Conversion of neopullulanase-α-amylase from Thermoactinomyces vulgaris r-47 into an amylopullulanase-type enzyme

Abstract

TVA I, an α-amylase from Thermoactinomyces vulgaris R-47, is a versatile enzyme which hydrolyzes the α-(1→4)-glucosidic linkages of pullulan to produce panose, known as neopullulanase activity, and the α-(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 a-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 α-(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 k(cat)/K(m) value of the mutated enzyme for α-(1→6)-glucosidic linkages was virtually unaltered, while that for α-(1→4)-glucosidic linkages was about 100 times smaller than that of the wild-type enzyme.