Study On Improving Gama-Aminobutyric Acid Production Of Lactic Acid Bacteria STX2by Physical And Chemical Mutagenesis

y-aminobutyric acid(GABA) is a kind of natural non-protein amino acid, and is the main inhibitory neurotransmitter of the central nervous system. It has several physiological functions such as regulating blood pressure, improving sleeping quality, promoting reproduction, preventing and treating epilepsy, and is widely used in food processing, medicine and agriculture. There are three methods to gain GABA:chemical synthesis, plant extraction and fermentation. The microbial fermentation is superior to others in that the culture conditions are mild, various strains are available and it is suitable for industrial production. It has stimulated many researchers’interests. In order to further improve the yield of GABA, the physical and chemical mutagenesis are used in the mutation breeding research of Streptococcus salivarius subsp. Thermophilus STX2, including UV mutagenesis, nitrogen ion beam implantation mutagenesis, NTG and LiCl mutagenesis and compound mutagenesis. Here the high-yielding mutant strains are obtained and its GABA bioconversion conditions are optimized. The results are as following:1. The paper chromatography(PC) for qualitative primary screening combined the high performance liquid chromatography(HPLC) quantitative accurate determination could screen high-producing GABA strains rapidly. The developing solvent in PC was V(n-butanol):V(acetic acid):V(water)=4:1:3and the chromogenic agent was0.4%ninhydrin. With this method, we could quickly ruled out the low-or non-producing GABA strains. Then, the HPLC quantitative method screened the high-producing strains, with Dansyl chloride (DNS-C1) as derivatization solvent, C18as separating column, and gradient elution was conducted under254nm detection wavelength. In this separating condition, GABA was separated from biotransformation solution quite well. Simultaneously, the GABA standard curve was established, and showed a good linear relationship at a range of0-50mmol/L. The regression equation was:y=898.31x+1275, R2=0.9963, and the relative error and average recovery were7.4%and98.861%, which proved that the HPLC detection method was stable and repeatable.2. Physical and chemical mutagenesis, including UV mutagenesis, N+implantation, NTG/LiCl and combined mutagenesis were used in the STX2mutation breeding processing. After PC primary screening and HPLC accurate determination, we screened67high-yielding GABA strains, and selected18strains to test hereditary stability. Among the high-yielding GABA strains, NTG-5was the highest one and its conversion ratio of GABA reached21.455±1.301mg/(g-h), and maximum mutation rate reached up to55.23%.3. NTG-5with high-yielding GABA production was screened by NTG mutagenesis. To further enhance its yield, firstly, three key factors, which affected the conversion ratio of GABA, were determined through single factor tests, including CuCl2concentration, L-glutamic acid sodium concentration and pH. The orthogonal experiment was conducted to study their interaction. The optimum bioconversion conditions were ascertained as:30mL centrifuged fermentation broth to obtain the cell, adding substrate buffer with initial pH4.5,10mmol/L CuCl2and200mmol/L L-MSG, then conducted in the conversion condition of45℃,180rpm for24h, the conversion ratio of GAB A reached46.611±0.540mg/(g-h),which was1.20times higher than the original condition, and2.36times higher than the original strains STX2.