烟草吡哆胺-丙酮酸转氨酶的部分纯化与表征

Purification and enzymatic characterization of pyridoxamine-pyruvate aminotrans-ferase from the Tobacco

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3h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6.337mmol·L-1和0.867mmol·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。

The pyridoxamin^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 ex- change chromatography. The enzymatic activity and properties were investigated with phenyl hydrazine method. The results showed that 92. 34-fold purification was obtained; the enzyme had an optimum temperature at 70℃ and pH at 9. 0,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.