灰平链霉菌Streptomyces griseoplanus S501产外切菊粉酶的分离纯化及酶学性质研究

Purification and characterization of exo-inulinase produced by S. griseoplanus S501

1株产外切菊粉酶的海洋放线菌灰平链霉菌Streptomyces griseoplanus S501发酵粗酶液,经硫酸铵分级沉淀、透析脱盐后,分别采用Sephadex G-75凝胶、DEAE-Sepharose CL-6B离子交换柱层析和SDS聚丙烯凝胶电泳(SDS-PAGE)分离纯化,得到菊粉酶不同活性组分。纯化倍数为10.42倍,比酶活249.5 U/mg(蛋白),相对分子质量为28.184 k Da。酶学性质研究结果表明,该菊粉酶的最适温度和p H值分别为50℃和5.0。在50℃和p H5.0条件下,以菊粉为底物时,该酶的Km值和vmax值分别为3.83 mg/m L和4.64 mg/(m L·min)。金属离子对酶活性影响实验表明,Mg2+、I-、Li+、Fe3+、Al3+和K+对该菊粉酶酶活有很强的抑制作用,Cu2+、Ca2+和Ag+对该菊粉酶活性具有一定促进作用。

Exo-inulinase was purified from the fermentation broth of Streptomyces griseoplanus S501 using the ammonium sulfate fractionation combined with Sephadex G-75 gel filtration chromatography and DEAE-sepharose CL-6B ion-exchange. The purification index of this enzyme was 10. 42 times and its specific activity was 249. 5 U / mg. The molecular weight of the purified exo-inulinase identified by SDS-PAGE was about 28. 184 k Da. The optimal temperature and p H for its reaction were 50 ℃ and 5. 0,respectively. Under this condition,its Kmand vmaxfor soluble inulin were 3. 83 mg / m L and 4. 64 mg /（ m L·min）. The effects of different metal ions on enzyme activity showed that the enzyme activity was strongly activated by Cu2 ＋,Ca2 ＋and Ag＋. However,Mg2 ＋,I-,Li＋,Fe3 ＋,Al3 ＋and K＋inhibited the enzyme activity at various extents.