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【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 SephroseCL 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1.7μmol/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,500μmol/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。
【Objective】 Isolation and purification of rice germ glutamate decarboxylase (GAD) and its enzymatic properties were studied. 【Method】 The GAD of rice germ was isolated and purified by (NH4) 2SO4 fractionation, DEAE-Sepharose FF ion exchange chromatography, Superdex 200 gel filtration chromatography and Glu Sephrose CL 4B affinity chromatography. The molecular weight of GAD in rice embryo was determined by size-exclusion high-performance liquid chromatography, and the relative molecular mass of its subunit was determined by SDS-PAGE. The optimum temperature and pH of rice embryo GAD were determined. The kinetic constants (Km) and maximum reaction velocity (Vmax) were determined by enzyme kinetics. The effects of chemical substances such as KCl and MgSO4 on the enzyme activity were also determined. 【Result】 The result showed that the GAD of rice embryo was obtained by purification and purification, with a specific activity of 65.7 and a specific activity of 223.4 U / mg. The enzyme activity recovery was 10.8%. The relative molecular mass of rice germ GAD was 78 ku and the molecular weight of the subunit was 40 ku, indicating that the rice embryo GAD is a dimer consisting of two identical subunits. The optimal reaction temperature of GAD was 40 ℃ and the optimum pH was 5.6. The thermal stability of GAD was poor and the enzyme was almost completely inactivated at 80 ℃. The GAD of rice embryo was stable at pH 4.5-8.0, More than 88% enzyme activity. The Km of rice GAD glutamate Glu was 32.3 mmol / L, Vmax was 1.159 mg / min, Km for pyridoxal phosphate (PLP) was 1.7 μmol / L and Vmax was 1.171 mg / min. K + and Ag2 + inhibited the activity of GAD in rice embryos. Mg2 +, Mn2 +, Al3 + and Li2 + had little effect on the activity of GAD. 500μmol / L Ca2 + had a strong activation on GAD of rice. 【Conclusion】 The characteristics of GAD in rice embryos are different from those in other plants.