Production Of Coenzyme A With Fermentation And Immobilization Of Brevibacterium Ammoniagenes

Coenzyme A(CoA),existing widely in animals,plants and microbes,takes part in acetylating reaction in organisms as a coenzyme,and plays an important role in the metabolism of carbohydrates,proteins and lipids.Present method for CoA production is fermentation with Brevibacterium ammoniagenes(B.ammoniagenes).Main factors which influence the yield of CoA are strain,culture medium and cultivation conditions.Because of the low yield of CoA in our country,we focused on strain and culture medium to increase fermentation level of CoA.Furthermore,we studied fundamentally on immobilization strain for CoA production.Prospective goals are all accomplished.1 Study on the fermentation process of B.ammoniagenes for CoA productionBy isolating and purifying initial strain,one stain with high yield was obtained as seed strain.Fermenting with this strain in basic medium,the yield of CoA reached 273 U/mL,which was 50%higher than that with initial strain(181 U/mL).By plotting growth curve,quality indexes of seed strain on subcultivation were determined.The optimal condition of seed cultivation was as follow:pH 6.4～6.6, strain density A 0.6～0.8,and seed age 18～20 h.By comparing different inoculation volume of seed,optimal inoculation volume was determined,that is 10%～12.5%(v/v).By trying different sterilization methods,best method was determined,that is 121℃15min for glucose+peptone+yeast powder+magnesium sulfate,100℃5min for phosphate+urea,cooling separately to 30～32℃and jointing them together.By this method,the color of the culture solution after sterilization is light,fermentation period is shorter and the yield of CoA is high.By combination experiment of organic nitrogen sources,best combination was peptone and com steep powder,which can shorten fermentation period and increase production of CoA.By serial experiments of components and concentrations of culture medium,best formula was:glucose 10%,peptone 1.2%,corn steep powder 0.8%,magnesium sulfate 1%,KH₂PO₄ 1%,K₂HPO₄ 1%,urea 0.7%.The yield of CoA with this medium reached 360 U/mL in flask,which was 30%higher than that with basic culture medium(273 U/mL).Addition of precursor was studied,which showed that addition in two times was better than in one time.Add 3/4 of total precursor for the first time,and 6h later,add the remaining 1/4.The yield of CoA reached 420 U/mL,which was 12%higher than that of addition in one time(372 U/mL)In experiment in 10 L fermentor,the yield of CoA reached 527 U/mL,which was 25%higher than that in flasks(420 U/mL).2 Study on the immobilization of B.ammoniagenes and its production of CoABy systematic investigation of performance of gel and CoA synthesis ability,the optimal carrier was screened for preparation of immobilization of B.ammoniagenes, which was the combination of carrageenan,konjak gum,and locust bean gum.By optimizing gel formula,best formula was determined,that was carrageenan 1.0%,konjak gum 0.4%,and robinia pseudoacacia gum 0.4%.By optimizing precursors formula,best formula was determined,that was ATP 2 %,calcium pantothenate 0.3%,cysteine 0.3%.In neutral or acid culture solution,immobilized B.ammoniagenes had stronger CoA synthesis ability.While the yield of CoA was less in alkaline culture solution.Addition of benzalkonium bromide to culture solution was better for CoA production,and the optimal concentration was 0.05%.By optimization of gel formula and precursor formula,the yield of CoA reached 360 U/mL.After used for 8 times,the yield of CoA was still more than 50%of that of initial immobilized cell.3 Outcome of the studyBy isolating and purifying strain of B.ammoniagenes,controlling quality indexes of strain,and choosing optimal culture medium and addition mode of precursor,the yield of CoA in flask reached 420 U/ml,which was 130%higher than that of initial strain in basic culture medium in flask(181 U/mL).In experiment in 10 L fermentor,the yield of CoA reached 527 U/ml,which was 25%higher than that in flasks(420 U/mL).Immobilized B.ammoniagenes cells were prepared with the best carrier combination of carrageenan,konjak gum and locust bean gum and the optimal synthesis condition of CoA was determined.Under the optimal condition,the yield of CoA by immobilized B.ammoniagenes cells reached 360 U/mL.