[Objective]The study aimed to separate and purify β-mannanase produced by A. Niger with solid state fermentation and study the enzymatic properties of β-mannanase. [Method] Crude enzyme liquid was prepared with solid state fermentation. Β-mannanase was separated and purified from the crude enzyme liquid by (NH4)2SO4 segmentation precipitation, acetone precipitation, and Sephadex gel chromatography, resp., and its purity was detected by PAGE. Enzymatic properties of purified β-mannanase were measured. [Result] The specific activity of β-mannanase could be increased to 1 180.9 U/mg through (NH4)2SO4 precipitation with saturation of 40%～90%; that could be increased to 1 847.0 U/mg through acetone precipitation with the volume ratio of 1.0:1.0-1.6:1.0; and finally, that could be increased to 7 950.4 U/mg and the purification fold was 8.67 through gel chromatography. It showed one single band on PAGE gel, which was purified β-mannanase.[Conclusion] The optimum pH value of purified β-mannanase had the enzymatic properties as follows: the optimum pH value of 4.2, the optimum reaction temperature of 60 ℃ and the michaelis constant Km of 2.67 mg/ml.%[目的]分离纯化黑曲霉固态发酵产生的β-甘露聚糖酶,研究β-甘露聚糖酶酶学性质.[方法] 黑曲霉经固态发酵制备粗酶液,分别采用硫酸铵分段沉淀法、丙酮沉淀法和Sephadex凝胶层析法对β-甘露聚糖酶进行分离纯化,用PAGE检验其纯度.同时测定纯化后的β-甘露聚糖酶酶学性质.[结果] β-甘露聚糖酶经40%～90%饱和度硫酸铵沉淀法纯化后比活力可提高到1 180.9 U/mg;经1.0 :1.0～1.6:1.0(V/V)丙酮沉淀法纯化后的比活力可提高到1 847.0 U/mg;最后经凝胶层析法纯化后的比活力可提高到7 950.4 U/mg,纯化倍数为8.67,在PAGE凝胶电泳图谱上得到单一条带,即纯化后的β-甘露聚糖酶.[结论] 纯化后β-甘露聚糖酶的酶学性质为:最适pH值4.2,最适反应温度60 ℃,米氏常数Km 2.67 mg/ml.
展开▼
