| Microorganism is one of the focus sources in the screening for natural lead compounds. Actinomyces are the important producing strains that maybe produce new bioactive secondary metabolites.With the discovery of a large number of common streptomyces from natural environment, the probability of screening news strains or new structural antibiotics has become smaller and smaller following the traditional isolation modle, so the researchers have taken steps to modify the methods of isolation and culture or to collect samples from special circumstances[0] to improve the capacity of screening new bioactive substances, the significant achievement has been obtained.This paper is about the research work based on one actinomycete strains No.313, which was screened through modified methods of isolation and culture, including strain identification, purification of the main active metabolite, structural identification, and evaluation of antimicrobial activity and optimization of separation processes. The primary results are as follows:(1) According to the results of morphological characteristics, physiological and biochemical measurement and analysis of 16S rDNA sequence, No.313 strain was highly homological (99.5%) with the type strain Streptomyces alboflavus NBRC-13196T, and both were similar features in majority except the milk coagulation property. Therefore, the No.313 strain was identified and designated as Streptomyces alboflavus 313.(2) Guided by the results of the bioassay and through enrichment of HPD400 macroporous resin, silica gel column chromatography and preparation of reversed-phase High Performance Liquid Chromatography, eight active compounds have been separated, four of them were cyclic hexadepsipeptide antibiotics named as NW-G01, NW-G03, NW-G06 and NW-G08. According to analysis of IR, HR-MS, 1D-NMR and 2D-NMR spectroscopy datas, the molecular structure of three compounds have been elucidated. Compound NW-G01 was comprised of Valine, three piperazine-3-carboxylic acid, 2-(methylamino)propanoic acid and 6-chlorl-3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid, the stereoscopic configuration of NW-G01 was determined by X-ray crystallographic analysis. NW-G03 was constited of Valine, two piperazine-3-carboxylic acid, 2,3,4,5-tetrahydro- pyridazine-3-carboxylic acid, 2-(methylamino)propanoic acid and 6-chlorl-3a-hydroxy- 1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid. NW-G06 was made of Valine, two piperazine-3-carboxylic acid, 4-hydroxy-6-methoxy-1,2,3,4-tetrahydropyridazine -3-carboxylic acid, 2-(methylamino)propanoic acid and 6-chlorl-3a-hydroxy-1,2,3,3a,8,8a -hexahydropyrrolo[2,3-b]indole-2-carboxylic acid.(3) Bioassay results in vitro showed that NW-G01 exhibited strong inhibitory effect on mycelial growth against Botrytis cinerea, Coniothyrium diplodiella and Sclerotinia sclerotiorum, the values of EC50 were 24.91,11.90 and 4.01μg?mL-1, respectively. It also had inhibitory effect on spore germination against Exserohlum turcium and Botrytis cinerea, the values of EC50 were 63.52 and 29.44μg?mL-1, respectively. Besides, compound NW-G01 showed strong antibacterial activity against Bacillus cereus, Bacillus subtilis, Staphylococcus aureus and methicillin-resistant Staphylococcus aureus(MRSA), with MIC values of 3.90, 3.90, 7.81 and 7.81μg?mL-1 (the MICs of ampicillin control were 25,12.5,25 and more than 100μg?mL-1), respectively. The MICs of compound NW-G03 against the four bacteria above were 12.25, 25, 3.07 and 6.13μg?mL-1 and those of NW-G06 were 50, 50, 6.13 and 12.25μg?mL-1, respectively. ( the MICs of ampicillin control were 50, 50, 6.13 and more than 100μg?mL-1). The MICs of three cyclopeptides against Escherichia coli and Pseudomonas aeruginosa were more than 250μg?mL-1.(4) A reversed-phase HPLC method for analyzing three cyclic hexadepsipeptide compounds was established. (Chromatographic parameter: Sinochrom ODS-BP column (5μm, 4.6mm×200mm), detection wavelength of 210 nm, the mobile phase of methanol:water (70:30, v/v), flowing rate of 1.00mL?min-1, injection volume of 10μL). The developed method was validated in terms of linearity, precision and accuracy. The linear regression data showed a good linear relationship for NW-G01, NW-G03 and NW-G06 over a concentration range of 0.3～10μg·mL-1. It showed a good precision that the RSD of NW-G01, NW-G03 and NW-G06 were 0.09%, 1.38% and 1.24%, respectively. When adding 10μg?mL-1, the recovery rates of NW-G01, NW-G03 and NW-G06 were 98.9～100.8%, 97.9～101.6% and 98.1～102.6%, respectively. When adding 1.0μg?mL-1, the recovery rates were 99.6～101.5%, 97.7～101.4% and 98.1～103.6%, respectively.(5) The studies on optimization of macroporous adsorption resin separation process were carried out. From six kinds of different models of HPD Series, HPD400 was selected as the adsorbent resin, the maximum dynamic adsorption capacity was 4300mL original fermentation broth (20mL wet resin volume). After optimization of elution conditions, use 40% methanol to elute firstly to clear most impurities and pigment and then with 100% methanol so that the active ingredient would be eluted down. From preliminary studies on the adding resin[0] into fermentation process, it was found that not only a large number of impurities were removed, but also the output of NW-G03 and NW-G06 were improved while adding 1% HPD400 resin in the initial fermentation period. Moreover,two substances, not been found from fermentation broth without adding resin, were obviously found from methanol eluate.
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