Optimization Of Acetyl Glucosamine Fermentation Conditions From Recombinant Escherichia Coli

N-Acetyl-D-glucosamine is a derivative of glucosamine acetylation,widely used in medicine,food,chemicals and cosmetics industry..At present,the production process is mainly through the hydrolysis of chitin to produce acetyl glucosamine monomer.The process is limited by raw materials,long production cycle,with the rapid growth of market demand and the strengthening of national environmental protection policy,the existing technology needs to be improved.This paper use recombinant E.coli(E.coli-glms-gnal-δnage-δmanxyz::kan)to product acetyl glucosamine by fermentation.The production has short production cycle,not affected by raw material of seasonal,and less environmental pollution.This paper investigated the genetic stability of recombinant Escherichia coli,optimized the fermentation conditions in 50 L fermentation tank including the inoculation,p H,temperature,inducers,and analyzed the reasons for improvement of fermentation titer.(1)The stability of the strains was investigated: The experimental strains had strong genetic stability and could meet the needs of production.(2)Seed age control and Inoculation quantity optimization: Take OD value as reference,choose to be in a few period of inoculation,according to 3%,10% of inoculation quantity to connect m9 a shaker and 50 L ferment tank respectively.(3)The fermentation control process was optimized: the IPTG(Isopropyl thiosulfate)was induced by adding 0.10mmol/l when the fermentation liquid od value reached 28,while the control condition of the induction period was changed,the ph was controlled at 7.2 and the fermentation temperature was controlled at 35 ℃.The results showed that the concentration of glutamic acid in the synthesis of 3.31g/L,reduced by 44.8%;and the activity of-6-phosphate synthase was 67.32u/mg,increased by 29.2%.(4)The glucose flow accelerated rate was regulated by 40% DO feedback,the result showed in the fermentation the glucose was controlled below0.5g/L,the acetic acid concentration was controlled below 0.3g/L,To maintain the normal carbon supply of the bacteria while avoiding the adverse effects of metabolic by-products acetic acid on normal metabolism.(5)Use single factor optimization experiment,the final fermentation control process was10% of the inoculation capacity of the fermentation tank,the IPTG of 0.10mmol/L was added when the fermentation liquid ODreached 28,before the incubation temperature was 37 ℃,p Hwas7.0;after the induction temperature was 35 ℃,p Hwas7.2,the dissolved oxygen content was controlled in 40%.(6)The results of verification experiment on 50 L fermentation tank showed that the yield of glucosamine was 108g/L,and the yield of glucosamine was increased by 28.6%.The average conversion rate reached 48.6% and increased by 17.7%,and the production cost was15.58 yuan/kg,reduced by 23.3%.This fermentation Technology of use recombinant Escherichia coli to product acetyl glucosamine in 50 L tank can be easily controlled,high fermentation titer,low production cost,environmental pollution,provides the basic technology for large-scale industrial production,which will have good economic and social benefits after industrial production.