来自嗜热栖热菌的一种耐热性外切多聚半乳糖醛酸酶的克隆表达及特性分析

Cloning expression and characterization of a thermostable exopolygalacturonase from Thermotoga maritima.

作者信息

Parisot Judicaël, Langlois Virginie, Sakanyan Vehary, Rabiller Claude

机构信息

Unité de Recherches en Biocatalyse (unité CNRS 2230), Faculté des Sciences et des Techniques, 2, rue de la Houssinière, BP, 92208 F-44322, Nantes, France.

出版信息

Carbohydr Res. 2003 Jun 16;338(12):1333-7. doi: 10.1016/s0008-6215(03)00165-4.

DOI:10.1016/s0008-6215(03)00165-4

PMID:12791288

Abstract

A gene encoding for a thermostable exopolygalacturonase (exo-PG) from hyperthermophilic Thermotoga maritima has been cloned into a T7 expression vector and expressed in Escherichia coli. The gene encoded a polypeptide of 454 residues with a molecular mass of 51,304 Da. The recombinant enzyme was purified to homogeneity by heat treatment and nickel affinity chromatography. The thermostable enzyme had maximum of hydrolytic activity for polygalacturonate at 95 degrees C, pH 6.0 and retains 90% of activity after heating at 90 degrees C for 5 h. Study of the catalytic activity of the exopolygalacturonase, investigated by means of 1H NMR spectroscopy revealed an inversion of configuration during hydrolysis of alpha-(1-->4)-galacturonic linkage.

摘要

编码来自嗜热栖热菌的一种耐热性外切多聚半乳糖醛酸酶（exo-PG）的基因已被克隆到一个T7表达载体中，并在大肠杆菌中表达。该基因编码一个由454个残基组成的多肽，分子量为51,304道尔顿。重组酶通过热处理和镍亲和层析纯化至同质。这种耐热性酶在95℃、pH 6.0时对多聚半乳糖醛酸具有最大水解活性，在90℃加热5小时后仍保留90%的活性。通过1H NMR光谱研究外切多聚半乳糖醛酸酶的催化活性，结果显示在α-(1→4)-半乳糖醛酸键水解过程中构型发生了翻转。