黑曲霉胞外β-葡萄糖苷酶的纯化及酶学性质的研究

Purification of β-glucosidase from Aspergillus niger and its Enzymological Character

利用硫酸铵盐析、Superdex 200凝胶柱层析等步骤,从黑曲霉发酵液中纯化得到两种β–葡萄糖苷酶蛋白,其相对分子质量分别为1.15×105和7.0×104左右,比活力分别为62.1,U/mg（相对分子质量约为1.15×10^5）和53.0,U/mg（相对分子质量约为7.0×10^4）,纯化倍数分别为1.54和1.31,回收率分别为19.6%,和19.1%,β–葡萄糖苷酶最适水解p H为4.6,最适反应温度为50~55,℃.在p H 4.2~4.8、温度40~60,℃下均能保持稳定.Mn2＋、Mg2＋和K＋对β–葡萄糖苷酶有不同程度的激活作用,而Ca^2＋、Fe^3＋、Zn^2＋、Cu^2＋和Fe^2＋会抑制β–葡萄糖苷酶的酶活,Na＋和Ba2＋对β–葡萄糖苷酶活力影响不明显.以p NPG为底物时,该酶的Km和vmax分别为2.33,mmol/L与3.14,mmol/（L·min）.

Two kinds of beta-glucosidase protein were separated and purified from Aspergillus niger through ammonium sulfate precipitation and Superdex 200 gel filtration chromatography. The enzyme molecular weights were about 1.15 × 10^5 and 7.0×10^4, which were identified by SDS-PAGE, The specific activities were 62.1 U/mg and 53.0 U/mg. And then they were purified to 1.54-fold and 1.31-fold with a recovery of 19.6% and 19.1%. The optimum reaction pH and temperature for beta-glucosidase were 4.6 and 50-55 ℃. The enzyme is stable in the pH value range of 4.2-4.8 and at the temperature be- tween 40 ℃ to 60 ℃. Mn^2＋, Mg^2＋ and K＋ have different degree of activation to the beta-glucosidase, while Ca^2＋, Fe^3＋, Zn^2＋, Cu^2＋ and Fe^2＋ can inhibit the activity of the beta-glucosidase. Na^＋ and Ba^2＋ have no obvious effect on the activity. The Km and vmax values of the enzyme are 2.33 mmol/L and 3.14 mmol/（L.min） respectively.