Genome Shulffling to Improve Antibacterial Substances Active of the Marine Streptomyces
|School||Nanjing Agricultural College|
|Keywords||marine Streptomyces genome shuffling inactivation protoplast fusion fermentation optimization|
The ocean bears a large number of microbial resources, which are widely distributed strains produce antimicrobial active substances, is an important potential source of antibacterial substances. Streptomyces sp. GB-2is a marine microbe with broadspectrum antimicrobial activity, isolated from soil taken from the coastal city Lianyungang in the JiangSu province of China. This bacteria have significant antagonism on five kinds of Gram-positive bacteria, four kinds of Gram-negative bacteria and18species of fungi. This indicates that it have potential applications in biocontrol, food and medicine. But the yield of wild strain is relatively low, can not neet industrialization requirements. The fermentation production of Streptomyces sp. GB-2has been greatly improved which induced by chemical mutagenesis, antibiotic mutagenesis and Ion Beam Implantation. Therefore, this strain has potential applications in industry.In order to further improve the comprehensive characters of the marine Streptomyces. In this paper, interspecific fusions of inactivated protoplasts of both parents Streptomyces FMBL-43and FMBL-42which handled with different mutation methods were studied. And also, study on the optimization of fermentation conditions for stable and high yield fusant PF-1. Achievements obtained primarily as follows:1. The effect of different media as well as concentration of GLY and sucrose on the mycelium was studied. The result indicated that in YEME medium the two parents grow rapidly and produce a high yield of mycelium, but in S medium are lower. The GLY concentration in mediun of FMBL-43and FMBL-42were0.5%,10%. Then the mycelium was produced appropriate amount and more sensitive. The sucrose concentration in medium of FMBL-43and FMBL-42were20%,20%. Then the amount and the loose of medium is the best.To unravel effects of different enzymes,enzyme concentration and enzymatic hydrolysis time on protoplast formation, the number of protoplast regenration and regeneration rate was measured. The result revealed that Lysozyme appeared to be more sutiable than Helicase for protoplast formation. And the suitable lysozyme concentrations of Streptomyces FMBL-43and Streptomyces FMBL-42were3mg/mL and4mg/mL. The results also show that when the hydrolysis time of Streptomyces FMBL-43and Streptomyces FMBL-42was2.Oh and1.5h, the number of the regeneration and the regeneration rate was the highest. The conditions of heat-inactived and UV-inactived were sdudied. The result illustrated that the protoplast of Streptomyces FMBL-43and Streptomyces FMBL-42were inactived by heating5min at100℃in water bath and ultraviolet irradiation180min (15W,10cm)Through screening and rescreening from the fusants, nine strains with high productivity of antibacterial substances were got by liquid fermentation. The nine strain were identified as Streptomyces PF-1PF-2PF-3PF-4PF-5PF-6, the PF-7PF-8, PF9. The stability test of the fusants from generation to generation was studied. Results showed that the fermentation yield of PF-1, PF-2, PF-4, PF-5, PF-6was stable, whose mutation was not restored apparently after ten generations. And the highest fermentation yield of strain is Streptomyces PF-1, its fermentation yield increased65.84%than parental Streptomyces FMBL-43.2. The fermentation conditions were further optimized. Screening of different carbon sources, The results showed that wheat flour and malt eatract powder as a suitable source of carbon. Screening of slowly available nitrogen and available nitrogen, the results show that the high-temperature crushing of soybean meal and peptone as nitrogen source. By screening species of the inorganic salt showed that, MgSO4/CaCO3is appropriate inorganic ions.Single factor experiments, the medium components, the The results showed that The wheat powder selected20g/L-40g/L to FFD for high-and low-level range. Malt extract to select2.5g/L-5g/L to FFD for high-and low-level range. Soybean meal, select20g/L-40g/L to FFD for high-and low-level range. The protein peptone selected1.25g/L-2.5g/L to FFD for high-and low-level range. Inorganic ions, including the appropriate range of CaCO3, MgSO4·7H2O, FeSO4·7H2O and CoCl2were3g/L-6g/L,24g/L-48g/L,7.5×10-4g/L-15×10-4g/L,0.75×103g/L-6×10-3g/L.The results of Fractional factorial design showed that significant factors in Streptomyces sp. PF-1medium are soybean meal, wheat flour, and MgSO4. Using the method of steepest ascent, The results showed that soybean meal, wheat flour and MgSO4·7H2O sulfate concentrations were35g/L,35g/L,30g/L, then the fermentation yield was the highestMedium for fermentation was optimized by central composite design. The results showed that the finally optimal medium condition was determined such as soybean meal32.92g/L, wheat flour36.35g/L, peptone2.5g/L, malt eatract powder2.5g/L, CaCO36g/L, MgSO4·7H2O30.81g/L, FeSO4·7H2O7.5×10-4g/L, CoCl23.75×10-3g/L. Verification test show that the fermentation yield of Streptomyces PF-1is7893.37U/mL under this conditions which was increased by16.72%over the original fermentation condition.