Study On The Secondary Metabolites Of Actinomycetes Based On HSQC-TOCSY Method

Actinomycete are a kind of extremely important microbial resources.In order to quickly screen strains with research is value,HSQC-TOCSY spectral screening method was established: MeOH was the extract solvent of samples;The NMR solvent is DMSO-d6;Experimental parameters: 8.4 ?s,901 h pulse scanning 16 times;Delay of internal scanning was 0.8 s;Increment 128;The spectral width is16.00 ppm.The f1 spectral width is 170 ppm,and the spin mixing time is 0.06 ?s;By HSQC-TOCSY detection of various structural types of compound samples,the structural regions were summarized,including fatty chain region,polyketone region,amino sugar region,sugar region,olefin region,peptide,alkaloid region,aromatic ring region,and aza-heterocyclic region.Two strains TRM 20105 and TRM 49605 with metabolic potential were screened by combining culture characteristic screening,ultraviolet spectrum screening and HSQC-TOCSY spectrum screening.By analyzing and comparing the colony morphology,culture characteristics and 16 s rDNA sequence of strain TRM 20105,TRM 20105 was preliminarily identified as a potential new species of Nocardia actinomycetes,and the most similar strain was Nocardiopsis dassonvillei subusp dassonvillei DSM 43111(98.34% similarity),and its optimal growth sodium chloride concentration was 3%.TRM 20105 was fermented in liquid state,and the fermentation broth was separated by various chromatographic methods to obtain 7 purified compounds.Through 1D&2D NMR identification,1 is a new diketopiperazine compound,1-demohyocazineA.Six known compounds were 1,6-dihydroxyphenazine(2),2-aminophenazine(3),coixlachryside A(4),neotigenin(5),yamogenin(6)and ?-sitosterol(7).In addition,the antibacterial activity of isolated compounds in vitro was determined by 96-well plate method,and the minimum inhibitory concentration(MIC)of 1against Candida albicans was 3.16 mM.Streptomyces luozhongensis TRM 49605 can produce tunicamycin VII and staurosporine,but under the initial fermentation conditions,the yield is low,so the optimal carbon and nitrogen source for TRM 49605 to produce staurosporine is glucose-yeast element through single factor experiments.The optimal carbon and nitrogen source to produce tunicamycin VII is malt extract powder-acid hydrolysis casein.Taking carbon source,nitrogen source and inoculum as independent variables,according to Box-behnken central composite experimental design,the optimal value of three factors was determined.The results showed that when glucose was 47.6 g/L,yeast extract was 26.5 g/L and inoculation amount was 6.8 %(v/v),the yield of staurosporine was 40.7 mg/L,which was seven times higher than that of the initial fermentation condition(5.7 mg/L).When malt extract powder was 33.9g/L,acid hydrolyzed casein was 23.6 g/L and inoculation amount was 5.3 %(v/v),the yield of tunicamycin VII was 39.4 mg/L,which was 21 times higher than that of the initial fermentation condition(1.9 mg/L).By means of HSQC-TOCHY and HPLC,it was analyzed that TRM 49605 can produce analogues of tunicamycin VII when the carbon source was malt extract powder,and the difference in the fatty chain in the molecular structure.When the carbon source is corn flour,TRM49605 can produce structural analogues of staurosporine.The results of HSQC-TOCCY sugar zone showed that when malt extract was used as carbon source,there were a lot of amino sugars(? c50,52,55,58 ppm)in the fermentation products,and strain TRM 49605 had the potential to produce aminoglycosides,which provided a guidance for the further discovery of secondary metabolites.