海藻糖高产株Brevibacterium sp SY361海藻糖磷酸化酶(EC2.4.1.64)的纯化

Study on the Purification of Trehalose Phosphorylase from a Rich Trehalose-producing Strain Brevibacterium sp SY361

目的研究分离纯化海藻糖高产株Brevibacterium sp SY361中海藻糖磷酸化酶的工艺,获得高纯度的海藻糖磷酸化酶。方法大量培养细菌Brevibacterium sp SY361,超声粉碎细胞,通过硫酸铵分级沉淀、DEAE-SephadexA-25柱层析和Sephadex G-200凝胶过滤等步骤纯化其海藻糖磷酸化酶,并通过SDS-PAGE检测纯化效果。结果经纯化后的海藻糖磷酸化酶纯度提高了35.6倍,比活力达到7.48U/mg,SDS-PAGE呈1条带。结论纯化后的海藻糖磷酸化酶已达到纯度要求,可用于酶性质的研究。

Objective To study the process of extracting and purifying the trehalose phosphorylase from a rich trehalose-producing strain Brevibacteriurn sp SY361. Methods The partial purified trehalose phosphorylase was prepared from a cell-free extract of Brevibacteriurn sp SY361 by precipitation with （NH4）2SO4, DEAE-Sephadex A-25 chromatography and Sephadex G-200 gel filtration successively. SDS-PAGE was applied to check the purity of the enzyme. Results The enzyme was purified about 35.6 fold from the cell-free extract, and the specific activity reached 7.48 U/mg. The enzyme showed a single band on SDS-PAGE. Conclusion The purified trehalose phosphorylase meets the requirements of the further research.