| Due to the unique environmental qualities such as high-pressure, high-salt, low-nutrient and low-temperature that the ocean has, the marine microorganisms have special physiological mechanismsand genotypes, and also possess great potential to produce new bioactive substances. The bioactive substances produced by the marine microorganisms include new antbiotics, antitumour agents, unsaturated fatty acids, enzymes, enzyme inhibitors, vitamins, toxins etc. Searching for new bioactive substances from marine microorganisms is becoming an important research field in many countries, but still in its infancy in our country.The strain GY-4 is a novel and highly antimicrobial strain which was isolated from coastal in Lianyungang. The aim of this research was to identify the strain, test the stability and physicochemical properties of the antibacterium substances produced by strain GY-4. At the same time purification and structural identification were also done. The main research findings are as follows:1.The strain GY-4 was identified with classical and molecular methods. According to the morphological and cultural characteristics, the physiological and biochemical properties, it was indicated that strain GY-4 was belong to Streptomyces. In the 16SrRNA sepuence analysis, the sequence fragment of 1396 bp 16Sribosomal RNA was amplified from total DNA of strain GY-4 by PCR, then the sequence analysis was down by the application of BLAST on the websites of NCBI and the phylogenetic tree was drawn by Bioedit 7.0T and TreedrawingT. The results showed that GY-4 shared 99% 16SrRNA homology with Streptomyces griseorubens. After comparing with the characteristics of Streptomyces griseorubens reported, strain GY-4 was identified as a varation species of Streptomyces griseorubens.2. Testing the broad-spectrum activities of strain GY-4 indicated that it displayed significant inhibitory effects not only on Staphylococcus aureus, Pseudomouas fluorescens, Micrococcus luteus, but also on Penicillium notatum, Penicillium expansum. Influence of different water of medium on the production of antimicrobial substances was examined. And the results showed the high production of antimicrobial substance was significantly associated with aritificial seawater, which revealed its marine speciality. The results of stability studies on the fermentation broth of strain GY-4 showed that it was very stable at acidiccondition, but became unstabile as pH ascended. The antimicrobial activity decreased after 60℃treatment for 30 minutes but had little change after ultraviolet treatment, which ruled out the possibility of polyene antibiotic. Solubility study showed that the antimicrobial substances were diffluent in methanol, alcohol and water, but insoluble in other organic solvents.3. A broad-spectrum antibacteria substance was separated and purified from strain GY-4 fermentation broth, organic extraction, vacuum concentration, silica gel chromatography and preparative high performance liquid chromatography(HPLC) were used. The substance was yellowish-brown in colour and its maximum absorption peak at UV spectrophotometer wavelength was 206nm. LC-MS showed that its molecular weight was 267.1. The results of IR indicated that-OH,-CH2,-CO-NH- were found. And the NMR indicated that the substance should be classified as a small peptide which contain-CH2-CH-CO-NH-CH-CH2-. Through the full scan MS/MS of the antibacteria substance, ion fragment peaks were observed at m/z250.1,232.1,226.1,208.1,191.1,180.1,148.1. 130.1. After comparing relevant literature and those results above, the molecular structure of the antibacteria substance was preliminarily determined as C13H17NO5. |
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