放射土壤杆菌M-1中甘露糖异构酶的纯化与特性分析

Purification and characterization of mannose isomerase from Agrobacterium radiobacter M-1.

作者信息

Hirose J, Maeda K, Yokoi H, Takasaki Y

机构信息

Department of Applied Chemistry, Faculty of Engineering, Miyazaki University, Japan.

出版信息

Biosci Biotechnol Biochem. 2001 Mar;65(3):658-61. doi: 10.1271/bbb.65.658.

DOI:10.1271/bbb.65.658

PMID:11330684

Abstract

A mannose isomerase from Agrobacterium radiobacter M-1 (formerly Pseudomonas sp. MI) was purified to electrophoretic homogeneity and characterized. A cell-free extract was separated by ammonium sulfate fractionation, Butyl-Toyopearl 650M, DEAE-Sepharose and hydroxylapatite column chromatography. Its molecular mass was estimated to be 44 kDa by SDS-PAGE and 90 kDa by gel filtration, in which the enzyme is most likely a dimer composed of two identical subunits. The purified enzyme had an optimum pH at 8.0, an optimum temperature at 60 degrees C, a pI of 5.2 and a Km of 20 mM, and specifically converted D-mannose and D-lyxose to ketose. The N-terminal amino acid sequence was identified.

摘要

对来自放射形土壤杆菌M-1（以前称为假单胞菌属MI）的一种甘露糖异构酶进行了纯化，使其达到电泳纯，并对其进行了表征。通过硫酸铵分级沉淀、丁基- Toyopearl 650M、DEAE-琼脂糖和羟基磷灰石柱色谱法对无细胞提取物进行分离。通过SDS-PAGE估计其分子量为44 kDa，通过凝胶过滤估计为90 kDa，其中该酶很可能是由两个相同亚基组成的二聚体。纯化后的酶的最适pH为8.0，最适温度为60℃，pI为5.2，Km为20 mM，并且能特异性地将D-甘露糖和D-来苏糖转化为酮糖。确定了其N端氨基酸序列。