conditions were determined by single factor experiments: the number of culture days was 11 days,the inoculation was 8%,the culture temperature was 30 ?,and the initial pH of the liquid medium was 8.The inhibition zone of Staphylococcus aureus,Aerobacter aerogenes and Proteus were improved 8 mm,12 mm and 7 mm respectively.The properties of the antibacterial active substances produced by Streptomyces griseorubens SSGRZ-F8 were studied,and the antibacterial active substances were determined to be substances with good pH stability,thermal stability,light stability and genetic stability.(3)Five compounds were isolated from the organic phase of the fermentation by using Sephadex LH-20 column chromatography,MPLC chromatography and HPLC chromatography.Five compounds were identified as: Compound 1 [cyclo(L-Val-L-Pro)],Compound 2 [cyclo(L-Pro-L-Leu)],Compound 3 [cyclo(L-Pro-L-Tyr)],Compound 4(N-acetyltyramine),Compound 5 [cyclo(L-Pro-L-Phe)].The complex Z3 was obtained from the aqueous phase of the fermentation by using 732 cation exchange resin column chromatography,AB-8 macroporous resin column chromatography and Sephadex G-25 column chromatography.(4)The biological activity of Compound 1-5 and Complex Z3 was determined.The MICs of compound 3 against Staphylococcus aureus,Proteus and Aerobacter aerogenes were 160 ?g/mL,120 ?g/mL,and 100 ?g/mL,respectively.The MICs of compound 5 against Staphylococcus aureus,Proteus and Aerobacter aerogenes were 180 ?g/mL,150 ?g/mL,and 130 ?g/mL,respectively.The MICs of Complex Z3 against Staphylococcus aureus,Proteus and Aerobacter aerogenes were 100 ?g/mL,30 ?g/mL,20 ?g/mL.Compounds 1,2,3 and 5 were also shown to inhibit the invasion and migration of A549 cells(lung cancer cells),and these compounds exhibited potential as novel anticancer drugs.