定向生物合成右旋糖酐的机理与过程调控研究

The control of Molecular Weight Distribution (MWD) and homogeneity of biomacromolecule is limited by the hysteretic research in process regulation and mechanism study of polyreaction. Method to obtain polysaccharides medicine is still followed by the route of "first synthesis, then hydrolysis". Side effects in clinic have been reported as a result of the poor quality of dextran produced by the multistep method. Directed biosynthesis of dextran is crucial to resolve the above questions. Based on the previous research in immobilization of dextransucrase and purification of dextran by membrane process, biosynthesis of dextran by immobilized enzyme system and enzyme-membrane reaction system is set up to simplify the polymerization process and avoid interference of other macromolecules. Membrane process is emploied to remove the desired dextran in order to improve the production rate of target dextran. High Performance Gel Permeation Chromatography (HPGPC), Liquid Chromatography-Electrospray Ionization Mass Spectrometry (LC-MS), Fourier Transform Infrared Spectrometer (FTIR) and Nuclear Magnetic Resonance(NMR) are used for the quality control of dextran. Variations of polymerization degree of dextran production and internal laws during the biosynthesis process are analyzed to elucidate the polyreaction mechanism and reveal the polymerization kinetics of dextran. There is no allied reports both at home and abroad.

生物合成大分子产物的分子量及均一度的有效控制受制于合成机理与过程调控研究的滞后，多糖类药物目前仍采用先合成再水解的制备路线，从蔗糖发酵产物中获得的药用右旋糖酐作为人工血浆的临床副作用主要来源于多步法制备所导致的质量问题，右旋糖酐定向生物合成是解决上述问题的有效途径。本项目基于右旋糖酐合成酶固定化、膜过程耦合等前期研究成果，拟构建酶膜反应系统以简化右旋糖酐合成体系的组成、避免其它大分子底物对右旋糖酐合成产物聚合度及均一度检测分析的干扰，通过膜分离不断移出符合聚合度要求的右旋糖酐合成产物，对合成过程进行调控，以实现右旋糖酐的生物合成的定向控制，利用HPGPC、LCMS、FTIR和NMR等技术手段对右旋糖酐生物合成过程的各个抽样进行检测分析，研究右旋糖酐定向合成过程聚合度的时效变化与内在规律，探明右旋糖酐生物合成机理及过程控制有效途径，研究右旋糖酐定向生物合成的过程动力学机制。相关研究未见报道。

本项目通过构建双酶合成体系及酶膜反应器以简化药用右旋糖酐聚合反应体系、避免其它大分子物质对右旋糖酐聚合度及均一度检测分析的干扰，通过膜过程移出符合聚合度要求的右旋糖酐产物对聚合反应过程进行调控以提高目标产物的产率，实现药用右旋糖酐的定向合成，探索药用右旋糖酐（分子量104<Mw<105 Da）高效制备新方法。基于右旋糖酐定向合成过程目标产物分子量的时效变化与内在规律，探明右旋糖酐合成反应机理及过程控制有效途径，揭示右旋糖酐定向生物合成的过程动力学问题。在右旋糖酐酶法合成的动力学、右旋糖酐酶法合成的过程调控和右旋糖酐定向生物合成过程的控制机制等环节均取得了阶段性研究成果。实验表明，利用双酶体系及耦合膜分离过程，可以显著提高药用右旋糖酐在产物中的比例，目标产物占合成产物百分比达90%以上，远高于传统“先发酵再水解、提纯”工艺，且制备流程更短、不涉及有机溶剂的使用。