枯草芽孢杆菌SA - 22内切 - 1,4 - β - D - 甘露聚糖酶的纯化及性质

Purification and properties of Bacillus subtilis SA-22 endo-1, 4-beta-D-mannanase.

作者信息

Yu Hong-Ying, Sun Yuan-Ming, Wang Wei-Jun, Yang Yue-Sheng, Yang You-Hui

机构信息

Faculty of Chemical Engineering and Light Industry, Guangdong University of Technology, Guangzhou 510090 2, China.

出版信息

Sheng Wu Gong Cheng Xue Bao. 2003 May;19(3):327-30.

PMID:15969016

Abstract

beta-mannanase (EC 3.2.1.78) from Bacillus subtilis SA-22 was purified successively by ammonium sulfate precipitation, hydroxyapatite chromatography, Sephadex G-75 gel filtration and DEAE-52 anion-exchange chromatography. Through these steps, the enzyme was concentrated 30.75-fold with a recovery rate of 23.43%, with a specific activity of 34780.56 u/mg. Molecular weight of the enzyme was determined to be 38 kD by SDS-PAGE and 34 kD by gel filtration. The results revealed that the optimal pH value for the enzyme was 6.5 and the optimal temperature was 70 degrees C. The enzyme is stable between pH 5 to 10. The enzyme remained most of its activity after a treatment of 4 h at 50 degrees C, but lost 25% of activity at 60 degrees C for 4 h, lost 50% of activity at 70 degrees C for 3 h. The enzyme activity was strongly inhibited by Hg2+. The Michaelis constants (Km) were measured as 11.30 mg/mL for locust bean gum and 4.76 mg/mL for konjac powder, while Vmax for these two polysaccharides were 188.68 (micromol x mL(-1) x min(-1)) and 114.94 (micromol x mL(-1) x min(-1)), respectively.

摘要

来自枯草芽孢杆菌SA-22的β-甘露聚糖酶（EC 3.2.1.78）先后通过硫酸铵沉淀、羟基磷灰石层析、Sephadex G-75凝胶过滤和DEAE-52阴离子交换层析进行纯化。通过这些步骤，该酶浓缩了30.75倍，回收率为23.43%，比活性为34780.56 u/mg。通过SDS-PAGE测定该酶的分子量为38 kD，通过凝胶过滤测定为34 kD。结果表明，该酶的最适pH值为6.5，最适温度为70℃。该酶在pH 5至10之间稳定。在50℃处理4小时后，该酶仍保留大部分活性，但在60℃处理4小时后活性丧失25%，在70℃处理3小时后活性丧失50%。Hg2+对该酶活性有强烈抑制作用。刺槐豆胶的米氏常数（Km）为11.30 mg/mL，魔芋粉的米氏常数为4.76 mg/mL，而这两种多糖的最大反应速度（Vmax）分别为188.68（微摩尔×毫升-1×分钟-1）和114.94（微摩尔×毫升-1×分钟-1）。