利用唾液链球菌嗜热亚种（Streptococcus salivarius subsp. thermophilus）Y-2生产γ-氨基丁酸的研究
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摘要
本文对利用唾液链球菌嗜热亚种生产γ-氨基丁酸(γ-aminobutyric acid，GABA)进行了系统研究，主要在高活力谷氨酸脱羧酶(glutamate decarboxylase，GAD)[EC4.1.1.15]菌株的筛选和鉴定、GAD的纯化和酶学特性、产酶条件的优化、细胞转化法生产GABA的条件优化、深层发酵生产GABA的条件优化以及GABA的纯化等方面进行了深入探讨，并建立了GAD产生菌快速筛选的pH指示剂法、高效液相色谱测定发酵醪中GABA的方法和GAD活力测定的Berthelot比色法，结果如下：
1．通过已知种属的Streptococcus salivarius subsp．thermophilus STX2，Eccherichia coli As1.487和Micrococcus luteus cMCC28001建立了GAD产生菌快速筛选的pH指示剂法，并利用该方法成功从84株不同微生物菌株中筛选到了25株GAD产生菌，其中以菌株Y-2的GAD活性最高，当菌体(按干重计)与1％谷氨酸一钠(monosodium glutamate，MSG)溶液按1：10混合，于37℃反应12 h，转化液中GABA浓度为14.52±0.93 mmol·L~(-1)。通过形态特征、生理生化特征和16s rDNA序列分析，将菌株Y-2鉴定为唾液链球菌嗜热亚种(Streptococcus salivarius subsp．thermophilus)。S．salivarius subsp．thermophilus Y-2粗GAD的最适反应温度和pH分别为45℃和5.0，在4～40℃和pH 4.75～5.25范围内较稳定，在0～6 h呈现一级反应。
2．建立了高效液相色谱测定发酵醪中GABA的方法。采用7％(V／V)乙酸水溶液对发酵醪进行预处理，以异硫氰酸苯酯作为衍生剂，用反相C_(18)柱为分离柱，柱温为27℃，阶段洗脱，在254nm下进行检测。结果发酵醪中的GABA获得了很好分离，GABA在0～1.5 mmol·L~(-1)范围内线性相关性好，其线性方程为y=14106.5713 x-258.24926(r=0.99928)。最小检测浓度为0.5μmol·L~(-1)(相对标准偏差≤10％)。组间样品测定相对误差为3.571％，加样平均回收率达到了99.038％。结果表明，所建方法稳定，灵敏，重现性好，可用于测定发酵醪中的GABA。
3．基于Berthelot反应的原理，建立了测定GAD活力的Berthelot比色法。通过对影响Berthelot比色法主要因素—次氯酸钠溶液的使用量进行详细研究，并模拟GAD催化反应体系中底物和产物变化过程，以L-Glu和GABA的混合溶液建立工作曲线，GABA在0～10 mmol·L~(-1)的范围内具有良好的线性，线性方程为：y=0.0893x+0.0158(R~2=0.9992)；精密度分析结果表明，测定相对误差和变异系数都＜5％；平均回收率为99.64％。Berthelot比色法测定操作程序为：取酶反应液0.4 mL，加入Na_2Co_3(1mol·L~(-1))0.1 mL、pH10.0硼酸盐缓冲液(0.2 mol·L~(-1))0.5 mL、6％苯酚1 mL，混匀，于室温下(20℃)在5 min内加入5.2％NaClO溶液1 mL，混匀后放置4～8 min，然后沸水浴10 min，立即冰浴20 min，待溶液出现蓝绿色后，加入2.0 mL 60％乙醇溶液，混匀后于20℃水浴中放置30 min，测定640 nm处的吸收值。
分别采用Berthelot比色法和高效液相色谱法对GAD活力进行测定，两种方法的测定结果相对误差为4.35％。
4．首次通过(NH_4)_2SO_4分级沉淀、等电点沉淀、DEAE-Sephadex A-50离子交换层析、HiPrep16／10 Phenyl FF疏水层析和Sephadex G-100凝胶层析对S．salivarius subsp。thermophilus Y-2 GAD进行了纯化，获得了电泳纯GAD，较粗酶液纯化了21.97倍，比酶活为103.92 U·mg~(-1)，酶活力回收率为7.84％。GAD的亚基和全酶相对分子量分别为46.85 kDa和103.57 kDa，表明GAD由2个大小相等的亚基组成的二聚体。GAD等电点为pH 4.2，最适反应温度和pH分别为55℃和pH 4.0，在4～60℃和pH3.75～4.5范围内稳定。在测试的19种氨基酸中只以L-Glu为底物，而对D-Glu等18种氨基酸没有催化作用，具有很强的底物特异性和立体结构选择性，对L-Glu的K_m和V_(max)分别为2.348 mmol·L~(-1)和4.627 mmol·L~(-1)·h~(-1)。GAD的N-末端氨基酸序列为NH_2-Met-Asn-GlU-Lvs-Leu-Phe-Arg-Glu-Ile-。该序列与报道的其他蛋白质序列均不一致，结果显示该GAD为新的GAD。
表面活性剂十二烷基硫酸钠、Tween 20、Tween 40、Tween 80和Triton X-100对GAD都有不同程度的抑制作用，分别抑制了91.61％，11.32％、13.28％、16.21％和15.86％的GAD活力。当终浓度为5 mmol·L~(-1)时，BaCl_2对GAD活性具有显著的促进作用，酶活力提高了22.53％，但是当终浓度为50 mmol·L~(-1)时，却对GAD活性具有抑制作用；低浓度和高浓度的FeSO_4、FeCl_3、ZnSO_4、MnSO_4、Pb(Ac)_2、AgNO_3、CoCl_2、和CuSO_4对GAD活性都有强烈的抑制作用；5 mmol·L~(-1)CaCl_2对GAD的活性影响不大，但50 mmol·L~(-1)CaCl_2却抑制了18.74％GAD活性；5 mmol·L~(-1)和50 mmol·L~(-1)MgCl_2对GAD均没有影响。低浓度(5 mmol·L~(-1))NaCl、KCl和LiCl对GAD活性有轻微的抑制作用或没有影响，但在高浓度(50 mmol·L~(-1))时却对GAD活性呈现促进作用，其机制主要是通过弱抑制作用、降低CO_2溶解度和增加酶蛋白的溶解性共同作用而影响GAD活性。
5．通过单次单因子实验法、Plackett-Burman设计法和Box-Behnken响应曲面法对S．salivarius subsp．thermophilus Y-2产GAD的适宜条件进行考察，结果表明最适产GAD的培养基组成为：蛋白胨15 g·L~(-1)，牛肉膏12.5 g·L~(-1)，蔗糖12.5 g·L~(-1)，柠檬酸二铵2.0 g·L~(-1)，乙酸钠5.0 g·L~(-1)，K_2HPO_4 1.03 g·L~(-1)，CaCl_2 2.12 g·L~(-1)，Tween 80 1.0mL·L~(-1)，pH 6.79；最适发酵条件为：接种量2％，发酵温度37℃，发酵时间12 h。在该优化条件下，200 mL发酵醪的菌体总GAD活力达到了257.46±5.12 U，较优化前(MRS培养基，177.31±9.33 U)提高了1.45倍。
6．通过对反应pH、反应温度、重金属盐、表面活性剂、底物浓度、菌体浓度和磷酸吡哆醛(pyrodoxal-5′-phosphate，PLP)等因素对S．salivarius subsp．thermophilus Y-2细胞转化法生产GABA的影响进行考察和分析，获得了反应体系的最佳组成为：湿菌体25 g·L~(-1)、BaCl_2 40 mmol·L~(-1)、Triton X-100 0.02％(V／V)、MSG 47.5 g·L~(-1)和L-Glu90.0 g·L~(-1)。当该体系在40℃、pH 4.5和搅拌速度100 rpm的最适转化条件下进行反应72 h，转化液GABA浓度达到了87.16±4.33 g·L~(-1)，细胞平均生产力为48.42±2.41mg_(GABA)·h~(-1)·g_(cells)~(-1)，摩尔转化率为97.60±4.71％。
7．通过对一步法发酵中S．salivarius subsp．thermophilus Y-2的生长情况、pH变化和细胞内外GABA的变化情况进行考察，首次发现S．salivarius subsp．thermophilus合成GABA的过程与GAD的生物化学性质有密切关系。基于GAD的性质，将S．salivarius subsp．thermophilus Y-2的最适产GAD条件与GABA的合成条件进行有机分离，建立了二步法发酵生产GABA，并对MSG的添加时间、MSG添加量、PLP的添加时间和不同中和剂对二步法发酵生产GABA的影响进行了考察，结果表明，S．salivarius subsp．thermophilus合成GABA的最适培养基为：MSG 12 g·L~(-1)，蛋白胨15g·L~(-1)，牛肉膏12.5 g·L~(-1)，蔗糖12.5 g·L~(-1)，柠檬酸二铵2.0 g·L~(-1)，乙酸钠5.0 g·L~(-1)，K_2HPO_41.03 g·L~(-1)，CaCl_2 2.12 g·L~(-1)，Tween 80 1.0 mL·L~(-1)，pH 6.79。最适发酵条件为：接种量2％(V／V)，在100 rpm搅拌和不通气的条件下于37℃发酵24 h，然后于40℃和pH4.5继续发酵48 h。在该条件下，通过75 L发酵罐进行发酵，发酵醪中GABA浓度达到了6271.79 mg·L~(-1)，较一步法(4534.03 mg·L~(-1)，84 h)提高了1.38倍，发酵时间缩短了12 h，摩尔转化率为85.75％。
8．通过等电点沉淀和732强酸阳离子交换树脂对S．salivarius subsp．thermophilus Y-2细胞转化液中的GABA进行纯化，GABA的总回收率达到了84.13％。纯化的GABA产品质量较好，GABA的外观为白色粉末状固体，GABA含量为97.81±0.67％，灰分为0.449±0.002％，干燥失重为1.79±0.06％，铅、砷和汞分别为0.4093±0.0001、0.0511±0.0001和0.0950±0.0000 mg·kg~(-1)，因此在食品行业中具有极好的应用前景。
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-C_(18) 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) Na_2CO_3 (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 NH_2-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 BaCl_2significantly increased the GAD activity, which promoted 22.53% of the activity, but 50mmol·L~(-1) of BaCl_2 inhibited the GAD activity. However, both 5 mmol.L~(-1) and 50 mmol·L~(-1)of FeSO_4, FeCl_3, ZnSO_4, MnSO_4, Pb(Ac)_2, AgNO_3, COCl_2 and CuSO_4, strongly inhibitedthe GAD activity. 50 mmol·L~(-1) of CaCl_2 inhibited 18.74% of GAD activity, but 5 mmol·L~(-1)of CaCl_2 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 CO_2 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); K_2HPO_4, 1.03g·L~(-1); CaCl_2, 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);BaCl_2, 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); K_2HPO_4,1.03 g·L~(-1); CaCl_2, 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.
1．通过已知种属的Streptococcus salivarius subsp．thermophilus STX2，Eccherichia coli As1.487和Micrococcus luteus cMCC28001建立了GAD产生菌快速筛选的pH指示剂法，并利用该方法成功从84株不同微生物菌株中筛选到了25株GAD产生菌，其中以菌株Y-2的GAD活性最高，当菌体(按干重计)与1％谷氨酸一钠(monosodium glutamate，MSG)溶液按1：10混合，于37℃反应12 h，转化液中GABA浓度为14.52±0.93 mmol·L~(-1)。通过形态特征、生理生化特征和16s rDNA序列分析，将菌株Y-2鉴定为唾液链球菌嗜热亚种(Streptococcus salivarius subsp．thermophilus)。S．salivarius subsp．thermophilus Y-2粗GAD的最适反应温度和pH分别为45℃和5.0，在4～40℃和pH 4.75～5.25范围内较稳定，在0～6 h呈现一级反应。
2．建立了高效液相色谱测定发酵醪中GABA的方法。采用7％(V／V)乙酸水溶液对发酵醪进行预处理，以异硫氰酸苯酯作为衍生剂，用反相C_(18)柱为分离柱，柱温为27℃，阶段洗脱，在254nm下进行检测。结果发酵醪中的GABA获得了很好分离，GABA在0～1.5 mmol·L~(-1)范围内线性相关性好，其线性方程为y=14106.5713 x-258.24926(r=0.99928)。最小检测浓度为0.5μmol·L~(-1)(相对标准偏差≤10％)。组间样品测定相对误差为3.571％，加样平均回收率达到了99.038％。结果表明，所建方法稳定，灵敏，重现性好，可用于测定发酵醪中的GABA。
3．基于Berthelot反应的原理，建立了测定GAD活力的Berthelot比色法。通过对影响Berthelot比色法主要因素—次氯酸钠溶液的使用量进行详细研究，并模拟GAD催化反应体系中底物和产物变化过程，以L-Glu和GABA的混合溶液建立工作曲线，GABA在0～10 mmol·L~(-1)的范围内具有良好的线性，线性方程为：y=0.0893x+0.0158(R~2=0.9992)；精密度分析结果表明，测定相对误差和变异系数都＜5％；平均回收率为99.64％。Berthelot比色法测定操作程序为：取酶反应液0.4 mL，加入Na_2Co_3(1mol·L~(-1))0.1 mL、pH10.0硼酸盐缓冲液(0.2 mol·L~(-1))0.5 mL、6％苯酚1 mL，混匀，于室温下(20℃)在5 min内加入5.2％NaClO溶液1 mL，混匀后放置4～8 min，然后沸水浴10 min，立即冰浴20 min，待溶液出现蓝绿色后，加入2.0 mL 60％乙醇溶液，混匀后于20℃水浴中放置30 min，测定640 nm处的吸收值。
分别采用Berthelot比色法和高效液相色谱法对GAD活力进行测定，两种方法的测定结果相对误差为4.35％。
4．首次通过(NH_4)_2SO_4分级沉淀、等电点沉淀、DEAE-Sephadex A-50离子交换层析、HiPrep16／10 Phenyl FF疏水层析和Sephadex G-100凝胶层析对S．salivarius subsp。thermophilus Y-2 GAD进行了纯化，获得了电泳纯GAD，较粗酶液纯化了21.97倍，比酶活为103.92 U·mg~(-1)，酶活力回收率为7.84％。GAD的亚基和全酶相对分子量分别为46.85 kDa和103.57 kDa，表明GAD由2个大小相等的亚基组成的二聚体。GAD等电点为pH 4.2，最适反应温度和pH分别为55℃和pH 4.0，在4～60℃和pH3.75～4.5范围内稳定。在测试的19种氨基酸中只以L-Glu为底物，而对D-Glu等18种氨基酸没有催化作用，具有很强的底物特异性和立体结构选择性，对L-Glu的K_m和V_(max)分别为2.348 mmol·L~(-1)和4.627 mmol·L~(-1)·h~(-1)。GAD的N-末端氨基酸序列为NH_2-Met-Asn-GlU-Lvs-Leu-Phe-Arg-Glu-Ile-。该序列与报道的其他蛋白质序列均不一致，结果显示该GAD为新的GAD。
表面活性剂十二烷基硫酸钠、Tween 20、Tween 40、Tween 80和Triton X-100对GAD都有不同程度的抑制作用，分别抑制了91.61％，11.32％、13.28％、16.21％和15.86％的GAD活力。当终浓度为5 mmol·L~(-1)时，BaCl_2对GAD活性具有显著的促进作用，酶活力提高了22.53％，但是当终浓度为50 mmol·L~(-1)时，却对GAD活性具有抑制作用；低浓度和高浓度的FeSO_4、FeCl_3、ZnSO_4、MnSO_4、Pb(Ac)_2、AgNO_3、CoCl_2、和CuSO_4对GAD活性都有强烈的抑制作用；5 mmol·L~(-1)CaCl_2对GAD的活性影响不大，但50 mmol·L~(-1)CaCl_2却抑制了18.74％GAD活性；5 mmol·L~(-1)和50 mmol·L~(-1)MgCl_2对GAD均没有影响。低浓度(5 mmol·L~(-1))NaCl、KCl和LiCl对GAD活性有轻微的抑制作用或没有影响，但在高浓度(50 mmol·L~(-1))时却对GAD活性呈现促进作用，其机制主要是通过弱抑制作用、降低CO_2溶解度和增加酶蛋白的溶解性共同作用而影响GAD活性。
5．通过单次单因子实验法、Plackett-Burman设计法和Box-Behnken响应曲面法对S．salivarius subsp．thermophilus Y-2产GAD的适宜条件进行考察，结果表明最适产GAD的培养基组成为：蛋白胨15 g·L~(-1)，牛肉膏12.5 g·L~(-1)，蔗糖12.5 g·L~(-1)，柠檬酸二铵2.0 g·L~(-1)，乙酸钠5.0 g·L~(-1)，K_2HPO_4 1.03 g·L~(-1)，CaCl_2 2.12 g·L~(-1)，Tween 80 1.0mL·L~(-1)，pH 6.79；最适发酵条件为：接种量2％，发酵温度37℃，发酵时间12 h。在该优化条件下，200 mL发酵醪的菌体总GAD活力达到了257.46±5.12 U，较优化前(MRS培养基，177.31±9.33 U)提高了1.45倍。
6．通过对反应pH、反应温度、重金属盐、表面活性剂、底物浓度、菌体浓度和磷酸吡哆醛(pyrodoxal-5′-phosphate，PLP)等因素对S．salivarius subsp．thermophilus Y-2细胞转化法生产GABA的影响进行考察和分析，获得了反应体系的最佳组成为：湿菌体25 g·L~(-1)、BaCl_2 40 mmol·L~(-1)、Triton X-100 0.02％(V／V)、MSG 47.5 g·L~(-1)和L-Glu90.0 g·L~(-1)。当该体系在40℃、pH 4.5和搅拌速度100 rpm的最适转化条件下进行反应72 h，转化液GABA浓度达到了87.16±4.33 g·L~(-1)，细胞平均生产力为48.42±2.41mg_(GABA)·h~(-1)·g_(cells)~(-1)，摩尔转化率为97.60±4.71％。
7．通过对一步法发酵中S．salivarius subsp．thermophilus Y-2的生长情况、pH变化和细胞内外GABA的变化情况进行考察，首次发现S．salivarius subsp．thermophilus合成GABA的过程与GAD的生物化学性质有密切关系。基于GAD的性质，将S．salivarius subsp．thermophilus Y-2的最适产GAD条件与GABA的合成条件进行有机分离，建立了二步法发酵生产GABA，并对MSG的添加时间、MSG添加量、PLP的添加时间和不同中和剂对二步法发酵生产GABA的影响进行了考察，结果表明，S．salivarius subsp．thermophilus合成GABA的最适培养基为：MSG 12 g·L~(-1)，蛋白胨15g·L~(-1)，牛肉膏12.5 g·L~(-1)，蔗糖12.5 g·L~(-1)，柠檬酸二铵2.0 g·L~(-1)，乙酸钠5.0 g·L~(-1)，K_2HPO_41.03 g·L~(-1)，CaCl_2 2.12 g·L~(-1)，Tween 80 1.0 mL·L~(-1)，pH 6.79。最适发酵条件为：接种量2％(V／V)，在100 rpm搅拌和不通气的条件下于37℃发酵24 h，然后于40℃和pH4.5继续发酵48 h。在该条件下，通过75 L发酵罐进行发酵，发酵醪中GABA浓度达到了6271.79 mg·L~(-1)，较一步法(4534.03 mg·L~(-1)，84 h)提高了1.38倍，发酵时间缩短了12 h，摩尔转化率为85.75％。
8．通过等电点沉淀和732强酸阳离子交换树脂对S．salivarius subsp．thermophilus Y-2细胞转化液中的GABA进行纯化，GABA的总回收率达到了84.13％。纯化的GABA产品质量较好，GABA的外观为白色粉末状固体，GABA含量为97.81±0.67％，灰分为0.449±0.002％，干燥失重为1.79±0.06％，铅、砷和汞分别为0.4093±0.0001、0.0511±0.0001和0.0950±0.0000 mg·kg~(-1)，因此在食品行业中具有极好的应用前景。
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-C_(18) 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) Na_2CO_3 (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 NH_2-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 BaCl_2significantly increased the GAD activity, which promoted 22.53% of the activity, but 50mmol·L~(-1) of BaCl_2 inhibited the GAD activity. However, both 5 mmol.L~(-1) and 50 mmol·L~(-1)of FeSO_4, FeCl_3, ZnSO_4, MnSO_4, Pb(Ac)_2, AgNO_3, COCl_2 and CuSO_4, strongly inhibitedthe GAD activity. 50 mmol·L~(-1) of CaCl_2 inhibited 18.74% of GAD activity, but 5 mmol·L~(-1)of CaCl_2 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 CO_2 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); K_2HPO_4, 1.03g·L~(-1); CaCl_2, 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);BaCl_2, 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); K_2HPO_4,1.03 g·L~(-1); CaCl_2, 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.
引文
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