| 摘要: |
| [目的]分离纯化米胚芽谷氨酸脱羧酶(Glutamate decarboxylase ,GAD),并对其酶学性质进行研究.[方法] 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,对米胚芽GAD进行分离纯化.用体积排阻高效液相色谱测定米胚GAD的相对分子质量,SDS-PAGE测定其亚基的相对分子质量.测定米胚GAD的最适温度和pH,通过酶动力学试验测定其动力学常数(Km)和最大反应速度(Vmax),并测定KCl和MgSO4等化学物质对酶活性的影响.[结果] 分离纯化得到了米胚GAD,其纯化倍数为65.7,比活达223.4 U/mg,酶活回收率为10.8%.米胚GAD的相对分子质量为78 ku,亚基相对分子质量为40 ku,说明米胚GAD是由2个相同亚基构成的二聚体.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,80 ℃时酶几乎完全失活;米胚GAD在pH 4.5～8.0较为稳定,能保持88%以上的酶活.米胚GAD对谷氨酸Glu的Km为32.3 mmol/L,Vmax为1.159 mg/min;对磷酸吡哆醛(PLP)的Km为1.7 μmol/L,Vmax为1.171 mg/min.K+ 和Ag2+ 对米胚GAD的活力有较大抑制,Mg2+ 、Mn2+ 、Al3+ 和Li2+ 等对活性影响不大,500 μmol/L Ca2+ 对米胚GAD有较强的激活作用.[结论] 米胚GAD的性质与其他植物GAD存在差异. |
| 关键词: 米胚芽 谷氨酸脱羧酶 酶学性质分析 |
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| 基金项目:国家自然科学基金项目(20671044)；新世纪优秀人才支持计划项目（NCET-06-0488） |
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| Purification and characterization of glutamate decarboxylase from rice germ |
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| Abstract: |
| 【Objective】 The study purified and researched the properties of rice germ glutamate decarboxylase(GAD). 【Method】 GAD was purified 65.7-fold from rice germ using a combination of ammonium sulfate fractionation,DEAE-Sepharose FF ion exchange chromatography,Superdex-200 gel filtration chromatography,and Glu-Sepharose CL 4B affinity chromatography.SE-HPLC was used to determine the relative molecular weight (Mr) of GAD,and SDS-PAGE was used to determine the Mr of the subunit of GAD.Then the optimum temperature and pH,Km of GAD was tested.【Result】 The purified preparation showed a single peak on SE-HPLC with an approximate molecular mass of 78 ku and a single band on SDS-PAGE with a subunit Mr of 40 ku.This indicated that the GAD from rice germ existed as a dimer of homological subunits.Rice germ GAD had an optimum pH at 5.6,and an optimum temperature at 40 ℃.Rice germ GAD inactived at 80 ℃,but it was stable at pH 4.5-8.0.Km values for glutamic acid and PLP were determined at 32.3 mmol/L and 1.7 μmol/L,respectively.Chemicals reagents such as HgCl2,KI and AgNO3 decreased the enzyme activity, but 500 μmol/L of CaCl2 at the optimum pH could increase the GAD activity greatly. 【Conclusion】 GAD of rice germ has some properties different from GAD of other plants. |
| Key words: rice germ glutamate decarboxylase enzymatic character |
