米胚谷氨酸脱羧酶性质及其富集γ-氨基丁酸研究
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摘要
本论文对米胚谷氨酸脱羧酶(GAD)的分离纯化、酶学特性、光谱性质和氨基酸残基的化学修饰等进行了深入的研究,并应用米胚GAD及其活性调节机制制备了高γ-氨基丁酸(GABA)米胚。
首先研究了米胚中GABA的测定方法,结果发现比色法误差较大,无法作为米胚中GABA的定量测定方法。而纸层析法精密度高,虽然比HPLC?法的测定结果略低,但差异不大,适合用于大量米胚样品的测定。
通过(NH4)2SO4分级沉淀、DEAE-Sephrose FF离子交换色谱、Superdex 200 凝胶过滤色谱和Glu Sephrose CL 4B亲和色谱等分离纯化技术对米胚GAD进行了分离纯化,从米胚中分离得到的GAD经 SDS-PAGE鉴定为单一组分。体积排阻高效液相色谱测得米胚GAD的相对分子质量为78k,SDS-PAGE测得其亚基的相对分子质量为40k,因此米胚GAD酶蛋白是由两个相同的亚基构成的二聚体。
米胚GAD的最适反应温度为40℃,最适反应pH为5.6。米胚GAD的热稳定较差, 75℃时酶基本完全失活。但米胚GAD在较宽的pH范围内(pH4.5-8.0)都较为稳定,能保持88%以上的酶活。米胚GAD对Glu的Km值为32.3mmol/L,Vmax为1.159mg/min;对PLP的Km值为1.7μmol/L ,Vmax为1.171mg/min。
Ca2+浓度达到500μmol/L时对米胚GAD的激活作用最强,可使酶的相对活性达到145%,GAD的表观米氏常数 Km’为25.9mmol/L,小于米胚GAD对 Glu的Km,说明Ca2+提高了酶与底物的亲和力,对米胚GAD的活性确有较强的激活作用。
在含PLP体系中,纯化的GAD的酶活性大大提高。GAD脱辅基后酶活完全丧失了,但是加入PLP后活性基本得以恢复,说明PLP从米胚GAD分子中的脱除是一个可逆过程。
米胚GAD的紫外-可见光谱特征为除280nm以下与蛋白质有关的吸收峰以外,420nm处有一个弱吸收峰,这是PLP与米胚GAD主链结合后产生的吸收峰。米胚GAD的荧光光谱分析表明,米胚GAD主要表现为Tyr残基的荧光特征,米胚GAD脱辅基前后的荧光发射光谱完全相似。
米胚GAD氨基酸残基的化学修饰研究表明,米胚GAD中的His残基与其酶活性有关,但可能不是其活性所必需的基团。Arg残基则是酶活性的必需基团,并且整个酶分子中有一个Arg残基为活性必需基团。底物GLu对Arg残基的修饰有着很强的保护作用,因而可推测说明Arg残基可能位于酶的底物结合位点。米胚GAD的His和Arg残基被修饰后,荧光发射光谱有着非常相似的变化,Trp残基的荧光发射基本被猝灭了,说明酶蛋白微区的
构象发生了变化。
CD光谱分析表明,米胚GAD的二级结构中, α-螺旋占13.2%,β-折叠占38.3%,是一种高β-折叠蛋白。米胚GAD脱辅基后蛋白质的二级结构的变化是部分β-折叠结构转变为了回转结构。His和 Arg残基被修饰后,酶蛋白的二级结构有较大的变化,底物对Arg残基修饰引起的结构变化有保护作用。分析米胚GAD二级结构的变化与酶活性的关系发现,米胚GAD二级结构中α-螺旋结构的维持对酶的活性可能更为重要。
通过对水浸泡工艺进行优化,并利用Ca2+激活GAD,米胚中GABA的量可以由未富集前的28mg/100g提高到615mg/100g左右,比资料报道有较大提高。 利用胰蛋白酶水解可以进一步提高GABA的富集量,在最佳条件下,GABA产量可达2.3g/100g,比GABA-RG1提高了2倍。
外加Glu和添加米胚GAD的激活剂是制备高浓度GABA的有效方法。最佳工艺条件是:pH5.6、0.08mol/L磷酸缓冲液, PLP和Ca2+的添加量分别为2 mmol/molGlu和20mmol/molGlu,米胚的用量为42.5U/mmolGlu,料液比为1/10,反应时间和温度为6h和40℃。产品中GABA含量为20.6g/100g,比GABA-RG2又提高了7.8倍,并且Glu的转化率可以达到100%。
动物实验表明, GABA-RG可显著促进小鼠GH的分泌和后腿肌重的增长,对小鼠力竭游泳时间和爬杆时间有显著地延长作用。还可使运动后小鼠血乳酸的含量明显下降,血清尿素氮含量明显降低,肝糖原含量的显著提高。这些结果表明 GABA-RG能促进小鼠GH分泌并提高运动能力。根据国家卫生部关于保健性食品的评价指标,可以判定GABA-RG具有抗疲劳的作用。
This research was conducted to purify and characterize the GAD from rice germ, to study the properties of GAD, and to produce rice germ with high GABA content.
The methods for GABA assay in rice germ was studied. It was suggested that the colorimetry method could not be used because of the determination error. Paper chromatography was a suitable method, and can be used in combination with HPLC for GABA assay in rice germ.
GAD was purified from rice germ by ammonium sulfate fractionation, DEAE- Sephrose FF chromatography, Superdex 200 gel filtration, and Glu- Sephrose CL 4B affinity chromatography. Single band from SDS-PAGE showed the subunit Mw was 40k. Similar molecular weight (78k) was obtained by SE-HPLC. It was suggested that the GAD from rice germ had two homological subunits.
The purified GAD showed its maximal activity at pH5.6 and 40℃. The enzyme was inactivated completely at 75 ℃. It was stable at pH4.5-8.0. The Km and Vmax of GAD for Glu was 32.3mmol/L and 1.159mg/min; The Km and Vmax of GAD for PLP was 1.7μmol/L and 1.171mg/min.
GAD could be activated by Ca2+, the comparative activity reached 145% when 500μmol/L Ca2+ was added, and the apparent Km’ was changed to 25.9mmol/L, less than the Km for Glu. This showed that the Ca2+ enhanced the affinity between enzyme and Glu, and activated the enzyme.
The purified GAD could be activated by PLP. The enzyme was inactivated by removal of the bond PLP, but it could be renewed completely after PLP adding.
Absorbance spectra of GAD showed one weak peak at 420nm, it was produced by the binding of PLP. Fluorescence emission spectra of GAD was characterized by a Tyr peak, and had no change after PLP removed.
The modification of DIC and DEPC resulted in a great loss of GAD activity, suggesting that the His and Arg involve in the enzyme active site. The number of the essential Arg was estimated as one. The Glu could protect the modification of DIC, suggesting that the essential Arg may be in the substrate binding site of the enzyme.
Circular Dichroism analysis of GAD showed that the ratios of α-helix and β-sheet in the secondary structures were 13.2% and 38.3%. The secondary structures had a little change after PLP removed. The modification of DIC and DEPC resulted in great changes of the secondary structures. The α-helix might be more important to GAD activity.
Optimizing the conditions of GABA - accumulation by water soaking and protein hydrolyzing by trypsin could increase the content of GABA in GABA-RG to 615mg/100g and 2.3g/100g. But
a higher GABA content must be obtained using Glu and GAD activator. The optimized conditions were 0.08mol/L phosphate buffer (pH5.6),adding 2 mmol/molGlu of PLP and 20mmol/molGlu of Ca2+ , 42.5U/mmolGlu of rice germ, and the ratio of rice germ to solvent was 1:10, reaction time and temperature were 6h and 40℃. The content of GABA in the final product (GABA-RG3) was 20.6g/100g,the convert rate of Glu was 100%.
Finally, the new function of GABA-RG on growth hormone (GH) secretion stimulation and exercise performance enhancing of rats were studied. It showed that the GABA-RG stimulated GH secretion and enhanced leg muscle weight of rats obviously. The swimming time burden with 5% weight and climb time of rats were prolonged. The biological index of rats including blood lactic acid , BUN and liver glycogen were also improved.
Judging by relevant specifications of governmental health food standards, we can conclude that GABA-RG has anti-fatigue activities.
首先研究了米胚中GABA的测定方法,结果发现比色法误差较大,无法作为米胚中GABA的定量测定方法。而纸层析法精密度高,虽然比HPLC?法的测定结果略低,但差异不大,适合用于大量米胚样品的测定。
通过(NH4)2SO4分级沉淀、DEAE-Sephrose FF离子交换色谱、Superdex 200 凝胶过滤色谱和Glu Sephrose CL 4B亲和色谱等分离纯化技术对米胚GAD进行了分离纯化,从米胚中分离得到的GAD经 SDS-PAGE鉴定为单一组分。体积排阻高效液相色谱测得米胚GAD的相对分子质量为78k,SDS-PAGE测得其亚基的相对分子质量为40k,因此米胚GAD酶蛋白是由两个相同的亚基构成的二聚体。
米胚GAD的最适反应温度为40℃,最适反应pH为5.6。米胚GAD的热稳定较差, 75℃时酶基本完全失活。但米胚GAD在较宽的pH范围内(pH4.5-8.0)都较为稳定,能保持88%以上的酶活。米胚GAD对Glu的Km值为32.3mmol/L,Vmax为1.159mg/min;对PLP的Km值为1.7μmol/L ,Vmax为1.171mg/min。
Ca2+浓度达到500μmol/L时对米胚GAD的激活作用最强,可使酶的相对活性达到145%,GAD的表观米氏常数 Km’为25.9mmol/L,小于米胚GAD对 Glu的Km,说明Ca2+提高了酶与底物的亲和力,对米胚GAD的活性确有较强的激活作用。
在含PLP体系中,纯化的GAD的酶活性大大提高。GAD脱辅基后酶活完全丧失了,但是加入PLP后活性基本得以恢复,说明PLP从米胚GAD分子中的脱除是一个可逆过程。
米胚GAD的紫外-可见光谱特征为除280nm以下与蛋白质有关的吸收峰以外,420nm处有一个弱吸收峰,这是PLP与米胚GAD主链结合后产生的吸收峰。米胚GAD的荧光光谱分析表明,米胚GAD主要表现为Tyr残基的荧光特征,米胚GAD脱辅基前后的荧光发射光谱完全相似。
米胚GAD氨基酸残基的化学修饰研究表明,米胚GAD中的His残基与其酶活性有关,但可能不是其活性所必需的基团。Arg残基则是酶活性的必需基团,并且整个酶分子中有一个Arg残基为活性必需基团。底物GLu对Arg残基的修饰有着很强的保护作用,因而可推测说明Arg残基可能位于酶的底物结合位点。米胚GAD的His和Arg残基被修饰后,荧光发射光谱有着非常相似的变化,Trp残基的荧光发射基本被猝灭了,说明酶蛋白微区的
构象发生了变化。
CD光谱分析表明,米胚GAD的二级结构中, α-螺旋占13.2%,β-折叠占38.3%,是一种高β-折叠蛋白。米胚GAD脱辅基后蛋白质的二级结构的变化是部分β-折叠结构转变为了回转结构。His和 Arg残基被修饰后,酶蛋白的二级结构有较大的变化,底物对Arg残基修饰引起的结构变化有保护作用。分析米胚GAD二级结构的变化与酶活性的关系发现,米胚GAD二级结构中α-螺旋结构的维持对酶的活性可能更为重要。
通过对水浸泡工艺进行优化,并利用Ca2+激活GAD,米胚中GABA的量可以由未富集前的28mg/100g提高到615mg/100g左右,比资料报道有较大提高。 利用胰蛋白酶水解可以进一步提高GABA的富集量,在最佳条件下,GABA产量可达2.3g/100g,比GABA-RG1提高了2倍。
外加Glu和添加米胚GAD的激活剂是制备高浓度GABA的有效方法。最佳工艺条件是:pH5.6、0.08mol/L磷酸缓冲液, PLP和Ca2+的添加量分别为2 mmol/molGlu和20mmol/molGlu,米胚的用量为42.5U/mmolGlu,料液比为1/10,反应时间和温度为6h和40℃。产品中GABA含量为20.6g/100g,比GABA-RG2又提高了7.8倍,并且Glu的转化率可以达到100%。
动物实验表明, GABA-RG可显著促进小鼠GH的分泌和后腿肌重的增长,对小鼠力竭游泳时间和爬杆时间有显著地延长作用。还可使运动后小鼠血乳酸的含量明显下降,血清尿素氮含量明显降低,肝糖原含量的显著提高。这些结果表明 GABA-RG能促进小鼠GH分泌并提高运动能力。根据国家卫生部关于保健性食品的评价指标,可以判定GABA-RG具有抗疲劳的作用。
This research was conducted to purify and characterize the GAD from rice germ, to study the properties of GAD, and to produce rice germ with high GABA content.
The methods for GABA assay in rice germ was studied. It was suggested that the colorimetry method could not be used because of the determination error. Paper chromatography was a suitable method, and can be used in combination with HPLC for GABA assay in rice germ.
GAD was purified from rice germ by ammonium sulfate fractionation, DEAE- Sephrose FF chromatography, Superdex 200 gel filtration, and Glu- Sephrose CL 4B affinity chromatography. Single band from SDS-PAGE showed the subunit Mw was 40k. Similar molecular weight (78k) was obtained by SE-HPLC. It was suggested that the GAD from rice germ had two homological subunits.
The purified GAD showed its maximal activity at pH5.6 and 40℃. The enzyme was inactivated completely at 75 ℃. It was stable at pH4.5-8.0. The Km and Vmax of GAD for Glu was 32.3mmol/L and 1.159mg/min; The Km and Vmax of GAD for PLP was 1.7μmol/L and 1.171mg/min.
GAD could be activated by Ca2+, the comparative activity reached 145% when 500μmol/L Ca2+ was added, and the apparent Km’ was changed to 25.9mmol/L, less than the Km for Glu. This showed that the Ca2+ enhanced the affinity between enzyme and Glu, and activated the enzyme.
The purified GAD could be activated by PLP. The enzyme was inactivated by removal of the bond PLP, but it could be renewed completely after PLP adding.
Absorbance spectra of GAD showed one weak peak at 420nm, it was produced by the binding of PLP. Fluorescence emission spectra of GAD was characterized by a Tyr peak, and had no change after PLP removed.
The modification of DIC and DEPC resulted in a great loss of GAD activity, suggesting that the His and Arg involve in the enzyme active site. The number of the essential Arg was estimated as one. The Glu could protect the modification of DIC, suggesting that the essential Arg may be in the substrate binding site of the enzyme.
Circular Dichroism analysis of GAD showed that the ratios of α-helix and β-sheet in the secondary structures were 13.2% and 38.3%. The secondary structures had a little change after PLP removed. The modification of DIC and DEPC resulted in great changes of the secondary structures. The α-helix might be more important to GAD activity.
Optimizing the conditions of GABA - accumulation by water soaking and protein hydrolyzing by trypsin could increase the content of GABA in GABA-RG to 615mg/100g and 2.3g/100g. But
a higher GABA content must be obtained using Glu and GAD activator. The optimized conditions were 0.08mol/L phosphate buffer (pH5.6),adding 2 mmol/molGlu of PLP and 20mmol/molGlu of Ca2+ , 42.5U/mmolGlu of rice germ, and the ratio of rice germ to solvent was 1:10, reaction time and temperature were 6h and 40℃. The content of GABA in the final product (GABA-RG3) was 20.6g/100g,the convert rate of Glu was 100%.
Finally, the new function of GABA-RG on growth hormone (GH) secretion stimulation and exercise performance enhancing of rats were studied. It showed that the GABA-RG stimulated GH secretion and enhanced leg muscle weight of rats obviously. The swimming time burden with 5% weight and climb time of rats were prolonged. The biological index of rats including blood lactic acid , BUN and liver glycogen were also improved.
Judging by relevant specifications of governmental health food standards, we can conclude that GABA-RG has anti-fatigue activities.
引文
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Hao R., Schmit J.C., Purification and characterization of glutamate decarboxylase from Neurospora crassa condia, J. Biol. Chem.,1991, Vol. 266: 5135-5149
Hao R., Schmit J.C.,Cloning of the gene for glutamate decarboxylase and its expression during condiation in Neurospora crassa, Biochem. J.,1993,Vol.293:735-738
Ueno Y., Hayakawa K., Takahashi S., and Oda K., Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005, Biosci. Biotech. Biochem., 1997,61(7):1168-1171
Sanders J.W.,Leenhouts K., Burghoorn J., et. al., A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation, Mole. Micrebio.,1998,Vol.27(2):299-310
Pamela L.C.Small and Scott R. Waterman, Acid stress, anaerobiosis and gadCB: lessons from Lactococcus lactis and Escherichia coli, Trends in Microbio., 1998,Vol.6(6):214-216
Lorca G.L., Devaldez G.F., A low-pH-indecible, stationary-phase acid tolerance response in Lactobacillus acidphilus CRL, Microbiology, 2001,Vol.42(1):21-25
Nomura M., Nakajima I., Fujita Y., et al. Lactococcus lactis contains only one glutamate decarboxylase gene, Microbiology, 1999, Vol.145:1375-1380
穆小民、沈黎明、吴显荣,林山生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究,生物化学杂志,1997,13(2):181-185
范军、李纯、朱苏文、程备久,小麦谷氨酸脱羧酶的分离纯化及部分性质的研究,中国生物化学与分子生物学学报,1998,14(5):641-644
Toshihiko Matsumoto, Izumi Yamaura and Masaru Funatsu, Purification and Properties Decarboxylase from Squash, Agric. Biol. Chem., 50(6):1413-1417
袁仕善、周智广, 谷氨酸脱羧酶若干研究进展,生理科学进展,1998,29(1):77-80
Areshev AG, Mamaeva OK, Andreeva NS, Sukhareva BS, Structure of glutamate decarboxylase and related PLP-enzymes: computer-graphical studies, J. Biomol. Struct. Dyn., 2000,18: 127-136
Salzmann D, Christen P, Mehta PK, Sandmeier E,Rates of evolution of pyridoxal-5'-phosphate-dependent enzymes, Biochem. Biophys. Res. Commun., 2000,270: 576-580.
Arazi T.,?Baum G.,?Snedden W.A.,?Shelp B.J., and?Fromm H., Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase, Plant Physiol.,?1995,108 (2): 551–561
Baum, G., Chen, Y., Arazi, T., Takatsuji, H., and Fromm, H., A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis, J. Biol. Chem., 1993,268, 19610-19617
Yevtushenko DP, McLean MD, Peiris S, Van Cauwenberghe OR, Shelp BJ Calcium/calmodulin activation of two divergent glutamate decarboxylases from tobacco, J. Exp. Bot., 2003,54: 2001-2002.
Yuan T, Vogel HJ,Calcium-calmodulin induced dimerization of the carboxyl-terminal domain from petunia: a novel calmodulin peptide interaction motif, J. Biol. Chem., 1998,273: 30328-30335.
Chen Y., Baum G., and?Fromm H., The 58-Kilodalton calmodulin-Binding glutamate decarboxylase is a ubiquitous protein in petunia organs and its expression is developmentally regulated, Plant Physiol., 1993,106:1381 -1387
Snedden WA, Arazi ., Fromm H., and Shelp BJ, Calcium/calmodulin activation of soybean glutamate decarboxylase, Plant Physiol., 1995, 108(2):543-549
Camas A, Cardenas L, Quinto C, Lara M , Expression of different calmodulin genes in bean (Phaseolus vulgaris L.): Role of nod factor on calmodulin gene regulation, Mol. Plant Microbe Interact, 2002,15: 428-436
Baum, G.,Lev-Yadum S., Fridmann Y., et al., Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.1996, EMBO J.,15:2988-2996
Catherine P., Scott-Taggart, et al., Regulation of γ- aminobutyric acid synthesis in situ by glutamate decarboxylase, Physiologia Plantarum, 1999,106:363-369
Schmidli RS, Colman PG, Bonifacio E, et al., Disease sensitivity and specificity of 52.assays for glutamic acid decarboxylase antibodies, Diabetes, 1995, 44:636-640
Kaufman DL, Clare-Salzler M, et al., Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature, 1993,
Tian J, Atkinson MA, Clare-Salzler M, et al., Nasal administation of glutamate decarboxylase(GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J Exp Med, 1996,183:1561-1567
章汝平,何立芳,用后道味精母液提取谷氨酸后的废液生产γ-氨基丁酸,长沙电力学院学报(自
然科学版),1998,Vol.13(4):433-435
余敦寿,崔俊鑫,在γ-氨酪酸生产中提高谷氨酸脱羧酶活力的研究,氨基酸杂志,1991,4:9-11
Rice E.W., Johnson M.E., and Reasoner D.J., Rapid glutamate decarboxylase assay for detection of Escherichia coli, Applied Environment Microbiology,1993, 59(12):4347-4349
Michael A., Grant, Stephen D.W., and Peter F., Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups, Applied Environment Microbiology,2001, 76(7):3110-3114
愛岩 世高等, Development of a super-GABA by Lactic Acid Fermentation,食品与开发,日刊,Vol.36(6):12-14
乃用,短乳杆茵用酒糟生产γ-氨基丁酸,工业微生物,2002, Vol.32 (3):62
Development of New Ingredients Riched with GABA, 食品与开发,日刊,Vol.36(6): 17-18
Omori M., Kato M., Tamma T., et al., Production of CTG gabaron black tea by modifying withering condition, Tea, 1998, 19(2):92-96
林智,大森正司,γ-氨基丁酸茶(Gabaron Tea)降血压机理的研究,茶叶科学,2001,21(2)153-156
林智,大森正司,γ-氨基丁酸茶成分对大鼠血管紧张素Ⅰ转换酶(ACE)活性的影响,茶叶科学,2002,22(1):43-46
杉下、朋子,The Research and Development of Rice Germ Enrich with GABA ,食品与开发,日刊,Vol.36(6) :10-11
伊藤,γ-氨基丁酸高含量米糠的制造方法,食品工业,日刊,2000(10):43-45
冈田忠司等,Physiological Function of Rice Germ Enriched with GABA, , 食品与开发,日刊,Vol.36(6):7-9
茅原等,Recent Studies on Biological Functions of GABA-on Improvements of Hypertension and Brain Function, 食品与开发,日刊,Vol.36(6): 4-6
冈田忠司等,Effect of the Deffatted Rice Germ Enriched with GABA for Sleeplessness,Depression,Autonomic Disorder by Oral Administration, Nippon Shokuhin Kagaku Kaishi, Vol.47(8): 596-603
Ogawa Y.,Yamaguchi H.Shimaoka Y,γ-aminobutyric acid-containing natural food material and method for manufacturing the same, United States Patent, 2002 ,20020106424
许仁溥,发芽糙米开发,粮食与油脂,2001,No.8:37-38
游祖持,日本对于超γ-氨基丁酸研究与利用,食品与发酵工业2002,No.3:-42-43
特開2001-014356 GABA含有飲食品の製造方法
杨柳,叶蕴华,袁洪生等,人参中γ-氨基丁酸的分离、鉴定及氨基酸的定量分析,科学通报,1991,No.7, 513-515
吴锦忠,李向高,鲜人参与红参氨基酸成分比较研究,中药材,1990,13(5):27-28
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Shukuya R. and Schwert G.W., Glutamic acid decarboxylase,Ⅱthe inactivation of the enzyme at low temperatures, J. Biol. Chem., 1960,Vol.235(6):1658-1661
Maras B., Sweeney G., Barra D., Bossa F., and John R. A., The amino acid sequence of glutamate decarboxylase from Escherichia coli, Evolutionary relationship between mammalian and bacterial enzymes, Eur. J. Biochem., 1992,Vol.204:93-98
Hao R., Schmit J.C., Purification and characterization of glutamate decarboxylase from Neurospora crassa condia, J. Biol. Chem.,1991, Vol. 266: 5135-5149
Hao R., Schmit J.C.,Cloning of the gene for glutamate decarboxylase and its expression during condiation in Neurospora crassa, Biochem. J.,1993,Vol.293:735-738
Ueno Y., Hayakawa K., Takahashi S., and Oda K., Purification and characterization of glutamate decarboxylase from Lactobacillus brevis IFO 12005, Biosci. Biotech. Biochem., 1997,61(7):1168-1171
Sanders J.W.,Leenhouts K., Burghoorn J., et. al., A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation, Mole. Micrebio.,1998,Vol.27(2):299-310
Pamela L.C.Small and Scott R. Waterman, Acid stress, anaerobiosis and gadCB: lessons from Lactococcus lactis and Escherichia coli, Trends in Microbio., 1998,Vol.6(6):214-216
Lorca G.L., Devaldez G.F., A low-pH-indecible, stationary-phase acid tolerance response in Lactobacillus acidphilus CRL, Microbiology, 2001,Vol.42(1):21-25
Nomura M., Nakajima I., Fujita Y., et al. Lactococcus lactis contains only one glutamate decarboxylase gene, Microbiology, 1999, Vol.145:1375-1380
穆小民、沈黎明、吴显荣,林山生山黧豆谷氨酸脱羧酶的分离纯化及部分性质的研究,生物化学杂志,1997,13(2):181-185
范军、李纯、朱苏文、程备久,小麦谷氨酸脱羧酶的分离纯化及部分性质的研究,中国生物化学与分子生物学学报,1998,14(5):641-644
Toshihiko Matsumoto, Izumi Yamaura and Masaru Funatsu, Purification and Properties Decarboxylase from Squash, Agric. Biol. Chem., 50(6):1413-1417
袁仕善、周智广, 谷氨酸脱羧酶若干研究进展,生理科学进展,1998,29(1):77-80
Areshev AG, Mamaeva OK, Andreeva NS, Sukhareva BS, Structure of glutamate decarboxylase and related PLP-enzymes: computer-graphical studies, J. Biomol. Struct. Dyn., 2000,18: 127-136
Salzmann D, Christen P, Mehta PK, Sandmeier E,Rates of evolution of pyridoxal-5'-phosphate-dependent enzymes, Biochem. Biophys. Res. Commun., 2000,270: 576-580.
Arazi T.,?Baum G.,?Snedden W.A.,?Shelp B.J., and?Fromm H., Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase, Plant Physiol.,?1995,108 (2): 551–561
Baum, G., Chen, Y., Arazi, T., Takatsuji, H., and Fromm, H., A plant glutamate decarboxylase containing a calmodulin binding domain. Cloning, sequence, and functional analysis, J. Biol. Chem., 1993,268, 19610-19617
Yevtushenko DP, McLean MD, Peiris S, Van Cauwenberghe OR, Shelp BJ Calcium/calmodulin activation of two divergent glutamate decarboxylases from tobacco, J. Exp. Bot., 2003,54: 2001-2002.
Yuan T, Vogel HJ,Calcium-calmodulin induced dimerization of the carboxyl-terminal domain from petunia: a novel calmodulin peptide interaction motif, J. Biol. Chem., 1998,273: 30328-30335.
Chen Y., Baum G., and?Fromm H., The 58-Kilodalton calmodulin-Binding glutamate decarboxylase is a ubiquitous protein in petunia organs and its expression is developmentally regulated, Plant Physiol., 1993,106:1381 -1387
Snedden WA, Arazi ., Fromm H., and Shelp BJ, Calcium/calmodulin activation of soybean glutamate decarboxylase, Plant Physiol., 1995, 108(2):543-549
Camas A, Cardenas L, Quinto C, Lara M , Expression of different calmodulin genes in bean (Phaseolus vulgaris L.): Role of nod factor on calmodulin gene regulation, Mol. Plant Microbe Interact, 2002,15: 428-436
Baum, G.,Lev-Yadum S., Fridmann Y., et al., Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants.1996, EMBO J.,15:2988-2996
Catherine P., Scott-Taggart, et al., Regulation of γ- aminobutyric acid synthesis in situ by glutamate decarboxylase, Physiologia Plantarum, 1999,106:363-369
Schmidli RS, Colman PG, Bonifacio E, et al., Disease sensitivity and specificity of 52.assays for glutamic acid decarboxylase antibodies, Diabetes, 1995, 44:636-640
Kaufman DL, Clare-Salzler M, et al., Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature, 1993,
Tian J, Atkinson MA, Clare-Salzler M, et al., Nasal administation of glutamate decarboxylase(GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J Exp Med, 1996,183:1561-1567
章汝平,何立芳,用后道味精母液提取谷氨酸后的废液生产γ-氨基丁酸,长沙电力学院学报(自
然科学版),1998,Vol.13(4):433-435
余敦寿,崔俊鑫,在γ-氨酪酸生产中提高谷氨酸脱羧酶活力的研究,氨基酸杂志,1991,4:9-11
Rice E.W., Johnson M.E., and Reasoner D.J., Rapid glutamate decarboxylase assay for detection of Escherichia coli, Applied Environment Microbiology,1993, 59(12):4347-4349
Michael A., Grant, Stephen D.W., and Peter F., Glutamate decarboxylase genes as a prescreening marker for detection of pathogenic Escherichia coli groups, Applied Environment Microbiology,2001, 76(7):3110-3114
愛岩 世高等, Development of a super-GABA by Lactic Acid Fermentation,食品与开发,日刊,Vol.36(6):12-14
乃用,短乳杆茵用酒糟生产γ-氨基丁酸,工业微生物,2002, Vol.32 (3):62
Development of New Ingredients Riched with GABA, 食品与开发,日刊,Vol.36(6): 17-18
Omori M., Kato M., Tamma T., et al., Production of CTG gabaron black tea by modifying withering condition, Tea, 1998, 19(2):92-96
林智,大森正司,γ-氨基丁酸茶(Gabaron Tea)降血压机理的研究,茶叶科学,2001,21(2)153-156
林智,大森正司,γ-氨基丁酸茶成分对大鼠血管紧张素Ⅰ转换酶(ACE)活性的影响,茶叶科学,2002,22(1):43-46
杉下、朋子,The Research and Development of Rice Germ Enrich with GABA ,食品与开发,日刊,Vol.36(6) :10-11
伊藤,γ-氨基丁酸高含量米糠的制造方法,食品工业,日刊,2000(10):43-45
冈田忠司等,Physiological Function of Rice Germ Enriched with GABA, , 食品与开发,日刊,Vol.36(6):7-9
茅原等,Recent Studies on Biological Functions of GABA-on Improvements of Hypertension and Brain Function, 食品与开发,日刊,Vol.36(6): 4-6
冈田忠司等,Effect of the Deffatted Rice Germ Enriched with GABA for Sleeplessness,Depression,Autonomic Disorder by Oral Administration, Nippon Shokuhin Kagaku Kaishi, Vol.47(8): 596-603
Ogawa Y.,Yamaguchi H.Shimaoka Y,γ-aminobutyric acid-containing natural food material and method for manufacturing the same, United States Patent, 2002 ,20020106424
许仁溥,发芽糙米开发,粮食与油脂,2001,No.8:37-38
游祖持,日本对于超γ-氨基丁酸研究与利用,食品与发酵工业2002,No.3:-42-43
特開2001-014356 GABA含有飲食品の製造方法
杨柳,叶蕴华,袁洪生等,人参中γ-氨基丁酸的分离、鉴定及氨基酸的定量分析,科学通报,1991,No.7, 513-515
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