ChBD和SUMO双功能融合肝素酶I的设计与表达

利用ChBD和SUMO标签构建4种不同的融合蛋白体系，分别为ChBD-Hep I、ChBD-SUMO-Hep I、SUMO-ChBD-Hep I和ChBD-Hep I-SUMO。经过发酵工艺优化实验，确定该4种酶在最优条件下的摇瓶发酵酶活分别为2.44IU/mL、6.92IU/mL、6.01IU/mL和4.51 IU/mL。同时，通过酶学性质研究，确定该4种重组酶的最适反应温度与pH分别为30℃和7.0，表明SUMO和ChBD标签不影响Hep I的催化反应。4种重组酶的热稳定性较天然Hep I明显提高，其中ChBD-SUMO-Hep I在30℃半衰期达50 min，由此，确定其为最优菌株。将ChBD-SUMO-Hep I进行5 L发酵罐扩大培养，酶活达到26 IU/mL，是摇瓶培养的5倍，表明其具有工业化生产的潜质。

Design and expression of recombinant heparanase I fused to chitin binding domain and SUMO-tag

Four fusion enzymes including ChBD-Hep I,ChBD-SUMO-Hep I,SUMO-ChBD-Hep I and ChBD-Hep I-SUMO were constructed by adjusting the relative location of SUMO and ChBD to Hep I.Their enzymatic activities were determined to be 2.44IU/mL,6.92IU/mL,6.01IU/mL and 4.51 IU/mL,respectively,under optimal fermentation conditions.Meanwhile,the optimal reaction temperature and reaction pH for all of the four fusion enzymes were 30℃ and 7.0,respectively,based on the integral study of enzymatic properties.It indicated that the fusion of SUMO and ChBD tags hardly affected the catalytic reaction of Hep I.Moreover,the thermal stability of fusion enzymes improved obviously compared with the natural Hep I.And the half-life of ChBD-SUMO-Hep I at 30℃ increased to 50 min.Hence,the optimal combination is that both of SUMO and ChBD located at the N-terminal of Hep I.Then,ChBD-SUMO-Hep I was further cultivated in a 5 L fermentor.It provided the enzyme activity of 26 IU/mL,which is four times higher than the shake-flask fermentation.The amplification experiment suggested that the fusion enzyme ChBD-SUMO-Hep I was suitable for industrial production.