苹果树腐烂病菌胞外果胶酶分离纯化及其性质

Isolation, purification and characterization of extracellular pectinase produced by Valsa mali

【目的】分离纯化苹果树腐烂病菌的果胶酶,明确其酶学性质。【方法】利用0.5%淀粉MS培养基对苹果树腐烂病菌分别进行不同天数发酵,DNS法定量测定果胶酶活性。通过硫酸铵梯度盐析、Sephacryl S-100凝胶过滤层析和阴离子交换层析DEAE-Sepharose Fast Flow分离纯化果胶酶,经SDS-PAGE检测样品纯度,并利用生物化学技术分析其酶学性质。【结果】发酵10 d的发酵液中果胶酶活性最高;分离得到的果胶酶为鼠李糖半乳糖醛酸酶,分子量为58.83 k D,等电点为6.03,最适反应温度为40°C,最适反应pH为3.5,在pH 2.0-5.5之间酶活性比较稳定。Ca^(2+)、Li^+、Co^(2+)对酶活力有激活作用,K^+、Fe^(2+)、Pb^(2+)、Zn^(2+)、Cu^(2+)、Mn^(2+)、Ni+对酶活有抑制作用,Ba^(2+)和Mg^(2+)对酶活性有钝化作用。酶动力学常数Km和Vm值分别是3.600 g/L和0.162 7 g/(L·min)。【结论】从苹果树腐烂病菌的发酵液中分离得到鼠李糖半乳糖醛酸酶并明确了其酶学性质,为果胶酶抗体的制备和细胞化学研究奠定基础。

[Objective] The purpose of this study was to separate and purify the pectinase of Valsa mali, and explore the corresponding enzymatic properties. [Methods] Valsa mali was cultured in MS medium containing 0.5% starch, and maintained for different days. The pectinase was extracted using ammonium sulphate gradient fractionation and purified by Sephacryl S-100 gel fitration and DEAE-Sepharose Fast Flow ion exchange chromatography. The purity was examined by SDS-PAGE. The properties of rhamnose galacturonic acid enzyme were studied using biochemical techniques. [Results] The result showed that the activity of the pectinase was highest after 10 days＇ fermentation. The pectinase was identified to be rhamnose galacturonic acid enzyme. The properties including the molecular mass, the isoelectric point, the optimal temperature and p H were 58.83 k D, 6.03, 40 ℃ and 3.5, respectively. The rhamnose galacturonic acid enzyme was stable when its p H varied from 2.0 to 5.5. Ca^2＋, Li＋, Co^2＋ could active the rhamnose galacturonic acid enzyme, while the metal ions of K＋, Fe^2＋, Pb^2＋, Zn^2＋, Cu^2＋, Mn^2＋, Ni＋ could inhibit it, especially Ba^2＋ and Mg^2＋. Besides, the kinetic constants was also detected, and the Km and Vm values were 3.600 g/L and 0.162 7 g/（L·min）, respectively. [Conclusion] The rhamnose galacturonic acid enzyme was isolated from the fermentation liquor of V. mali, and enzymatic properties were explored. The results will lay the foundation for the preparation of antibody to pectinase and studies of cytochemistry.