Selective Separation And Identification Of Micromonospora And Mining Of Their Secondary Metabolites

Actinomycetes are the main source of microbial antibiotics,among which Streptomyces are the most common.With the emergence of drug-resistant strains,people's demand for antibiotics is increasing,and it is increasingly difficult to mine new antibiotics from Streptomyces,so people focus their attention on rare actinomycetes,especially Micromonospora.This article was took the red willow forest silt in the Tarim Basin in Xinjiang as a sample,we have established a pretreatment method for adding amikacin to the soil for original culture,at the same time,traditional pretreatment methods of phenol and dry heat treatment were also used.We used P₃ and HV two medias for selective separation of Micromonospora,16S rRNA identification after pure culture.A total of 59 strains of actinomycetes were isolated,42 strains of Micromonospora,71%of the total.We used the"strain confrontation method"for the primary screening of the isolated micromonospora,the indicated strains were Escherichia coli ATCC 25922,Staphylocccus aureus ATCC 6538 and Candida albicans ATCC 64550,the results showed that a total of 11 strains of Micromonospora had antagonistic activity against the three indicator strains,and they were all separated from the soil sample with amikacin added.Then,we fermented 11 active Micromonospora,fermentation products using"filter paper sheet method"for the active rescreening of three indicator strains,the results showed that the fermentation extracts of 11strains of Micromonospora had antagonistic activity against Staphylococcus aureus,so we selected these 11 active Micromonospora for follow-up research.We fermented 11 active Micromonospores with Gaoshi No.1,screening by culture characteristics,combined method of HPLC screening and HSQC-TOCSY spectroscopic screening,screened 1 strain with large growth,UV absorption had a conjugated system,in-depth study of strain TRM 66019A which was rich in metabolites.We fermented Micromonospora TRM 66019A with four kings of fermentation medium respectively,through the color of the fermentation broth,mycelium density observation and TLC,HPLC detection,screen out the dark color of the fermentation broth,fermentation medium No.2with dense mycelium and abundant metabolites for subsequent large-scale fermentation.We used No.2 fermentation medium for TRM 66019A mass fermentation,adsorption of fermentation products by macroporous resin,silica gel column chromatography,separation and purification by gel column chromatography and efficient preparation of liquid phase.Structural was analysised by¹H NMR,¹³C NMR,HSQC and HMBC spectral data,we isolated four compounds.They were daizein,geldanamycin,17-O-desmethylgeldanamycin and Cyclic?leucine-isoleucine?dipeptide.