Optimization Of Fermentation For Producing Glutamic Acid Decarboxylase(GAD) By The Construction Of Recombinational Bacillus Subtilis

Objectives:Glutamic acid decarboxylase(GAD) was a key enzyme involved in the biosynthesis of y-aminobutyric acid(GABA).Nowadays,the main researches about the production of GAB A by GAD are focus on Lactic acid bacteria-LAB and Escherichia Coli. Lactic acid bacteria fermentation is very safe, but the process and production of fermentation is difficult,production is lower and the cost is high. Escherichia Coli fermentation is easy.production is higher and the cost is lower. However, The E.coli of fermentation could be produce many endotoxin and other chemical substrate. This bacteria existence of security risks.So the two kinds of microorganism are not appropriate for GAB A fermentation. Although, Bacillus subtills 168 is less study on the fermentation for GAB A, Bacillus subtills 168 has be widely used in medicine and food industrial. And Bacillus subtills 168 is a microorganism safety which was approval by U.S.Food and Drug Administration,FDA. In our research, we design a series of experiments for fermenting GAB A. It can provide a basis research and experiment for industrial manufacture.Methods:In this paper, an Bacillus subtilis strain over-expressing GAD was constructed, and a compositional optimization method which aimed to optimize producing GAD was performed. One factor at a time was utilized to evaluate the best carbon and nitrogen sources.Two orthogonal design usal was used to evaluate these factors, sucrose, soybean meal, ammonium acetate, magnesium sulfate heptahydrate is MgSO4.7H2O, potassium dihydrogen phosphate is KH2PO4 and initial is pH, on fermentation production of glutamic acid decarboxylase and then three major relevant factors were obtained, which were ammonium acetate,sucrose, potassium dihydrogen phosphate The composition of the best medium was further optimized which can be based on Box-Behnken Design (BBD), Next is Response Surface Methodology (RSM). Finally, Running Design-Expert8.0.6 software and GAD activity is response value, we can acquire the optimum liquid medium.Conclusions:1.On the basis of constructing the gadB gene segment of E.coli’s GAD,a genetically engineered bacteria is Bacillus subtilis strain over-expressing GAD was constructed successful. We call it Bacillus subtilis 168/pHT01-gadB.2.Formula of fermentation medium is researched by flask fermentation. One time at a time design to get the optimum carbon sources:sucrose, the optimum organic nitrogen source:soybean,the optimum inorganic nitrogen source:ammonium acetate.the basic culture medium are sucrose level, soybean meal, ammonium acetate, magnesium sulfate heptahydrate is MgSO4.7H2O, potassium dihydrogen phosphate is KH2PO4.3.Two orthogonal design usually was used to evaluate these factors, such as sucrose level, soybean meal, ammonium acetate, magnesium sulfate heptahydrate is MgSO4.7H2O, potassium dihydrogen phosphate is KH2PO4 and initial is pH, Finally,on fermentation production of glutamic acid decarboxylase and then three major relevant factors were obtained, which were three factors:Sucrose, potassium dihydrogen phosphate and ammonium acetate.4.Finally, the composition of the best medium was further optimized based on Box-Behnken Design (BBD) and Response Surface Methodology (RSM). Running Design-Expert8.0.6 software and GAD activity is response value, we can acquire the optimum liquid medium.It is sucrose 23.5g/L,soybean meal 10g/L,ammonium acetate 8.5g/L,KH2PO44.1g/L,MgSO4.7H2O 0.5g/L.initial pH7.0 and culture temperature 37℃.GAD activity reached 0.428U/mL under the optimum fermentation conditions compared with before 0.143U/ml. The result of GAD activity increased by approximately 199.3%,a perfected prediction model.5. We can produce GAB A by using whole cell transformation,GAD can catalyze the substrate Glu generate GABA. In 5L fermentation tank,there are 60g fermented bacteria,that is 3% content,2L tap water, total Glu amount is 1189g. After 48h, stop the reaction, the residual volume is 2.7L. Measuring the amount of GABA is 239,91g by HPLC, the percent conversion of Glu is 54.48%.6.1n our research, the result of GAD activity is improved and decided the optimum liquid medium with fermentation optimization.We study the fermentation technology of Bacillus subtilis strain for GABA production, the traditional technology can resolve the endotoxin of E.coli and the high cost of Lactic acid bacteria. It also improve the GAD activity of Bacillus subtilis168 strain by fermentation optimization,whcih can prompt the industrial GABA by Bacillus subtilis strain.