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【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】 To isolate and purify pectinase from apple tree rot fungi and clarify its enzymatic properties. 【Method】 The apple tree rot bacteria were fermented at different days using 0.5% starch MS medium, and the pectinase activity was quantitatively determined by DNS. Pectinase was purified by ammonium sulfate gradient salting-out, Sephacryl S-100 gel filtration chromatography and anion exchange chromatography DEAE-Sepharose Fast Flow. The purity of pectinase was determined by SDS-PAGE and its enzymatic properties were analyzed by biochemistry . 【Result】 The results showed that the pectinase activity was the highest in the fermentation broth for 10 days. The isolated pectinase was rhamnogalacturonase with a molecular weight of 58.83 kD, an isoelectric point of 6.03 and an optimum reaction temperature of 40 ° C, the optimum reaction pH is 3.5, the enzyme activity is stable at pH 2.0-5.5. Ca 2+, Li 2+, Co 2+ could activate the enzyme activity, and K +, Fe 2+, Pb 2+, Zn 2+, Cu 2+, ~ (2 +), Mn ~ (2 +) and Ni + can inhibit the activity of enzyme, while Ba ~ (2+) and Mg ~ (2+) passivate the enzyme activity. The kinetic constants Km and Vm were 3.600 g / L and 0.162 7 g / (L · min), respectively. 【Conclusion】 Rhamnose galacturonase was isolated from the fermentation broth of apple tree rot fungi and its enzymatic properties were clarified, which laid the foundation for the preparation of pectinase antibody and cytochemistry.