Production Of Gamma-Aminobutyric Acid By Streptococcus Salivarius Subsp. Thermophilus Y-2

In this paper, the production ofγ-aminobutyric acid (GABA) by Streptococcussalivarius subsp, thermophilus Y-2 was investigated systemically respect to the screeningand identification of glutamate decarboxylase (GAD) [EC 4.1.1.15] producing strain,purification and characterization of novel GAD, culture conditions for GAD production,biotransformation conditions for GABA production using cells, preparation of GABA bysubmerged fermentation and the purification of GABA. Three methods, i.e., the pHindicator method for rapid screening of bacteria with GAD activities, the improved methodfor determination of GABA in the broth of fermentation by high-performance liquidchromatography and the colorimetric method for determination of GAD activity, were alsoestablished. The main results are as following:1. A pH indicator method for rapid screening of strains with GAD was established viaS. salivarius subsp, thermophilus STX2, Eccherichia coli As1.487 and Micrococcus luteusCMCC28001. Twenty-five strains with GAD activities were screened form eighty-fourdifferent strains by the pH indicator method, in which the strain Y-2 showed the highestGAD activity. When the biotransformation was conducted with the ratio of wet cells and1% of monosodium glutamate (MSG) at 1:10 under 37℃for 12 h, the GABA ofbiotransformation solution of strain Y-2 was 14.52±0.93mmol.L^-1. Based on morphological,physiological and biochemical characteristics, 16S rDNA and phylogenic analysis, thestrain Y-2 was classified to Streptococcus salivarius subsp, thermophilus. The properties ofits crude GAD were also examined, and the optimal temperature and pH value for GADactivity was 45℃and pH 5.0, respectively. The crude GAD activity was stable at 4～40℃and pH 4.75～5.25. The crude GAD activity was kept linear in 6 h.2. A method for the determination of GABA in the broth of fermentation by HPLC wasdescribed. The GABA formed was derivatized to PTC-GABA; the latter was subsequentlyseparated and assay by HPLC (Agilent:ZORBAX.Eclips XDB-C₁₈ column; elution with pH5.8 acetate buffer in acetonitrile-water) with UV absorbance detection at 254 nm. Under theconditions described above, HPLC separation gave a well-resolved and symmetricalPTC-GABA peak, the retention times (18.3 min) was reproducible when temperature variations were≤1℃. Peak area values showed good reproducibility with relative standarddeviation (R.S.D) values at 1.019%. The calibration curve for PTC-GABA was calculatedby linear least-squares regression. The regression equation was y=14106.5713 x-258.24926 (r=0.99928) when the initial GABA concentration in the range 0～1.5mmol·L^-1. The limit of quantitation was 0.5μmol·L^-1 (R.S.D.≤10%). The result indicatesthe method described is a sensitive, reproducible and specific assay useful for thedetermination of GABA in the broth of fermentation.3. A colorimetric method of determining GAD activity assay with high sensitivity,accuracy and reproducibility was proposed, and the standard procedure leading to theestablishment of the method was described. The reaction mixture (0.4 mL) of GAD andL-Glu was mixed with 1 mol·L^-1 Na₂CO₃ (0.1 mL), 0.2 mol·L^-1 borate buffer (0.5 mL) and6% phenol (1.0 mL), then adding 1 mL of 5.2% sodium hypochlorite solution at 20℃within 5 min. After kept at 20℃for 4～8 min, the mixture was heated by boiling water bathfor 10 min and promptly cooled in ice bath for 20 min, then' adding 2 mL of 60% ethanol,and kept at 20℃for 30 min.The optical density of the mixture was measured by theabsorbance at 640 nm. The accuracy and sensitivity of the determination method were alsodiscussed, indicating that the relative error was 4.35% in comparison with the results ofHPLC.4. GAD was firstly purified to homogeneity from a cell-free extract of S. salivariussubsp, thermophilus Y-2 by ammonium sulfate fraction, isoelectric precipitation, DEAE-Sephadex A-50 column chromatography, HiPrepl6/10 Phenyl FF column chromatography,and Sephadex G-100 column chromatography. GAD was purified 21.97-fold from crudeprotein extracts with a yield of 7.84% and specific activity of 103.92 U·mg^-1. The finalpreparation gave a single band on sodium dodecyl sulfate polyacrylamide gels. The subunitand native molecular weights of purified GADwas 46.85 kDa and 103.57 kDa respectively,indicating that GAD from S. salivarius subsp, thermophilus Y-2 exists as a dimmer as adimmer of homological subunits. The isoelectric point of GAD was pH 4.2 tested byDEAE-Sephadex A-50 and N-Methyl piperazine buffer at various pHs. The optimumtemperature and pH of the purified GAD was at 55℃and pH 4.0 respectively. The purifiedGAD activity was stable at 4～60℃and pH 3.75～4.5. The enzyme was reacted only withL-glutamate among 19α-amino acids with apparent K_m and V_max at 2.348mmol·L^-1 and4.627 mmol·L^-1·h^-1 respectively, and couldn't react with D-glutamic acid. The resultsindicated the enzyme has high substrate specificity and stereospecificity. The N-terminal amino acid sequence of the purified GAD was NH₂-Met-Asn-Glu-Lys-Leu-Phe-Arg-Glu-Ile-. This sequence had no close similarity with any other proteins reported so far, whichshown it was a novel GAD.SDS, Tween 20, Tween 40, Tween 80 and Triton X-100, inhibited the purified GADactivity at 91.61%, 11.32%, 13.28%,16.21% and 15.86% respectively. 5 mmol·L^-1 of BaCl₂significantly increased the GAD activity, which promoted 22.53% of the activity, but 50mmol·L^-1 of BaCl₂ inhibited the GAD activity. However, both 5 mmol.L^-1 and 50 mmol·L^-1of FeSO₄, FeCl₃, ZnSO₄, MnSO₄, Pb(Ac)₂, AgNO₃, COCl₂ and CuSO₄, strongly inhibitedthe GAD activity. 50 mmol·L^-1 of CaCl₂ inhibited 18.74% of GAD activity, but 5 mmol·L^-1of CaCl₂ hardly affected GAD activity. It was interesting that NaCl, KCl and LiCl, slightlyaffect the GAD activity at 5 mmol·L^-1, but significantly increased the GAD activity at 50mmol·L^-1. It was found that the results of the effects of salts on GAD activity was caused bythe coaction of weak inhibition, reducing the dissolving of CO₂ and increasing thesolubility of GAD protein.5. The culture medium and conditions of S. salivarius subsp, thermophilus Y-2 for theGAD-production were investigated through one-factor-at-a-time experimental design,Plackett-Burman experimental design and Box-Behnken design. The results indicated thatthe optimal components of medium for GAD-production by S. salivarius subsp.thermophilus Y-2 was as following: peptone, 15 g·L^-1; beef extract, 12.5 g·L^-1; sucrose,12.5 g·L^-1; ammonium dibasic citrate, 2.0 g·L^-1; sodium acetate, 5.0 g·L^-1; K₂HPO₄, 1.03g·L^-1; CaCl₂, 2.12 g·L^-1, Tween 80, 1.0 mL·L^-1 and initial pH, 6.79. The optimal inoculationpercentage, temperature and time for the fermentation were 2% (V/V), 37℃and 12 h,respectively. Under the optimal culture medium and conditions, the total GAD activity ofthe cells from 200 mL of cultures was 257.46±5.12 U, which was 1.45-fold than that ofnon-optimization (MRS, 177.31±9.33 U).6. The technology of GABA synthesis by treated L-Glu or MSG with the cells of S.salivarius subsp, thermophilus Y-2 was developed. The effects of pH, temperature, time,salts, supernatants, pyrodoxal-5'-phosphate (PLP), and the concentrations of MSG andcells on the biotransformation were systemically investigated. As a result, the optimal pH,temperature and time for the biotransformation was 4.5, 40℃and 72 h respectively under100 rpm, and the optimal reaction system was composed as following: wet cells, 25 g·L^-1;BaCl₂, 40 mmol·L^-1; Triton X-100, 0.02% (V/V); MSG, 47.5 g·L^-1 and L-Glu, 90.0 g·L^-1.Under above conditions, 97.60±4.71% (mole rate) of L-GIu were converted to GABA, and the final concentration of GABA in the reaction medium was 87.16±4.33 g·L^-1. The totalproductivity of the cells was 48.42±2.41 mg_GABA·h^-1·g_cells^-1.7. Through the investigation of the growth of cells, changes of pH of the culture broth,intracellular and extracellular GABA content in the submerged fermentation, it was foundthat the GABA synthesis was closely related to the biochemical characteristics of GAD.Therefore, two-steps fermentation strategy, i.e., the culture conditions were firstly adjustedto the condition suitable for GAD-production and then turned to the optimal conditions ofbiotransformation reaction of cells, was proposed. It was investigated systematically on theeffect of different neutralizers, the MSG and PLP additions at different time and amount forthe GABA production by two-steps fermentation. The results indicated the optimal mediumfor the GABA production was: MSG, 12 g·L^-1; peptone, 15 g·L^-1; beef extract, 12.5 g·L^-1;sucrose, 12.5 g·L^-1; ammonium dibasic citrate, 2.0 g·L^-1; sodium acetate, 5.0 g·L^-1; K₂HPO₄,1.03 g·L^-1; CaCl₂, 2.12 g·L^-1; Tween 80, 1.0 mL·L^-1 and initial pH, 6.79. The optimalconditions for the two-steps fermentation were: After 2% (V/V) of seminal broth of S.salivarius subsp, thermophilus Y-2 were inoculated to the fermentation medium, thefermentation was firstly conducted at 37℃for 24 h, then the fermentation was furtherconducted with pH and temperature regulation at 40℃and 4.5 respectively for 48 h. Whenthe fermentation was conducted in 75 L of fermentor with agitation at 100 rpm and withoutair addition under above conditions for 72 h, 85.75% (mole rate) of MSG were converted toGABA, and the final concentration of GABA in the broth was 6271.79 mg·L^-1, which was1.38-fold than that of one-step fermentation (4534.03 g·L^-1, 84 h) and saved 12 h.8. GABA was purified from the biotransformation medium of S. salivarius subsp.thermophilus Y-2 cells by isoelectric precipitation and type 732 cation-exchanged resinscolumn chromatography with a final yield of 84.13%. The purified preparation was whitepowder with the quality as following: GABA, 97.81±0.67%; ash, 0.449+0.002%; loss ofweight in drying, 1.79±0.06%; Pb, 0.4093±0.0001 mg·kg^-1; As, 0.0511±0.0001 mg·kg^-1; Hg,0.0950±0.0000 mg·kg^-1. The results indicated that the preparation could be use as foodadditive.