类芽孢杆菌环果寡糖糖基转移酶的分离纯化和酶学性质

Purification and characterization of cycloinulooligosaccharide fructanotransferase from Paenibacillus sp. Lfos16

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℃,p H 7. 0;温度稳定范围为30℃～45℃,p H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。

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.