洋葱蔗糖：蔗糖 1-果糖基转移酶的表达和活性分析。

Expression and activity analysis of sucrose:sucrose 1-fructosyltransferase from onion.

机构信息

Zhengzhou University of Light Industry, Henan Province, 450002, China.

出版信息

N Biotechnol. 2010 Sep 30;27(4):324-9. doi: 10.1016/j.nbt.2010.02.004. Epub 2010 Feb 25.

DOI:10.1016/j.nbt.2010.02.004

PMID:20188876

Abstract

This study was designed to express the onion fructosyltransferase by Escherichia coli DH5alpha, and obtain the optimal conditions of FST-1 activity. Thereby, fructosyltransferase gene was obtained by RT-PCR from onion in this experiment, and named FST-1. The expressed proteins were analyzed by SDS-PAGE. FST-1 activity was identified by the high performance liquid chromatography (HPLC). The optimal conditions of FST-1 were analyzed by the dinonylnaphthalene sulfonic acid (DNS) and orthogonal test. Results revealed that FST-1 was identified to 98% similarity with fructosyltransferase mRNA of onion (accession number: AJ006066). FST-1 was successfully expressed in E. coli DH5alpha. HPLC results indicated that the expressed protein from FST-1 had a good transferring activity for fructose. The optimal conditions of FST-1 in catalyzing reaction were the pH 5.0, 45 degrees C and 60% sucrose substrate. The results in this experiment would lay the foundation for the large-scale of kestose by bio-catalysis method.

摘要

本研究旨在通过大肠杆菌 DH5alpha 表达洋葱果糖基转移酶，并获得 FST-1 活性的最佳条件。因此，本实验通过 RT-PCR 从洋葱中获得了果糖基转移酶基因，并将其命名为 FST-1。通过 SDS-PAGE 分析表达的蛋白质。通过高效液相色谱法（HPLC）鉴定 FST-1 活性。通过二壬基萘磺酸（DNS）和正交试验分析 FST-1 的最佳条件。结果表明，FST-1 与洋葱果糖基转移酶 mRNA （登录号：AJ006066）具有 98%的相似性。FST-1 成功在大肠杆菌 DH5alpha 中表达。HPLC 结果表明，FST-1 表达的蛋白质对果糖具有良好的转移活性。FST-1 催化反应的最佳条件为 pH5.0、45°C 和 60%蔗糖底物。本实验的结果将为大规模生物催化法生产昆布糖奠定基础。