【作者】 张晖；

【导师】 姚惠源；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2004， 博士

【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<WP=10>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。更多还原

【Abstract】 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 <WP=12>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.更多还原