嗜碱芽孢杆菌N16-5 β-甘露聚糖酶的纯化与性质

PURIFICATION AND PROPERTIES OF β-MANNANASES FROM ALKALOPHILIC BACILLUS N16-5

嗜碱芽孢杆菌(Bacillus sp.)N16-5产生的三种胞外碱性β-甘露聚糖酶(β-mannana-se)经硫酸铵沉淀、两次 DEAE-Sephadex A-25离子交换柱层析、羟基磷灰石吸附柱层析及制备 PAGE 等步骤,得到了凝胶电泳均一的样品。用 SDS-PAGE 测得纯化的β-甘露聚糖酶M-1、M-2和 M-3的分子量分别为51000、38000和34700道尔顿。用 PAGEIEF 测得等电点 pI 分别为4.3、2.5和2.5。酶反应的最适 pH 为9.0(M-1)和10.0(M-2和 M-3);三种酶的最适温度均为70℃;稳定 pH 为10.0左右(M-1和 M-2)、8.0—10.0(M-3);M-1、M-2和 M-3的稳定温度分别为40、55和50℃。金属离子 Ag^+、Hg^(2+)和 Mn^(2+)对三种酶均有抑制作用。β-甘露聚糖酶 M-1、M-2和 M-3对魔芋葡萄甘露聚糖作用的 K_m 分别为2.9、1.7和12.5mg/ml,V_(max)值分别为27500、47500和15700mol·min^(-1)·mg^(-1)。β-甘露聚糖酶 M-1水解魔芋葡萄甘露聚糖产生单糖、双糖、三糖和四糖等低聚糖。

Three extracellular alkline β-mannanases from art alkalophilic Bacillus N16-5 were puri-fied to electrophoretic homogeneity by(NH_4)_2SO_4 precipitation,DEAE-Sephadex A-25 chroma-tography,hydroxyapatite chromatography and preparative electrophoresis.Molecular weightsand pI values of the β-mannanases(M-1, M-2 and M-3)were 51000,38000 and 34700 by SDS-PAGE and 4.3,2.5 and 2.5 by PAGEIEF,respectively.The optimum pH for enzyme catalysis were 9.0 for M-I and 10.0 for M-2 and M-3,res-pectively.All of three enzymes were the most active at 70℃.The activities of three enzymeswere strongly inhibited by Ag^(1+),Hg^(2+) and Mn^(2+).Michaelis constants(K_m)of the enzyme M-1,M-2 and M-3 for mannase from konjak were 2.9,1.7 and 12.5 mg/ml,maximum velocities(V_(max))for the saccharide were 27500,47500 and 15700 μmol·min^(-1)·mg^(-1),respectively Kon-jak was hydrolyzed by the enzyme M-1,and major components in digests were mono-,di-,tri- and tetra-saccharides.