类芽孢杆菌环果寡糖糖基转移酶的分离纯化和酶学性质

摘要

The cycloinulo-oligosaccharide fructanotransferase (CFTase) was purified from the cultured medium of Paenibacillus sp. Lfos16 to electrophoretic homogeneity by ammonium sulfate sedimentation, column chromatographies on phenyl-sepharose and DEAE-sepharose. The molecular mass of the enzyme was estimated to be 120 kDa by SDS-PAGE and Native-PAGE, indicating a double-subunit structure. The characterization of CFTase was explored. Maximal activity was observed at 40 ℃ ～ 45 ℃ and pH 7.0. The enzyme was active from pH 5.0 to pH 9.0, at temperatures between 30 and 45 ℃. The catalytic mechanism of CFTase was analyzed and it showed that the CFTase could splite the terminal 6 fructoses from inulin and cyclized them into CFs.%采用硫铵沉淀、疏水层析和DEAE离子交换层析对产自类芽孢杆菌Paenibacillus sp.Lfos16的环果寡糖糖基转移酶(Cycloinulooligosaccharide Fructanotransferase,CFTase)进行分离纯化,得到电泳纯的CFTase,最终纯化倍数为13.9,比活力为33.3 U/mg.纯化的CFTase经SDSPAGE和Native-PAGE电泳分析得出其相对分子量大小约为120 000,且是双亚基蛋白.探究了CFTase的酶学性质,最适反应条件为:40℃～45℃,pH 7.0;温度稳定范围为30℃～45℃,pH稳定范围为5.0～9.0.并对CFTase降解菊糖的催化机制进行了分析,初步确定为外切加环化作用形成环果寡糖.