Analysis of the active center of Bacillus stearothermophilus neopullulanase.
The active center of the neopullulanase from Bacillus stearothermophilus was analyzed by means of site-directed mutagenesis. The amino acid residues located in the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanse, Taka-amylase A, and other amylolytic enzymes. When amino acid residues Glu and Asp, corresponding to the putative catalytic sites, were replaced by the oppositely charged (His) or noncharged (Gln or Asn) amino acid residue, neopullulanase activities toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages disappeared. When the amino acids corresponding to the putative substrate-binding sites were replaced, the specificities of the mutated neopullulanases toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of neopullulanase participated in the dual activity toward alpha-(1----4)- and alpha-(1----6)-glucosidic linkages. Pullulan is a linear glucan of maltotriosyl units linked through alpha-(1----6)-glucosidic linkages. The production ratio of panose from pullulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the alpha-(1----4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanse which exhibited higher specificity toward the alpha-(1----6)-glucosidic linkage.
Biochemical Research Laboratories, Ezaki Glico Co., Ltd., Osaka, Japan.
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Kuriki
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Takata
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Okada
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Imanaka
D000595:Amino Acid Sequence; D001665:Binding Sites; D002236:Carbohydrate Conformation; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D005936:Glucans / Q000096:biosynthesis / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D005936:Glucans; D005960:Glucosides; C008763:panose; C009109:pullulan; D006026:Glycoside Hydrolases; C058484:neopullulanase