重组大肠杆菌生产肝素酶的培养基优化及酶活快速监测新方法研究.pdfVIP

重组大肠杆菌生产肝素酶的培养基优化及酶活快速监测新 方法研究 叶逢春 陈银 况莹 邢新会 (清华大学化工系生物化工研究所) 摘要 肝素酶Ⅰ是降解大分子肝素制备低分子量肝素(Low molecular weight heparin，LMWH) 的重要生物催化 剂。本研究采用大肠杆菌重组表达肝素酶Ⅰ的策略，成功地构建了由肝素酶Ⅰ（hepA ）和麦芽糖结合蛋白 (MBP)融合的表达体系（pMHS ）。结果表明，MBP 的融合极大地提高了肝素酶Ⅰ的可溶性表达。以M9 培 养基为基础进一步对培养基组成进行了优化，获得了酶活高达15928IU/L，比酶活为10.340IU/mg 总蛋白的 培养结果。为实现适于酶生产及其应用过程控制和优化的酶活的快速监测技术，通过构建绿色荧光蛋白GFP 与肝素酶I （hepA ）融合表达体系（pGFP-hepA ），研究了利用绿色荧光蛋白GFP 构建快速定量肝素酶活方 法的可行性。结果表明，GFP 的融合也可以提高肝素酶I 的可溶性表达，其荧光量与肝素酶活之间具有良 好的线性关系。 关键词：肝素酶I 、麦芽糖结合蛋白、绿色荧光蛋白、可溶表达，酶工程 MEDIA OPTIMIZATION AND RAPID DETECTION OF RECOMBINANT SOLUBLE HEPARINASE BY E. COLI Ye Fengchun, Chen Yin, Kuang Ying, Xing Xinhui (Department of Chemical Engineering, Tsinghua University, Beijing 100084, China) Abstract Heparinase is the most useful enzyme in production of LMWH (Low molecular weight heparin). In order to realize an efficient production process of the heparinase, heparinase I was fused with the maltose binding protein (MBP) and the green fluorescent protein (GFP), to construct the respective expressing vectors of pMHS and pGFP-hepA. The results showed that the fusion strategy by using maltose binding protein (MBP) was effective to significantly enhance the solubility of heparinase I in recombinant E. coli . Further more, by optimizing the components of M9 modified medium, the enzymatic activity of heparinase I reached as high as 15928IU/L by shake flask culture with the specific activity of 10.340IU/mg-total protein. In order to establish a rapid detection method of heparinase activity for the bioprocess control and optimization of the enzyme production, fusion of green fluorescent protein (GFP) to hepA was studied. The results showed that not only there was a linear relationship between th