Study On Optimization And Metabolic Analusis Of γ-polyglutamic Acid Synthesis

γ-poly-glutamic acid(γ-PGA)is a mutil-role and biodegradable bio-polymers.It has a wide range of application in medicine,agricultural,environmental protection,food,cosmetic industries and other fields.However,only a few companies could produce y-PGA in small scale all over the world,and the cost for producing y-PGA is so high.In this study,using existing laboratory y-PGA producing strain Bacillus licheniformis as the research object by replacing the inorganic nitrogen source,the fermentation medium were optimized by using shake flask and process parameters of 30L fementation tank.And the pilot scale of 200L femetor were exceuted.We analysed the metabolic changes after adding Icucinc by using GC-MS and protein mass spectrometry.And what we got from the conclusion is finally used in industrial production.Main results of this thesis are as follows:(1)Single factor experiment was used to select inorganic nitrogen source which could replace ammonium nitrate and finally the ammonium chloride was determined as a substitute object.We optimized the content of other media components after substitution.The result showed that when yeast extract was treated as the single nitrogen source、the highest concentration was 40 g/L,and the yield could be 27.5 g/L.The yield of yeast extract and ammonium chloride in the case of 1:1 compound was 26.6 g/L.When the ammoniurn chloride concentration was 6 g/L.the yield was 29.2 g/L.(2)Plackett-Burman experiments were carried out by shaker fermentation.The fermentation medium was optimized by 4-factor 3 level experiment with y-PGA yield as the response value.When the concentration of ammonium chloride was 6 g/L,the yield was 29.2 g/L.The yield of y-PGA was increased greatly,and the yield in 72 h y-PGA was 28.15 g/L.(3)Optimization of Fermentation carried out in 30 L fermentor with parameter of agitation and aeration rate.The range of agitater speed is 300 r/min,400 r/min,500 r/min,600 r/min and 700 r/min.And rate of aeration change was 1.0 vvm,1.5 vvm and 2.0 vvm.Results showed that two factors had profound impact on y-PGA production.After analysis of those femetation results,the concentration of y-PGA reached to 33.2 g/L under optimal condition which was agitater speed of 500 r/min and volume of 1.5vvm aeration in diferent phase depending on the cell growth.The pilot scale utilizing 200L fermentor was tried and the yield of y-PGA was finally 28.6 g/L.(4)In this paper,we investigated the changes of metabolic processes of Bacillus licheniformis after adding leucine by GC-MS and protein mass spectrometry.The accumulation of oxaloacetic acid,succinic acid and fumaric acid in the intermediate metabolites was significantly higher than that in the control group by GC-MS.Biosynthesis and degradation of the ornithine cycle,leucine,valine and isoleucine were observed to varying degrees,which could promoting the TCA cycle and providing more prerequisites for the synthesis of polyglutamic acid.Those theory may provides a basis for future strain improvement and fermentation optimization.(5)Industrial production found that after adding leucine,the fermentation cycle has been shortened,which could save costs.