多形拟杆菌肝素酶Ⅰ的SUMO融合表达及酶学特性分析  

SUMO-Fused expression and characterization of Heparinase Ⅰ from Bacteroides thetaiotaomicron

作　　者：张川 张悦 丁啸虎[1,2] 李中媛 宋亚囝 罗学刚[1,2] Chuan Zhang;Yue Zhang;Xiaohu Ding;Zhongyuan Li;Yajian Song;Xuegang Luo(Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, TianjinUniversity of Science and Technology, Tianjin 300457, China;National Demonstration Center for Experimental Bioengineering Education, Tianjin University of Science and Technology,Tianjin 300457, China)

机构地区：[1]天津科技大学生物工程学院,工业发酵微生物教育部重点实验室暨天津市工业微生物重点实验室,天津300457 [2]天津科技大学,生物工程国家级实验教学示范中心,天津300457

出　　处：《微生物学报》2019年第7期1318-1330,共13页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2017YFD0400303);国家“863计划”(2012AA021505)~~

摘　　要：【目的】克隆多形拟杆菌(Bacteroides thetaiotaomicron) Heparinase I基因,在大肠杆菌(Escherichiac。")中进行基因工程表达获得重组酶SUMO-Bt-HepI和Bt-HepI,并研究其酶学特性。【方法】对B.thetaiotaomicron肝素酶I (Bt-HepI)的基因序列进行密码子优化,PCR扩增得到目的基因,构建表达载体pET-28a-Bt-HepI和pE-SUMO-Bt-HepI,并转化至E. colt Rosetta (DE3)进行表达,分别得到重组产物Bt-HepI和SUMO-Bt-HepI,以肝素钠为底物研究两者的酶学性质。【结果】SDS-PAGE检测显示Bt-HepI和SUMO-Bt-HepI的分子量大小分别约为42.5 kDa和55 kDa。与Bt-HepI相比,融合SUMO-Tag后的肝素酶I比酶活提高了 48.9%。酶学性质表明:Bt-HepI和SUMO-Bt-HepI的最适pH和温度均为pH 9、45℃,二者在pH 5-9都具有很好的稳定性,但pH<5时,SUMO-Bt-HepI的耐酸性明显高于Bt-HepI0同时,在温度低于50 ℃时,SUMO-Bt-HepI的比酶活高于Bt-Hepl。此外,Ca^2+和Mg^2+对重组肝素酶I具有明显的促进作用,而CU^2+、Mr^2+、Zt^2+则表现出一定的抑制作用,提示在多形拟杆菌肝素酶I的结构中除了存在已知的Ca2+结合位点外,可能还存在Mg2+的结合位点。【结论】本研究首次将多形拟杆菌来源的肝素酶I和SUMO-Tag进行了融合表达,使其比酶活得到了显著的提高,为其生产应用奠定了基础。[Objective] To clone and recombinant express the gene Heparinase I from Bacteroides thetaiotaomicron, and then characterize the recombinant SUMO?Bt-HepI and Bt-HepI.[Methods] Codon optimization was done on the gene sequence of B. thetaiotaomicron heparinase I. The target gene was obtained by PCR amplification, inserted into the expression vectors pET-28a and pE?SUMO, and then transformed into E. coli Rosetta (DE3) to obtain recombinant products Bt-HepI and SUMO-Bt-HepI. Heparin sodium was used as substrate to study the enzymatic properties of the recombinant proteins.[Results] SDS-PAGE analysis showed that the molecular weights of Bt-HepI and SUMO-Bt-HepI were about 42.5 kDa and 55 kDa, respectively. Compared with Bt-HepI, the specific enzyme activity of Heparinase I increased by 48.9% after fusion SUMO-Tag. The enzymological properties showed that the optimum pH and temperature of Bt-HepI and SUMO-Bt-HepI were pH 9 and 45 °C, and both recombinant enzymes were stable at pH 5-9, while the acid resistance of SUMBt-HepI was obviously higher than Bt-HepI when the pH value was lower than 5. Besides, SUMO?Bt-HepI also showed higher activities than Bt-HepI under 50 °C. In addition, Ca2+ and Mg2+ have obvious promoting effect on the recombinant heparinase I, while Cu2+, Mn2+ and Zn2+ show certain inhibiting effect, suggesting that in addition to the well-known Ca2+ binding site, Mg2+ binding sites may aslo exist in the structure of B. thetaiotaomicron Heparinase I.[Conclusion) Recombinant Heparinase I in B. thetaiotaomicron using SUMO fusion system significantly improved its specific enzyme activity for potential production and application.

关 键 词：多形拟杆菌 肝素酶I SUMO-Tag 融合表达 酶学性质 

分 类 号：Q936[生物学—微生物学] Q78