金属离子及保护剂对深绿木霉T155产芥子酶的活性影响及芥子酶的分离纯化

Effects of metal ions and protectants on myrosinase activity and purification of myrosinase from Trichederma atroviride T155

摘要:

【目的】探求金属离子及保护剂对深绿木霉T155产生的粗芥子酶液活性的影响以及芥子酶的分离纯化方法。【方法】通过添加不同浓度的金属离子及保护剂研究对芥子酶活性的影响并通过细胞破壁、硫酸铵沉淀、透析、Sephadex G-100柱层析、DEAE-52离子交换层析、Sephadex G-200柱层析等方法提取到纯的芥子酶，最后通过电泳检测芥子酶的纯度和分子量。【结果】研究发现Ag+、Zn2+、Pb2+、Mg2+、Hg2+、Fe3+等金属离子在低浓度时对芥子酶均表现为一定的促进作用，但达到一定浓度后则起抑制作用，Ca2+浓度在0.069–17.000 g/L范围内均对酶活性起到促进作用，且促进效果基本与Ca2+浓度成正比；添加1 mmol/L EDTA和3 mmol/L DTT对芥子酶的保护作用最好，4 °C保存26 d后芥子酶能够保持85%活性；电泳分析该芥子酶的分子量大约在150 kD左右，并且是一个二聚体。【结论】Ca2+在实验浓度内主要是提高芥子酶活性，而其他金属离子对芥子酶活性主要起抑制作用；EDTA或者EDTA与DTT协同能够较好保持芥子酶活性的稳定性；通过一系列分离纯化步骤得到了纯的二聚体芥子酶，研究结果为挖掘产芥子酶的微生物资源提供了新的途径和思路。

Abstract:

[Objective] To examine the effects of metal ions and protectants on activity of myrosinase and explore purification method of myrosinase produced by Trichederma atroviride T155. [Methods] By concentration of metal ions and protectants to study of the myrosinase activity was measured by adding different ions with different concentration and three protectants, respectively. The pure myrosinase was obtained by cell disruption, ammonium sulfate precipitation, dialysis, Sephadex G-100 chromatography, DEAE-52 chromatography and Sephadex G-200 chromatography in order. [Results] The results showed that Ag+, Zn2+, Pb2+, Mg2+, Hg2+, Fe3+ promoted enzyme activity of myrosinase at low concentration and inhibited enzyme activity at high concentration, Ca2+ promoted enzyme activity at the concentration of 0.069-17.000 g/L. 1 mmol/L EDTA associated with 3 mmol/L DTT was the best treatment for keeping myrosinase activity, and 85% enzyme activity was retained after 27 days of preservation at 4 °C. Molecular weight of the myrosinase produced by T155 was about 150 kD by SDS-PAGE, and this myrosinase was a dimer. [Conclusion] The tested metal ions inhibited myrosinase activity except for Ca2+, EDTA or associated with DTT showed preferable effect on maintaining myrosinase activity. A dimer myrosinase was obtained by separation and purification technology. This study intended to provide a new way and idea for mining myrosinase producing microorganism.