Production Of γ-aminobutyric Acid By Lactobacillus Brevis CCTCCM208054and Analysis Of Its GAD System Key Genes

Gamma-aminobutyric acid (GABA) is a naturally non-protein acid that is widely distributed in nature. GABA acts in animals as a major inhibitory neurotransmitter, and has several important physiological functions and the potential as a bioactive component in foods, pharmaceuticals and feeds. GABA has beeen approved as a new resource food for food processing in China. This dissertation was focused on the bioconversion of GABA with lactic acid bacteria (LAB), including the isolation and identification of a high-yielding GABA producer; the methods for the analysis of GABA; the establishment and optimization of fermentation process for GABA production; the isolation and purification of GABA; and the analysis of GAD system key genes in CCTCCM208054. The main results are as follows.1. Pre-staining paper chromatography was developed for qualitative analysis of amino acids. Pre-staining paper chromatography-vis-spectrophotometry was developed for quantitative analysis of GABA. Operating conditions for pre-staining paper chromatography were as follows:the chromatography paper was developed in a developing solvent (n-butanol:acetic acid:water=5:3:2, v/v/v) containing ninhydrin, then the paper was directly dried for color yield. Glutamate and GABA were completely separated with condense spots in the pre-staining paper chromatography while the spots in classical method were partially overlapped and with long tails. The effects of ninhydrin concentration, color temperature and time on the color yield in the ninhydrin reaction, and the effect of Cu2+concentration on the stability of GABA-ninhydrin compound were optimized. The optimized pre-staining paper chromatography coupled with vis-spectrophotometry could be applied to gamma-aminobutyric acid quantification as follows. Appropriate2μL of samples were spotted onto the chromatography paper. The paper was developed at30℃with n-butanol-acetic acid-water (5:3:2) containing1.2%of ninhydrin. After development, the paper was directly heated for color yield at70℃for80min. Then the GABA-ninhydrin spots were cut out from the paper and were extracted with5.0mL eluent (75%ethanol:0.6%CuSO4·5H2O=38:2,v/v) at40℃50rpm for60min. The absorptions were read in a UV-vis spectrophotometer at512nm. The results indicated that the linear range of the developed method was from0.5to20.0mg/mL. Furthermore, an excellent correlation coefficient was observed with an R2=0.998. The method is accurate (RSD<2.64%), and has good recoveries (102.7-103.9%). The concentrations of GABA determined by the current method and HPLC were quite close.2. A high GABA-producing strain, Lactobacillus brevis NCL912was isolated from Chinese traditional paocai. More than1000strains of lactic acid bacteria from paocai samples were screened by the ability in production of GABA, analysed with paper chromatography and HPLC, and23suspicious bacteria were obtained. Among them, strain NCL912exhibited the highest conversion ability (15.37g/L). The molecular weight of the suspicious product was almost the same to that of GABA standard determined by LC-MS. It showed that strain NCL912could indeed convert glutamate to GABA. Strain NCL912was identified as Lactobacillus brevis according to its phenotye, physiological and biochemical characteristics and full16S rNDA sequence alignment. This strain has been deposited in China Center for Type Culture Collection with the accession number CCTCCM208054(=NCL912).3. Fermentation medium for the production of GABA by CCTCCM208054was optimized. Single-dimensional search method was first adopted to select the key factors that impact the GABA production to preliminarily determine the suitable concentration ranges of the key factors. Then response surface methodology was applied to analyze the optimal contents of the key factors glucose, soya peptone, Tween-80and MnSO4·4H2O, and their optimal levels were55.25g/L,30.25g/L,1.38mL/L and0.0061g/L, respectively. The production of GABA was predicted as36.06g/L under the optimized conditions with this model. While the measured GABA content was35.66g/L in the verification test, which was basically identical with the predicted value. GABA production of CCTCCM208054in optimized medium was130%higher than that in the initial medium.4. The impacts of pyridoxal-5'-phosphate, pH, temperature and initial glutamate concentration on the GABA production and cell growth of CCTCCM208054were investigated. Pyridoxal-5'-phosphate did not affect the cell growth and GABA production of CCTCCM208054. Temperature, pH and initial glutamate concentration had significant effects on the cell growth and GABA production of CCTCCM208054. The optimal temperature, pH and initial glutamate concentration were30～35℃,5.0and0.25～0.50M. According to the data obtained in the above, a fed-batch fermentation process was developed to produce GABA as follows.The seed medium was composed of (g/L):glucose,50; soya peptone,25; MnSO4·4H2O,0.01; L-glutamate,0.15M; and Tween80,2mL/L; pH5.0. The fermentation medium was the same to the seed medium except for glucose50g/L and glutamate0.4M. CCTCCM208054was cultured in the seed medium at32℃for about10h till A600between4.0and6.0and then used for seed culture inoculation. The specific fed-batch fermentation parameters were:fermentation medium3L, seed culture300mL, temperature32℃, stirring speed100rpm, fermentation period48h,280g and112g glutamate were supplemented into the bioreactor at12h and24h, respectively. pH value of the fermentation broth was controlled at5.0with5M H2SO4during the whole process. The GABA concentration reached102.78±5.30g/L while no glutamate and glucose remained at48h.5. Centrifugation, activated carbon decoloration,70%ethanol desalination, refining by ion exchange chromatography and crystallization from ethanol were successively conducted for the purification GABA from the fermented broth. The recovery rate for the whole purification process was about50%. The purified product displayed a single spot in TLC chromatogram. Its purity reached98.66±2.36%through HPLC determination.6. gadA and its flanking region in CCTCCM208054was cloned. Gene order of the cloned region is (from upstream to downstream):acetyltransferase gene(act), PgadR, gadR, Pgad, gadC, gadA and glutamyl-tRNA synthetase gene (gts). This is almost the same to that in Lactobacillus brevis ATCC367. However, NADPH:quinone reductase related Zn-dependent oxidoreductase gene is located immediately upstream of PgadR in ATCC367. The homologous coefficients of gadR, gadC and gadA in CCTCCM208054with those in ATCC367are66%,79%and79%, respectively; and those of the encoded proteins are66%,91%and91%, respectively. Intergenic regions of between act and gadR, gadR and gadC, and gadC and gadA are278,210and59bp, respectively in CCTCCM208054, while they are270,193, and55bp, respectively in ATCC367. In the two microbes, the homologous coefficients of the above intergenic regions are43%,58%and62%, respectively. No possible transcription signals could be identified in or near the59bp intergenic region between gadA and gadC. We could not clone gadB by using primer pairs for direct amplification it. We amplified aldo-keto reductase gene (akr) and walked into downstream of akr for3027bp but did not find gadB although it locates downstream only1003bp of akr in ATCC367. This suggests that CCTCCM208054maybe contain no gadB.7. Real-time fluorescence quantitative PCR was applied to analyze the transcriptional levels of gadA, gadC and gadR in CCTCCM208054during the fermentation process in the fermentation media supplemented with or without glutamate. The results showed that glutamate induced their expression. The transcriptional level of gadC is identical to that of gadA. Different from constitutive transcription of gadR in Lactococcus lactis, transcription of gadR in CCTCCM208054is synchronous with gadCA, and its transcriptional level is14-156times of that of gadCA. Sequence analysis and quantitative PCR results suggested that gadCA maybe form an operon structure. The high GABA-producing ability of CCTCCM208054maybe derived from three reasons:first, transcriptional level of gadCA and activity of the enzymes maybe enhanced via optimizing functional gene and regulatory sequences of GAD system; second, the synchronous expression of gadC and gadA via forming an operon is conducive to coordinating the decarboxylation and antiport; third, high expression of gadR guarantees normal transcription of gadCA.