Antimicrobial Activity And Antimicrobial Components From Fermentation Broth Of Streptomyces Djakartensis NW35

Acyinomyces is one of the most important microorganism resources, which can produceantibiotics and other bioactive substances, the discovery of novel bioactive compounds isbecoming difficult with a large number of bioactive strain have been isolated by traditionaltactics. Exploration of rare actinomycete has become an important direction to discover newactive substance.This paper is about the research work on the actinomycete strain NW35, which wasisolated from a pesticide-polluted soil, including strain identification, antimicrobial activity offermentation broth, purification and structural identification, mechanism and optimizationfermentation conditions. The primary results are as follows:1. According to the results of morphological and culture characteristics, physiology andbiochemical measurement and16S rDNA sequence analysis, NW35strain was highlyhomological（up to99%） with Streptomyces djakartensis NBRC15409, except for culturecharacteristics on glucose asparagines agar and potato, gelatin liquefaction and starchhydrolysis properties. The NW35strain was identified and designated as Streptomycesdjakartensis NW35. The strain is deposited in the center of the China GeneralMicrobiological Culture Collection Center, November13,2012, with the registration numberof CGMCC No.6817.2. The antimicrobial activity of fermentation broth from strain NW35was tested.Bioassay results in vitro showed that the fermentation broth exhibited different antimicrobialactivity against9species of tested bacteria and8species of tested plant pathogens. Thefermentation broth had strong inhibition against Bacillus cereus, Bacillus subtilis,Staphylococcus aureus, methicillin-resistant Staphylococcus aureus with transparentinhibition zone, and inhibition against Escherichia coil, Pseudomonas aeruginosa, Erwiniacarotovora, Pseudomonas syringae pv.actinidiae, Ralstonia solanacearum with clearinhibition zone. The fermentation broth possessed good inhibition against Sclerotiniasclerotiorum（73.68%） and certain degree inhibition against the other tested plant pathogens,The protective efficacy and curative efficacy of the fermentation broth against Blumeria giamiais were41.89%and38.41%, respectively.3.Four compounds have been separated through bioassay guided fraction, includingenrichment by HPD100macroporous resin, and separation on silica gel column chroma-tography and preparation reversed-phase high performance liquid chromatography. Accordingto spectroscopy datas, the separated four compounds have been elucidated as:（E）-2-methoxy1-4-naphthoquinone1-oxime（Z-4-1）,2-（hydroxymethyl）-2,3-dihydrobenzo[e][1,4]oxazepin-5-（1H）-one（Z-4-2）,2-（2-Hydroxy-1-hydroxymethyl-ethylamino）-benzoic acid （Z-8-2） arenovel compounds. N-Acetyl-tryptamine （Z-9-2） are known compounds. Compound Z-4-2isthe main bacteriostatic active ingredients of the fermentation broth, which is a new antibiotic,and named as Xinongmycin.According to microdilution method, the bioassay results in vitro showed that Xinongmycinexhibited antibacterial activity against Pseudomonas syringae pv.actinidiae with MIC valuesof7.8μg/mL, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, methicillin-resistantStaphylococcus aureus, Ralstonia solanacearum with MIC values of15.6μg/mL, Escherichiacoil, Pseudomonas aeruginosa, Erwinia carotovora with MIC values of31.3μg/mL. Sporegermination demonstrated Xinongmycin showed strong inhibition against Fusariumoxysporum f.sp.vasinfectum, Curvularia lunata, Colletotrichum orbiculare, Colletotrichumgloesporioides, with the inhibition rate of above90%at the concentration of500μg/mL.Z-4-1，Z-9-2also showed certain degree antibiotic activity against Bacillus cereus, Bacillussubtilis, Erwinia carotovora, Staphylococcus aureus, Escherichia coil, Pseudomonasaeruginosa, methicillin-resistant Staphylococcus aureus, Pseudomonas syringae pv. actinidiae,Ralstonia solanacearum.4. The preliminary mechanism of Xinongmycin against Bacillus cereus was investigated.When the bacteria was treated with Xinongmycin at MIC with different time, the releasementof reducing sugar, alanine aminotransferase（ALT）, and aspartate aminotransferase （AST）were detected and the relative permeability of cell membrane was higher than control.Together with scanning electron microscopy, further detection of the decrement, in activitiesof of succinate dehydrogenase （SDH） and malate dehydrogenase （MDH） demonstratedXinongmycin might damage the cell membrane, enhance the cell membrane relativepermeability, and inhibit protein synthesis leading to cell metabolism disorder and cell deathfinally.5. The optimized fermentation for strain NW35was obtained by response surfacemethodology and the orthogonal experiment design. The best formula is: sucrose10g/L,lactose,10g/L, maltose,10g/L, wheat bran,27.68g/L, peptone4.5g/L, NH₄Cl3g/L and （NH₄）₂SO₄3g/L, CaCO₃,0.87g/L, FeSO40.0073g/L. Best fermentation condition:60mLmedium in250mL flask,6×10⁷cfu/mL bacteria amount,28℃,144h. In the optimal culturemedium and fermentation conditions, the content of Xinongmycin（12.86μg/mL） is8.03timesof the original content（1.60μg/mL）. The influence of feeding precursor on the production ofXinongmycin showed that neither single precursor nor composite precursor can significantlyimprove the content of Xinongmycin.