摘要
［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。
[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/rag 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 miehaelis constant Km of 2.67 mg/ml.
出处
《安徽农业科学》
CAS
北大核心
2009年第20期9366-9368,9374,共4页
Journal of Anhui Agricultural Sciences
基金
宝鸡文理学院校级重点项目(ZK08151)
关键词
Β-甘露聚糖酶
纯化
酶学性质
β-manuanase
Purification
Enzymatic properties
