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A Thermophilic Alkalophilic α-Amylase from Bacillussp. AAH-31 Shows a Novel Domain Organization among Glycoside Hydrolase Family 13 Enzymes

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Title: A Thermophilic Alkalophilic α-Amylase from Bacillussp. AAH-31 Shows a Novel Domain Organization among Glycoside Hydrolase Family 13 Enzymes
Authors: Saburi, Wataru Browse this author →KAKEN DB
Morimoto, Naoki Browse this author
Mukai, Atsushi Browse this author
Kim, Dae Hoon Browse this author
Takehana, Toshihiko Browse this author
Koike, Seiji Browse this author
Matsui, Hirokazu Browse this author →KAKEN DB
Mori, Haruhide Browse this author →KAKEN DB
Keywords: α-amylase
glycoside hydrolase family 13
carbohydrate-binding module family 20
starch-binding domain
Issue Date: Sep-2013
Publisher: Taylor & Francis
Journal Title: Bioscience, Biotechnology, and Biochemistry
Volume: 77
Issue: 9
Start Page: 1867
End Page: 1873
Publisher DOI: 10.1271/bbb.130284
PMID: 24018662
Abstract: α-Amylases (EC hydrolyze internal α-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic α-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial α-amylases. In this study, the AmyL gene was cloned to determine its primary structure, and the recombinant enzyme, produced in Escherichia coli, was characterized. AmyL shows no hydrolytic activity towards pullulan, but the central region of AmyL (Gly395-Asp684) was similar to neopullulanase-like α-amylases. In contrast to known neopullulanase-like α-amylases, the N-terminal region (Gln29-Phe102) of AmyL was similar to carbohydrate-binding module family 20 (CBM20), which is involved in the binding of enzymes to starch granules. Recombinant AmyL showed more than 95% of its maximum activity in a pH range of 8.2-10.5, and was stable below 65 °C and from pH 6.4 to 11.9. The kcat values for soluble starch, γ-cyclodextrin, and maltotriose were 103 s(-1), 67.6 s(-1), and 5.33 s(-1), respectively, and the Km values were 0.100 mg/mL, 0.348 mM, and 2.06 mM, respectively. Recombinant AmyL did not bind to starch granules. But the substitution of Trp45 and Trp84, conserved in site 1 of CBM20, with Ala reduced affinity to soluble starch, while the mutations did not affect affinity for oligosaccharides. Substitution of Trp61, conserved in site 2 of CBM20, with Ala enhanced hydrolytic activity towards soluble starch, indicating that site 2 of AmyL does not contribute to binding to soluble long-chain substrates.
Rights: This is an Accepted Manuscript of an article published by Taylor & Francis in Bioscience biotechnology and biochemistry on 2013 Sep, available online:
Type: article (author version)
Appears in Collections:農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 佐分利 亘

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