重组大肠杆菌右旋糖酐蔗糖酶的纯化及其性质

Purification and characterization of recombinant Escherichia coli dextransucrase

【目的】研究工程菌E.coli BL21(DE3)/pET28-dexYG产右旋糖酐蔗糖酶的纯化和酶学性质。【方法】工程菌经过IPTG诱导后生产含His-tag融合蛋白的右旋糖酐蔗糖酶,通过硫酸铵沉淀、Ni-NTA亲和层析纯化,得到纯度较高的酶蛋白,并对纯酶进行了酶学性质及动力学研究。【结果】经过SDS-PAGE测得该酶的分子量约为170kDa,与理论推测值基本相同。以蔗糖为底物,酶促反应的最适温度为25～30℃,最适pH值为5.4,动力学常数Km值为10.43mmol/L;酶活在pH5.0～8.0较为稳定,在室温(25℃)保藏4天仍有59%的酶活力,4℃保存7周酶活力仅下降一半,但在35℃以上失活很快;Ca2+对催化作用有较大的促进,Mg2+有微弱的促进作用,K+对催化反应无影响,Cu2+的抑制作用最强。其他试剂对重组酶的活性有不同程度的影响,其中SDS抑制作用很强。【结论】研究为重组右旋糖酐蔗糖酶纯酶的获取、得到稳定性好、活性高的酶反应体系及利用该酶进行催化反应和工业化应用提供了重要参数。

[Objective] To purify and characterize recombinant dextransucrase expressed in engineered strain BL21 （DE3）/pET28-dexYG. [Methods] The dextransucrase gene （dexYG） was expressed in engineered strain after IPTG induction and the crude enzyme was obtained by sonication. We purified the recombinant dextransucrase by using ammonium sulfate precipitation and metal chelate affinity chromatography on a Ni-NTA column. Then we characterized catalytic kinetic parameter of purified enzyme. [Results] This purification protocol resulted in a 11.4-fold purification with a yield of 37.5%. The molecular weight of dextransucrase measured by SDS-PAGE was 170 kDa ,which was similar to the enzyme from Leuconostoc mesenteroides. The enzyme had an optimum temperature between 25and 30℃ and an optimum pH of 5.4. It was relatively stable in the range of pH 5.0 to 7.0, but the stability declined rapidly as soon as the temperature rose over 35℃.The enzyme activity remained 59% after stored for 4 days at room temperature （25℃）, and lost 50% activity after stored for seven weeks at 4℃. Ca^2＋ of 0.5 mmol/L could strongly activate the enzyme, Mg^2＋ of 1 mmol/L had little effect, Cu^2＋ and SDS could greatly inhibit the enzyme. [Conclusion] These results may provide an important basis for industrial applications of the recombinant dxtransucrase.