Bacteria Screening And Condition Optimization To Produce γ-aminobutyric Acid With Biological Methods

γ-aminobutyric acid (GABA) is an important inhibitory neurotransmitter of brain tissue,with the function of self-possession, lowering blood pressure, treatment of epilepsy, anti-aging, In addition, GABA is sweet, able to adjust the taste of food, reacting with alcohol of the body, with the function of sobering up and deodorant, In medicine, for the treatment of disorders, medicines which are used in the treatment of uremia, CO poisoning also contain GABA. The synthesis methods of GABA are mainly chemical synthesis and biological synthesis, in the process of chemical synthesis of GABA, Strong acid or alkali and other corrosive solvents are needed,with severe reaction conditions, toxic materials, expensive price, more security risks and so on. In actual industrial production, the synthesis of GABA is mainly biological methods, biosynthesis is mainly microbiological method. Microbiological method includes the traditional fermentation method and new microbial transformation in recent years.Early fermentation production of GABA is mainly E. coli (Escherichia coli) fermentation.For reasons of food safety, the food safety grade microbes of yeast, lactic acid bacteria and aspergillus which contain glutamic acid decarboxylase are used in fermentation preparation of GABA.But the fermentation method has some disadvantages, such as polluted easily, with poor reproducibility and long cycle in production, so it is abandoned gradually. This experiment used microbial transformation mainly study on the strain improvement condition of enzyme production and transformation conditions. The results are as follows:Eleven mold were separated and purified and obtained two high-producing strains (MZ-2 and MZ-7) by primary screening. After secondary screening, using Berthelot colorimetry as measurement method, the yield of GABA, L-Glu negative and the molar yield of L-Glu as measurement index, finally determined MZ-2 as starting strain. Determined the optimum irradiation time was 4-5min after ultraviolet mutagenesis. Through transformation experiment obtained a strain(number UV-5), the yield of GABA achieved 1.95 g/L and the molar yield of L-Glu reached 17.5%. Through Genetic stability test proof that its characters was stably inherited and can be used as original strain for successive test. Qualitative and quantitative analysis of the transformation liquid by paper chroma,tography, SBA-40D biosensor analyzer and colorimetry. During the transformation the content of L-Glu decreased and the content of GAB A increased. The results show that UV-5 has GAD activity, which can transform L-Glu into GABA, and its activity is higher than any other strains.Studying the various components of the strain UV-5, we can find the best medium for enzyme production by the single factor experiment. Components of the best enzyme production medium,2% glucose as carbon source,1.2% corn syrup as nitrogen,0.75% of calcium chloride,0.5g/L dipotassium hydrogen phosphate,0.4% L-glutamic acid. Optimizing the culture conditions by single factor and orthogonal experiments. The best culture conditions for enzyme production, inoculation time 20 h, inoculation volume 6 %, pH 5, fermentation temperature 30℃, fermentation time 72 h.With the optimal medium and culture conditions, the molar conversion rate of UV-5 conversion of L-glutamic is 27.13%.To optimize the mutant UV-5 conversion conditions, the initial conversion conditions was determined by the best single factor experiment, acetic acid-sodium acetate system was selected as buffer, optimum conversion temperature 36℃,optimum pH 5.0, cell concentration 6%,best shaking speed 180 r/min, substrate concentration of L- glutamic 0.1mol/L, conversion time 14h. Based on the results of single factor experiment, using the Response Surface to optimize glutamate conversion conditions.Transformation experiments was selected by the PB experiments and three factors which affect the moore conversion rate apparently was determined,temperature, time,and pH.By the response surface optimization results and the actual situation, we select the best pH 5.07, temperature 36℃, time 14.2h,as a result, the production of GABA reached 0.59 g/L.