利用甘露聚糖-琼脂糖4B亲和层析法从蜂房芽孢杆菌中纯化内切-β-N-乙酰氨基葡萄糖苷酶。

The use of mannan-Sepharose 4B affinity chromatography for the purification of endo-beta-N-acetylglucosaminidase from Bacillus alvei.

作者信息

Morinaga T, Kitamikado M, Iwase H, Li S C, Li Y T

出版信息

Biochim Biophys Acta. 1983 Dec 12;749(2):211-3. doi: 10.1016/0167-4838(83)90255-8.

Abstract

In order to facilitate the isolation of endo-beta-N-acetylglucosaminidase for the structural analysis of glycoconjugates, we have isolated a strain of Bacillus alvei which produces a high level of endo-beta-N-acetylglucosaminidase. We have also devised a simple procedure for the purification of endo-beta-N-acetylglucosaminidase from B. alvei using mannan-Sepharose affinity chromatography. By using this method, endo-beta-N-acetylglucosaminidase was purified 3300-fold with 85% yield from the crude enzyme obtained by ammonium sulfate precipitation of the culture medium. The molecular weight of this enzyme was estimated to be about 66 000 by gel filtration. When using (Man)6(GlcNAc)2-Asn-Dns as substrate, the optimal activity occurs at pH 6.5 with Km of 1.9 mM. The action of endo-beta-N-acetylglucosaminidase toward several glycopeptides was also studied.

摘要

为便于分离用于糖缀合物结构分析的内切-β-N-乙酰氨基葡萄糖苷酶，我们分离出了一株产高水平内切-β-N-乙酰氨基葡萄糖苷酶的蜂房芽孢杆菌。我们还设计了一种简单的方法，利用甘露聚糖-琼脂糖亲和色谱从蜂房芽孢杆菌中纯化内切-β-N-乙酰氨基葡萄糖苷酶。通过这种方法，从经硫酸铵沉淀培养基获得的粗酶中，内切-β-N-乙酰氨基葡萄糖苷酶得到了3300倍的纯化，产率为85%。通过凝胶过滤估计该酶的分子量约为66000。以(Man)6(GlcNAc)2-Asn-Dns为底物时，最佳活性出现在pH 6.5，Km为1.9 mM。还研究了内切-β-N-乙酰氨基葡萄糖苷酶对几种糖肽的作用。