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

摘要:

[目的]克隆多形拟杆菌（Bacteroides thetaiotaomicron）Heparinase I基因，在大肠杆菌（Escherichia coli）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. coli 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-HepI。同时，在温度低于50℃时，SUMO-Bt-HepI的比酶活高于Bt-HepI。此外，Ca²⁺和Mg²⁺对重组肝素酶I具有明显的促进作用，而Cu²⁺、Mn²⁺、Zn²⁺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。

关键词:多形拟杆菌;肝素酶I;SUMO-Tag;融合表达;酶学性质

Abstract:

[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℃, and both recombinant enzymes were stable at pH 5-9, while the acid resistance of SUMO-Bt-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℃. 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.

Key words:Bacteroides thetaiotaomicron;heparinase I;SUMO-Tag;fusion expression;enzymological properties