Process Study On The Preparation Of γ-Aminobutyric Acid By Lactobacillus Brevis

Gamma-aminobutyric acid (GABA), a four-carbon nonprotein amino acid, serves as a major inhibitory neurotransmitter in mammalian nervous systems. GABA has several physiological functions such as hypotensive activity, treatment of epilepsy, tranquilizing and allaying excitement, enhancing memory, controlling asthma, regulating hormone secretion, promoting reproduction and activating liver and kidney function. Preparation and application of GABA are concerned.Preparation of GABA by Lactobacillus brevis was studied in detail in this dissertation. Based on determination of GABA analytical method, GABA-producing mutant strain was bred; the fermentation conditions in shake flask and fermenter were optimized; biosynthesis of GABA using free and immobilized Lactobacillus brevis cells was investigated. Furthermore, purification and characterization of glutamate decarboxylase(GAD) which is a unique enzyme synthesizing GABA were studied.First, paper and thin-layer chromatography were used as qualitative analysis of GABA, and high performance liquid chromatography(HPLC) as quantitative determination of GABA. With Dansyl chloride (DNS-Cl) as pre-column derivatization, derivatization reaction was stable when pH of sample was higher than 7.5 and reaction time exceeded 20 min. Peak area (A) had good linearity with GABA concentration(C) when GABA concentration ranged from 0 to 2 mM. The regression equation was A=313.242·C+8.314 and correlation coefficient was 0.9997. Average recoveries were in the range of 95.30%～106.34%.Second, a GABA-yielding strain (hjxj-01) was isolated from fresh milk without pasteurization, and GABA production reached 6.9 g/L after 72h fermentation. The strain hjxj-01 was named as Lactobacillus brevis according to its colony morphology as well as physiological and biochemical properties. Applying ultraviolet and ⁶⁰Co γ-ray to mutagenize Lactobacillus brevis hjxj-01, a mutant strain hjxj-08119 was selectively bred by GABA resistance selection. After 72h fermentation, the mutant strain gave a GABA output of 17 g/L, 140% higher than that of the parent strain (hjxj-01). After 12 generation, the mutant strain had stable yield of GABA and no reverse mutation. The mutant strain is kept China General Microbiological Culture Collection Center (CGMCC) as Lactobacillus brevis CGMCC NO.1306.Third, artificial neural network (ANN) and particle swarm optimization (PSO) were used to optimize GABA production by Lactobacillus brevis CGMCC NO.1306 in shake flask. Firstly, glucose, sodium glutamate(L-MSG) and MnSO₄·4H₂O, which influencedGABA production positively were screened from 15 related factors by using Plackett-Burman design. The reasonable ranges of these three factors were determined by single factor experiment. Then experimental samples of hybrid design were selected for training ANN, and the ANN was modeled. Finally, based on the ANN model, the optimized condition was predicted by particle swarm optimization(PSO) algorithm. The optimal medium composition in shake flask was determined as follows (g/L): glucose 17.6, yeast extract 15, peptone 5, CH₃COONa 3, MgSO₄·7H₂O 0.03, MnSO₄·4H₂O 0.02, NaCl 0.001, FeSO₄·7H₂O 0.001, L-MSG 73.3. The fermentation should be performed at 30℃, pH 6.8, and medium volumetric ratio 20%. After 72h fermentation, the yield of GABA reached 33.42 g/L, 97% higher than fermentaion conditions before optimization.Fourth, the effects of operation conditions (aeration and pH) on GABA batch fermentation by Lactobacillus brevis hjxj-08119 in a 3.7 liters stirred fermenter were investigated. The results showed that operation conditions (dissolved oxygen and pH) had the significant effects on GABA production. The aerobic cultivation was advantageous to high level of dry cell weight (2.78 g/L), but the anaerobic cultivation was advantageous to GABA accumulation that GABA yield reached 23.94 g/L at 72h. To assess the effects of pH on GABA production, three batch processes with pH control at 4.5, 5.0 and 5.5 respectively, were conducted in facultative anaerobic cultivation. The yield of GABA was the highest at pH 5.0 and reached 40.73 g/L at 72h. The kinetic models for two stages were established based on the Logistic and modified Luedeking-Piret equations involving cell growth, product formation and substrate consumption for GABA fermentation process with pH control at 5.0. The parameters of the models were obtained by using Matlab 6.0 software with experimental data and the models. With the evaluated models parameters, the calculated values of the models and experimental data are in a good agreement. The fed-batch fermentation of GABA was preliminarily studied. After four times L-MSG addition, the yield of GABA reached 76.36 g/L at 108h, and the yield which fed-batch fermentation significantly enhanced GABA yield were 128.6%, 219% and 87.5% respectively higher than shake flask fermentation, anaerobic fermentation and fermentation with pH control at 5.Fifth, ANN and PSO were applied to the biotransformation of L-MSG to GABA catalyzed by the free cells of Lactobacillus brevis hjxj-08119. The modeled maximum GABA yield reached 9.4 g/L under the following optimal conditions: 25 mL Na₂HPO₄-citric acid buffer (100 mM, pH 4.23), 120 mM L-MSG, 0.83 g/LFeSO₄·7H₂O, 10 μM 5'-pyridoxal phosphate(PLP), the resting cells obtained from a 60-h culture broth, 2.68 g dry cell weight (DCW)/L and without agitation at 40℃ for 5 h. The average value of the four experimentally tested GABA yield was 9.34±0.22 g/L compared with a value of 9.4 g/L by ANN coupling PSO. The prediction capacity between ANN and response surface methodology (RSM) was compared. The results demonstrated a slightly higher prediction accuracy of ANN compared to RSM.Sixth, by entrapping the Lactobacillus brevis cells into Ca-alginate gel beads, the biotransformation conditions of L-MSG to GABA were optimized with the immobilized cells. The optimal cell density in gel beads, reaction pH and temperature were 11.2 g DCW/L, 4.4 and 40℃ respectively. GABA yield still reached more than 50% and Ca-alginate gel beads kept integrity after ten-time recycling (80 h) of the immobilized cells.Finally, effective isolation and purification procedure of GAD from Lactobacillus brevis hjxj-08119, including lysozyme treatment, French press disruption, 30～90% saturation (NH₄)₂SO₄ fractional precipitation, Q Sepharose FF anion-exchange chromatography, Sephacryl S-200 gel filtration chromatography and Resource Q anion-exchange chromatography, was brought forward. Using this protocol, the purified GAD was demonstrated to possess electrophoretic homogeneity via SDS-PAGE. The purification fold, activity recovery and specific activity of GAD were 43.78, 16.95% and 3.94 U/mg(protein), respectively. The molecular weight of the purified GAD was estimated to be approximately 62 kDa via SDS-PAGE. The optimum pH and temperature of the purified GAD were 4.4 and 37℃, respectively. The V_max and K_m value of the GAD enzyme from Lineweaver-Burk plot was found to be 6.59 U/mg and 8.22 mM. PLP had little effect on the regulation of its activity.