| Isolated from Streptomyces 702 fermented product extract antifungal active substances for polyene macrolide antibiotics,named as "antifungalmycin 702".The components are significantly inhibited,but the productivity of antifungalmycin 702 is very low.In this paper,by adding different elicitors to improve the synthesis of antifungalmycin 702,that the selection,optimization,preparation methods and the effect of the best elicitor on the cell structure and fermentation process of Streptomyces 702 were studied:(1)Promotion effects on synthesis of antifungalmycin 702 by Streptomyces 702 were conducted by adding abiotic and biological elicitors.The results showed that promotion effect was obvious by adding threonine and sorbitol into Streptomyces 702 cultures at 32 and 64 h,in which the inhibition zone diameter of adding threonine was 30.25%higher than the control.Biological elicitors,Staphylococcus aureus had obvious promotion effect on antifungalmycin 702 synthesis at both 64 and 96 h,especially at 64 h,and Bacillus subtilis had promotion effect at only 64 h.While,Aspergillus niger and Penicillium orange had no effect.E.coli elicitor had promotion effect when adding at 0,32,64 and 96 h,and the best was at 32 h whose inhibition zone diameter was 39.67%higher than the control.The optimum adding concentrations of threonine and E.coli were 50 and 3 ?g/mL,respectively,and the inhibition zone diameters were 25.80%and 51.13%higher than the control.On the basis of single factor test,the time of adding the elicitor,adding the concentration and the culture time were designed by the three factor.The antibacterial activity response value of Streptomyces 702 and the culture conditions was optimized.Verify the consistency between the response surface prediction and the actual value.The results showed that the addition time of Escherichia coli elicitor was 35.75 h,the concentration of reducing sugar was 3.09 ?g/mL,and the incubation time was 146.26 h.The diameter of the inhibition zone was 23.75 mm,and the actual value fine agreement with the predicted value.It is feasible to optimize the culture conditions of antifungalmycin 702.(2)In order to the preparation method screening of high efficiency,through the different physical and chemical methods for broken the E.coli bacteria.There was preparation of the elicitors,and determination of the reducing sugar content in its inducer.The Streptomyces 702 was added the elicitors of sugar content and the final concentration of 3.1 ?g/mL,after the fermentation,measuring its antifungalmycin 702 content.The fractions and the main function of preliminary analysis by the organic solvent extraction separation test.The results showed that the grinding method and the freezing and thawing method combined,the elicitors of sugar content reached a maximum of 1.46 mg/mL.In comparison with the control group was increased by 245.15%.When the lysozyme concentration was 0.8 mg/mL,and holding time was 24 h,whose antifungalmycin 702 content reached a maximum of 380.88 ?g/mL,and its was 39.70%higher than the control.Finally,we were showed that the bacteria elicitors of plays a main inducer inducing activity of polysaccharide,protein precipitation.And some non protein,non polysaccharide,and small molecules have induced effect for the synthesis of the antifungalmycin 702,and its similar polarity and ethyl acetate.(3)The mycelial morphology was observed by light microscope and electron scanning microscope,and the activities of ICDHc dehydrogenase,acetyl coenzyme A carboxylase(ACC)and pyruvate dehydrogenase(PDH)were determined.Gel electrophoresis was used to analyze the variation of the mass spectrum.The results are as follows:compared with the control elicitor concentration was 3.1 ?g/mL,it slow Streptomyces 702 mycelium aging,or ICDHc,ACC,and PDH activity,respectively,1.15,0.17 and 0.75 U,significantly increased.The pretreatment method of sample protein was phenol extraction,which compared with TCA/acetone precipitation method and acetone method,the total protein concentration in control group was increased by 5.61,and 14.12 times,respectively.In the protein molecular weight was 15?99 kDa,phenol extraction hair of protein electrophoresis was 27 bands,acetone method and TCA/acetone precipitation method,electrophoresis having 23 and 15 bands,in which all bands than acetone and TCA/acetone precipitation method was significantly enhanced.(4)The biochemical parameters of Streptomyces 702 fermentation were determined by the addition of E coli inducer into it.That compared with the control,the addition of elicitor concentration was 3.1 ?g/mL promote antifungalmycin 702 synthesis in advance.The stable yield,and the yield of antifungalmycin 702 was 47.18%high than the control at 144 hours of fermentation.To sum up,the effect of E coli promoter on the synthesis of antifungalmycin 702 was best.The results provide an experimental basis for the application of bacterial elicitors.The synthesis of antifungalmycin 702 was further influenced by the change of mycelial morphology,the key enzyme activity of antifungalmycin 702 synthesis pathway,and the metabolic synthesis of Streptomyces 702 extracellular protein and bacterial protein.The mechanism of Escherichia coli elicitor to Streptomyces 702 was studied,and the experimental evidence was provided for the application of bacterial elicitors. |
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