Sheperd V, Montgomery R
Biochim Biophys Acta. 1976 May 13;429(3):884-94. doi: 10.1016/0005-2744(76)90334-x.
Alpha-D-Mannosidase (alpha-D-mannoside mannohydrolase, EC 3.2.1.24) has been purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight of the enzyme is approx. 200000; the protein appears to contain 4 subunits, with molecular weights of 66000 and 44000. The enzyme was immobilized on Sepharose and the properties of the coupled and free enzyme were compared. Both were stable up to 70 degrees C with rapid loss of activity between 75-80 degrees C; both retained 25-30% activity in 6 M urea and 65% of the original activity could be restored in the coupled preparation by removal of the urea. The pH maximum of each form was approximately the same, with the maximum of the immobilized enzyme shifted slightly to a lower pH. The coupled alpha-D-mannosidase presented in this report offers the possibility of digesting high molecular weight substrates, such as glycoproteins, with the advantages of (1) recovering large quantities of digested substrate; (2) recovery of the active glycosidase; and (3) digestion at high temperatures and under conditions that denature many proteins.
α-D-甘露糖苷酶（α-D-甘露糖苷甘露水解酶，EC 3.2.1.24）已通过聚丙烯酰胺凝胶电泳和超速离心证明纯化至同质。该酶的分子量约为200000；该蛋白质似乎包含4个亚基，分子量分别为66000和44000。将该酶固定在琼脂糖凝胶上，并比较了偶联酶和游离酶的性质。两者在高达70℃时都稳定，在75-80℃之间活性迅速丧失；两者在6M尿素中都保留25-30%的活性，并且通过去除尿素，偶联制剂中65%的原始活性可以恢复。每种形式的最适pH值大致相同，固定化酶的最适pH值略有向较低pH值的偏移。本报告中呈现的偶联α-D-甘露糖苷酶提供了消化高分子量底物（如糖蛋白）的可能性，具有以下优点：（1）回收大量消化后的底物；（2）回收活性糖苷酶；（3）在高温和使许多蛋白质变性的条件下进行消化。
