项目名称: 基于C5-异构酶-2-O-硫酸基转移酶双功能融合酶的高艾杜糖醛酸含量肝素类似物的人工合成、结构调控及活性评价
项目编号: No.21306066
项目类型: 青年科学基金项目
立项/批准年度: 2014
项目学科: 化学工业
项目作者: 程咏梅
作者单位: 江南大学
项目金额: 25万元
中文摘要: 酶法合成肝素(HP)是糖生物学领域的热点问题。在目前以K5PS为前体酶法或化学-酶法合成HP的研究中,普遍发现C5-异构酶(C5-epi)催化葡萄糖醛酸(GlcA)异构化成艾杜糖醛酸(IdoA)效率低,严重影响后续反应中硫酸基转移酶的催化活性,导致合成HP在结构和功能上与天然肝素存在较大差异。针对这一问题,本项目拟借助2-O-硫酸基转移酶(2-OST)与C5-epi天然协同催化的特性,构建高异构化活性C5-epi-2-OST双功能融合酶,提高C5-epi的催化效率,以合成高IdoA含量HP类似物。项目将主要研究包括:(1)高异构化活性C5-epi-2-OST双功能融合酶的构建策略、筛选和优化;(2)2-OST对C5-epi增强的分子机制探讨;(3)高IdoA含量HP类似物的合成及结构表征;(4)IdoA含量对HP结构和生物活性的影响等。
中文关键词: 融合酶;肝素类似物;艾杜糖醛酸;结构调控;C5异构化
英文摘要: Enzymatic Synthesis of heparin is a "hot" spot in Glycobiology field. Current studies found that the L-idopyranosyluronic acid (L-IdoA) content in enzymatic or chemoenzymatic synthesized heparin was much lower than that of natural heparin due to the low conversion efficiency of C5-epimerase (C5-epi) catalyzed epimerization, causing the structure and biological functions gap between synthetic heparin and natural heparin. This work aims to construct C5-epi-2-OST bifunctional fusion enzyme in order to enhance the epimerization activity and increase the IdoA content based on the known intimate synergetic effects of C5-epi and 2-O-sulftransferase (2-OST). The majority of this work includes: i) construction, expression, selection and optimization of C5-epi-2-OST bifunctional fusion enzyme with high epimerization activity; ii) study the molecular mechanism of the enhancement of C5-epi activity by 2-OST fusion; iii) synthesis of high IdoA content Heparin-like molecules and their structure characterization; and iv) study the effects of IdoA content on the overall structure, sulfation pattern as well as biological function of HP.
英文关键词: Fusion enzyme;heparin-like molecules;iduronic acid;structure regulation;C5 epimerization