摘要
使用木霉TP09固态发酵,提纯并鉴定了β-1,3-葡聚糖酶的部分性质。采用饱和度为70%的(NH4)2SO4及DEAE-琼脂糖凝胶CL-6B柱层析纯化,β-葡聚糖酶相对粗酶溶液纯化了28.7倍,酶回收率为45.2%。经SDS-PAGE分析,该酶分子量近似54.6KD。酶最适反应pH为5.0,最适温度为50℃。在pH3.0-5.0、温度30℃-70℃之间酶活相对稳定。Fe3+、Mg2+、Mn2+以及Cu2+对该酶有抑制作用,Zn2+、Ca2+和Fe2+则有激活作用。底物选择性研究表明该酶为β-1,3、β-1,4-葡聚糖酶。该酶可作用于含β-1,3、β-1,4糖苷键的底物,对含α-1,4和α-1,6糖苷键的底物无作用。以上表明分离自啤酒废水的木霉TP09生产的β-1,3-葡聚糖酶可增强不溶性β-1,3-葡聚糖的可溶性,促进了其在免疫疗法中的应用。
As an initial investigation to improve the insoluble yeast β-1, 3-glucan solubility, a novel β-glucanase from Trichoderma viride TP09 was purified in the culture supernatant and partially characterized. By 70% saturation ammonium sulfate and chromatography on DEAE-Sepharose CL-6B column, β-glucanase was purified 28.7-fold, with recovery of 45.2% of the initial activity. The molecular weight of this enzyme was estimated to be 54.6 KD by SDS-PAGE. The optimum pH and the optimum temperature for the enzyme were 5.0 and 50 ℃, respectively. The enzyme showed high stability within the range of pH 3.0-5.0 and thermostability between 30 and 70 o C. The enzyme activity was inhibited by Fe3＋, Mg2＋, Mn2＋, Cu2＋, and stimulated by Zn2＋, Ca2＋, Fe2＋. Substrate specificity studies revealed the enzyme to be a β-1, 3-1, 4-glucanase. The β-glucanase showed preference for β--1, 3 linkage and β--1, 4 linkage, but had no activity on α -1, 4 and a -1, 6 linkage. The above results indicated that the enzyme extracted from T. viride TP09 of the beer-making sewage could be used as a potential predominant tool to enhance solubility of the insoluble yeast β-1, 3-glucan. These findings may lead to an enhanced solubility and expedite the progress of application in immunotherapy.
出处
《山东食品发酵》
2008年第4期46-50,共5页
Shandong Food Ferment
关键词
木霉
Β-葡聚糖酶
酶提纯
Trichoderma viride- β-Glucanase-Enzyme purification
