智能多功能肝素酶生产低分子量肝素工艺研究.pdf

智能多功能肝素酶生产低分子量肝素工艺研究 1 1,2 3 1 1 叶逢春 、东乡昌宏 、陈银 、张翀 、邢新会 1 清华大学化工系生物化工研究所 2 日本东京工业大学生命理工学研究科 3 英国华威大学生物科学系 摘要：低分子量肝素（Low molecular weight heparin, LMWH）作为抗凝试剂和抗栓试剂 肝素理想替代品，目前已在临床得到了广泛的应用。但清洁高效低分子量肝素制备技术的缺 乏却极大地限制其应用。在众多低分子量肝素制备技术中，酶降解法以其反应条件温和性、 无毒且易实现生产连续化的优势脱颖而出。本实验室前期从一株肝素黄杆菌中克隆得到肝素 酶I 的基因，利用融合蛋白技术构建了高效生产可溶性肝素酶I 的基因工程菌株，为酶法制 备低分子肝素技术的发展提供了基础。一方面，麦芽糖结合蛋白（MBP）的融合可以稳定高 效地实现肝素酶Ⅰ的可溶性表达，并利用其亲和性能实现酶的分离纯化及固定化；另一方面， 绿色荧光蛋白（GFP）的引入，利用荧光量与肝素酶活之间良好的线性关系可以对反应过程 中的酶活进行实时监测。两种功能的结合很好地实现了肝素酶的智能多功能化目标。 关键词：低分子量肝素、肝素酶I、麦芽糖结合蛋白、绿色荧光蛋白、生物催化、酶工程 Production of Low Molecular Weight Heparin (LMWH) by Intelligent Multifunctional Fusion Heparinase I Ye Fengchun, Masahiro Togo, Chen Yin, Zhang Chong, Xing Xinhui (Department of Chemical Engineering, Tsinghua University, Beijing 100084, China) Abstract : For the side effects of heparin as the anticoagulant and antithrombotic agent, low molecular weight heparin (LMWH) capable of maintaining the anticoagulant activity while showing less side effect has been widely used and replaced heparin in clinics. However, the preparation of LMWH remains a problem due to the low efficiency and severe pollution. Compared to other methods, enzymatic depolymerization of heparin by heparinase I has been paid more and more attention due to its mild reaction conditions, high selectivity and environment friendship. In order to achieve an efficient production process of heparinase I, the fusion protein strategy was used. The fusion of MBP can realize the soluble expression, separation, purification and immobilization potentially based on the affinity adsorption, while the direct proportional relationship between the activity of heparinase I