Hamdy Hossam S
Biological Sciences & Geology Department, Faculty of Education, Ain Shams University, Roxy, Heliopolis, Cairo, 11757, Egypt.
Indian J Exp Biol. 2002 Dec;40(12):1393-8.
Protein precipitate of cell-free dialysate of extracellular inulinase (2,1-beta-fructan fructanohydrolase, EC 3.2.1.7) of A. alternata was maximally obtained by methanol. Such protein was fractionated by using 2-step column chromatography on Sephadex G150 and DEAE-cellulose. The partially purified enzyme had activity of 81 x 10(3) U/mg protein, with a yield of 69% of the original activity and the fold of purification was 62. Optimum temperature and pH for the activity of the purified enzyme were found to be 55 degrees C and 4.5, respectively. The enzyme was found to be stable up to 55 degrees C and in pH range of 4 to 5. Ba2+ and Ca2+ were found to stimulate the enzyme activity while Cu2+, Fe3+, Hg2+, and iodoacetate were recorded as strong inhibitors. T(1/2) of the enzyme was estimated to be two weeks and its apparent Km was calculated to be 0.066 M. The enzyme recorded hydrolyzing activity against sucrose and raffinose recording I/S ratio of 0.50. Molecular mass of the enzyme preparation was estimated by gel filtration and found to be 115 +/- 5 kDa.
通过甲醇可最大程度地获得链格孢菌胞外菊粉酶（2,1-β-呋喃果糖苷酶，EC 3.2.1.7）无细胞透析液的蛋白质沉淀。使用Sephadex G150和DEAE-纤维素进行两步柱色谱法对该蛋白质进行分级分离。部分纯化的酶的活性为81×10³ U/mg蛋白质，产率为原始活性的69%，纯化倍数为62。发现纯化酶活性的最适温度和pH分别为55℃和4.5。该酶在高达55℃以及pH 4至5的范围内稳定。发现Ba²⁺和Ca²⁺可刺激酶活性，而Cu²⁺、Fe³⁺、Hg²⁺和碘乙酸被记录为强抑制剂。该酶的半衰期估计为两周，其表观Km计算为0.066 M。该酶对蔗糖和棉子糖具有水解活性，I/S比为0.50。通过凝胶过滤估计酶制剂的分子量，发现为115±5 kDa。
