Conversion of neopullulanase-alpha-amylase from Thermoactinomyces vulgaris R-47 into an amylopullulanse-type enzyme.
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.
Ibuka
A
A
Department of Biotechnology, University of Tokyo.
Tonozuka
T
T
Matsuzawa
H
H
Sakai
H
H
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D004789:Enzyme Activation; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008848:Micromonosporaceae / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013213:Starch / Q000378:metabolism; D014312:Trisaccharides / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D005936:Glucans; D009844:Oligosaccharides; D014312:Trisaccharides; C031146:maltoheptaose; C008317:maltotriose; C009109:pullulan; D013213:Starch; D006026:Glycoside Hydrolases; D000516:alpha-Amylases; C058484:neopullulanase; C082507:amylopullulanase