| 摘要: |
| 通过冷冻干燥、DEAE Sepharose Fast Flow阴离子交换柱层析、Sephadex G 100凝胶过滤柱层析，SP葡聚糖凝胶C 25阳离子交换柱层析等分离纯化技术，对烟草吡哆胺 丙酮酸转氨酶进行分离纯化。采用苯肼衍生化方法检测活性，并对其基本酶学性质进行分析。结果显示：该酶被纯化了92.34倍；最适温度为70 ℃，最适pH为9.0。在pH7.0～9.0内稳定且热稳定性较好，80 ℃保温3 h仍有51.55%的酶活力；在最适反应条件下，测得反应底物吡哆胺和丙酮酸的Km值分别为6.337 mmol•L 1和0.867 mmol•L 1。该结果为进一步研究烟草体内VB6代谢机制奠定了基础。 |
| 关键词: 烟草 吡哆胺 丙酮酸转氨酶 纯化 性质分析 |
| DOI： |
| 分类号:Q945.14 |
| Fund project: |
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| Purification and enzymatic characterization of pyridoxamine pyruvate aminotrans ferase from the Tobacco |
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WU Qiong1， HUANG LongQuan1， ZHANG JianYun2*
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1.College of Tea & Food Sciences， Anhui Agricultural University， Hefei 230036， China;2. 2.College of Life Sciences， Anhui Agricultural University， Hefei 230036， China
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| Abstract: |
| The pyridoxamine pyruvate aminotransferase was purified from the tobacco leaf by freeze drying，DEAE Sepharose Fast Flow ion exchange chromatography，Sephadex G 100 gel filtration and SP Sephadex C 25 ion exchange chromatography. The enzymatic activity and properties were investigated with phenyl hydrazine method. The results showed〖JP3〗 that 92.34 fold purification was obtained；the enzyme had an optimum temperature at 70 ℃ and pH at 90，and it was stable at pH7.0-9.0；the enzyme had good thermal stability which remained about 51.55% of its activity after being treated at 80 ℃ for 3 h；under optimal conditions，the Km values for pyridoxamine and pyruvate were 6.337 mmol•L 1 and 0.867 mmol•L 1. The results provided basis for the metabolic mechanism of VB6 in tobacco plants. |
| Key words: tobacco pyridoxamine pyruvate aminotransferase purification properties |
