荔枝果皮过氧化物酶的纯化及部分酶学性质研究

Purification and Some Properties of Peroxidase from Pericarp of Litchi (Litchi chinensis Sonn.)

摘要:

经硫酸铵分级盐析、DEAE-Sepharose和SephadexG-75柱层析分离,从荔枝果皮中分离提纯了过氧化物酶(POD),该酶被纯化了12.5倍,产率为1.9%。经SDS-PAGE确定为单一条带。该酶最适反应温度为35℃,对热具有较强的稳定性,经75℃处理30min,酶活性只损失50%。最适pH约为6.5,但在pH4.0-8.0范围内活力仍比较稳定。该酶在25℃和0.05 mol/L磷酸缓冲液(pH7.0)条件下对愈创木酚、邻苯二酚和没食子酸的Km分别是2.75、12.4和12.8 mmol/L。二硫苏糖醇和抗坏血酸能完全抑制POD活性,L-半胱氨酸、柠檬酸、FeSO₄、GSH、SDS和ZnSO₄对POD活性有一定的抑制作用,而FeCl₃和CuSO₄对POD则有较好的激活作用。

Abstract:

Peroxidase(POD) from pericarp of litchi(Litchi chinensis Sonn.) was purified by 12.5-fold and a 1.9% yield using ammonium sulfate, DEAE-Sepharose and Sephadex G-75. The enzyme was determined to be homogenous by SDS-PAGE, which was relatively heat stable with a optimum temperature of 35℃, requiring a time of at least 30 min at 75℃ for 50% loss of the activity. Litchi POD had a optimum pH at 6.5, but it was stable within a range of pH 4.0-8.0. The apparent Km values with guaiacol, catechol and pyrogallol as substrates were 2.75, 12.4 and 12.8 mmol/L at 25oC and pH 7.0，respectively．The presence of Fe³⁺ and Cu²⁺ enhanced POD activity．L-cysteine，citrate acid,FeSO₄，SDS，GSH and ZnS0₄ partially inhibited POD activity,but it was completely inhibited by dithiothreitol and ascorbate.