Henson C A, Livingston D P
Agricultural Research Service, United States Department of Agriculture.
Plant Physiol. 1996 Feb;110(2):639-44. doi: 10.1104/pp.110.2.639.
Oat (Avena sativa cv Fulghum) fructan hydrolase was purified by ammonium sulfate precipitation and anion-exchange, hydrophobic interaction, and size-exclusion chromatography. The enzyme was purified to homogeneity as determined by the presence of a single band (43 kD) on a silver-stained sodium dodecyl sulfate-polyacrylamide gel. A mixture of beta-2,6-linked fructan (neokestin) isolated from oat was used as the substrate to purify fructan hydrolase. Neokestin and small degree of polymerization fructan isomers were used to characterize the substrate specificity of the purified enzyme. The purified fructan hydrolase catalyzed hydrolysis of the terminal beta-2,6 linkage of 6G,6-kestotetraose 3.5 times more rapidly than it hydrolyzed the terminal beta-2,6 linkage of 6G-kestotriose and approximately 10 times faster than it hydrolyzed the terminal beta-2,1 linkage of chicory inulin. Sucrose and 1-kestose were not substrates. The Km for neokestin (beta-2,6-linked fructans with a degree of polymerization of 7-14) hydrolysis was 2.8% (w/v), and the Vmax was 0.041 mumol min-1 mL-1. The Km for hydrolysis of 6G,6-kestotetraose was 5.6% (w/v), and the Vmax was 0.138 mumol min-1 mL-1. Catalysis was exolytic and by multiple chain attack. Hydrolysis of neokestin was maximal at pH 4.5 to 5.0.
燕麦（燕麦品种富尔格姆）果聚糖水解酶通过硫酸铵沉淀以及阴离子交换、疏水相互作用和尺寸排阻色谱法进行纯化。通过银染十二烷基硫酸钠-聚丙烯酰胺凝胶上出现的单一条带（43 kD）确定，该酶已纯化至同质。从燕麦中分离得到的β-2,6-连接果聚糖（新凯斯汀）混合物用作底物来纯化果聚糖水解酶。新凯斯汀和低聚合度果聚糖异构体用于表征纯化酶的底物特异性。纯化的果聚糖水解酶催化6G,6-蔗果四糖末端β-2,6键的水解速度比催化6G-蔗果三糖末端β-2,6键的水解速度快3.5倍，比催化菊苣菊粉末端β-2,1键的水解速度快约10倍。蔗糖和1-蔗果三糖不是底物。新凯斯汀（聚合度为7-14的β-2,6-连接果聚糖）水解的Km为2.8%（w/v），Vmax为0.041 μmol min-1 mL-1。6G,6-蔗果四糖水解的Km为5.6%（w/v），Vmax为0.138 μmol min-1 mL-1。催化作用是外切型的且通过多链攻击。新凯斯汀的水解在pH 4.5至5.0时达到最大值。
