肝素黄杆菌肝素酶Ⅲ基因hepC的可溶表达体系构建及酶学性质研究

生物技术通报 ›› 2013, Vol. 0 ›› Issue (6): 133-139.

肝素黄杆菌肝素酶Ⅲ基因hepC的可溶表达体系构建及酶学性质研究

李晔^{1, 2}, 吴敬君², 叶逢春², 苏楠², 张翀², 邢新会²

(1.北京电子科技职业学院, 北京 100029;2.清华大学化学工程系生物化工研究所, 北京 100084)

收稿日期:2013-06-20 修回日期:2013-06-20 出版日期:2013-06-20 发布日期:2013-06-20
作者简介:李晔, 女, 博士, 讲师, 研究方向: 基因工程药物;E-mail: liyeyashi@163.com
基金资助:
国家自然科学基金重点项目(20836004), 中国博士后科学基金项目(20100470140), 北京电子科技职业学院院内重点课题(YZKB20-11003)

Clonging and Expression of Heparinase III Gene from Flavobacterium heparinum and Characterization of the Recombinant Fusion Enzyme

Li Ye^{1, 2} Wu Jingjun² Ye Fengchun² Su Nan² Zhang Chong² Xing Xinhui²

(1. Beijing Polytechnic, Beijing 100029;2. Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Beijing 100084)

Received:2013-06-20 Revised:2013-06-20 Published:2013-06-20 Online:2013-06-20

摘要/Abstract

摘要： 肝素酶III(HepC) 是肝素结构分析及酶法制备低分子量肝素的重要生物催化剂。为实现HepC在大肠杆菌中的可溶表达, 通过PCR方法从肝素黄杆菌(Flavobacterium heparinum) 的基因组中扩增得到HepC基因, 采用融合蛋白方法构建了麦芽糖结合蛋白(MBP) 与HepC融合的表达载体和体系, 并在低温(15℃) 下诱导重组大肠杆菌, 显著提高了MBP-HepC重组表达的可溶性。通过摇瓶培养发酵液的HepC比酶活达到46.41 IU/mg, 超过目前报道的最高水平。通过MBP与直链淀粉的亲和吸附实现了MBP-HepC的亲和分离和纯化, 一步亲和纯化后蛋白的纯度即可达95%以上。该融合酶基本特性研究表明, 融合肝素酶III(MBP-HepC) 的最适作用pH7.3, 最适作用温度为42℃-48℃。热稳定性较好, 其30℃时的稳定性高于融合肝素酶I(MBP-HepA) 。

关键词: 肝素酶III, 麦芽糖结合蛋白, 重组表达, 纯化, 酶学性质

Abstract: Heparinase III is an important enzyme for heparin structure analysis and low molecular weight heparin production. To achieve the soluble expression of HepC with high activity in recombinant E. coli, the heparinase III gene(hepC)from Flavobacterium heparinum was amplified by the PCR method, and the fusion expression vector and system of expressing fusion protein of a maltose binding protein(MBP)and HepC were constructed. The results showed that the fusion strategy by using MBP was effective to enhance solubility of the MBP-HepC when induced at low temperature of 15℃. By shake flask cultivation, the specific enzyme activity of MBP-HepC could reach about 46.41 IU/mg, which was the highest among the values reported so far. By one-step affinity purification with amylose resin, the MBP-HepC fusion protein was easily purified to more than 95% purity. The enzyme characteristic study showed that the optimum pH value of MBP-HepC was 7.3, the optimum reaction temperature was 42℃-48℃, and the thermal stability of MBP-HepC is better than Heparinase I(MBP-HepA)at 30℃.

Key words: Heparinase III, Maltose binding protein(MBP), Recombinate expression, Purification, Characterization

李晔, 吴敬君, 叶逢春, 苏楠, 张翀, 邢新会. 肝素黄杆菌肝素酶Ⅲ基因hepC的可溶表达体系构建及酶学性质研究[J]. 生物技术通报, 2013, 0(6): 133-139.

Li Ye, Wu Jingjun, Ye Fengchun, Su Nan, Zhang Chong, Xing Xinhui. Clonging and Expression of Heparinase III Gene from Flavobacterium heparinum and Characterization of the Recombinant Fusion Enzyme[J]. Biotechnology Bulletin, 2013, 0(6): 133-139.

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