关键词: 肝素酶I, 酶活, ChBD标签, 低分子量肝素

Abstract: 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.

Key words: heparanase I, enzyme activity, chitin binding domain, low molecular weight heparin