Ibuka A, Tonozuka T, Matsuzawa H, Sakai H
Department of Biotechnology, University of Tokyo.
J Biochem. 1998 Feb;123(2):275-82. doi: 10.1093/oxfordjournals.jbchem.a021933.
TVA I, an alpha-amylase from Thermoactinomyces vulgaris R-47, is a versatile enzyme which hydrolyzes the alpha-(1-->4)-glucosidic linkages of pullulan to produce panose, known as neopullulanase activity, and the alpha-(1-->6)-glucosidic linkages of certain oligosaccharides. We modified the Ala-357, Gln-359, and Tyr-360 residues located in region II, one of the four regions conserved in alpha-amylase family enzymes, and deleted 11 consecutive amino acid residues located after the C-terminus of region II of the TVA I sequence by means of site-directed mutagenesis. The action pattern of the mutated enzyme for pullulan was greatly altered and it hydrolyzed mainly the alpha-(1-->6)-glucosidic linkages of pullulan to produce maltotriose, while the action patterns for starch and maltooligosaccharides were almost identical to those of the wild-type enzyme. This means that the mutated TVA I has lost the neopullulanase activity, and thus can be designated as an amylopullulanase-type enzyme. The kcat/Km value of the mutated enzyme for alpha-(1-->6)-glucosidic linkages was virtually unaltered, while that for alpha-(1-->4)-glucosidic linkages was about 100 times smaller than that of the wild-type enzyme.
TVA I是一种来自嗜热放线菌R-47的α-淀粉酶，是一种多功能酶，它能水解支链淀粉的α-(1→4)-糖苷键生成潘糖，这被称为新支链淀粉酶活性，还能水解某些寡糖的α-(1→6)-糖苷键。我们通过定点诱变修饰了位于区域II（α-淀粉酶家族酶中四个保守区域之一）的丙氨酸-357、谷氨酰胺-359和酪氨酸-360残基，并删除了TVA I序列区域II C末端之后的11个连续氨基酸残基。突变酶对支链淀粉的作用模式发生了很大改变，它主要水解支链淀粉的α-(1→6)-糖苷键生成麦芽三糖，而对淀粉和麦芽寡糖的作用模式与野生型酶几乎相同。这意味着突变后的TVA I失去了新支链淀粉酶活性，因此可以被指定为一种支链淀粉酶型酶。突变酶对α-(1→6)-糖苷键的kcat/Km值实际上没有改变，而对α-(1→4)-糖苷键的kcat/Km值比野生型酶小约100倍。
