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

Saburi, Wataru; Morimoto, Naoki; Mukai, Atsushi; Kim, Dae Hoon; Takehana, Toshihiko; Koike, Seiji; Matsui, Hirokazu; Mori, Haruhide

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

Saburi, Wataru; Morimoto, Naoki; Mukai, Atsushi et al.

Bioscience, Biotechnology, and Biochemistry, 2013, 77(9), 1867-1873

Permalink : https://hdl.handle.net/2115/67547

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アクセス権	open access
URI	https://hdl.handle.net/2115/67547
タイトル	A Thermophilic Alkalophilic α-Amylase from Bacillussp. AAH-31 Shows a Novel Domain Organization among Glycoside Hydrolase Family 13 Enzymes
著者	Saburi, Wataru ; Morimoto, Naoki ; Mukai, Atsushi ; Kim, Dae Hoon ; Takehana, Toshihiko ; Koike, Seiji ; Matsui, Hirokazu ; Mori, Haruhide
e-Rad_Researcher 00598089 著者名 Saburi, Wataru
著者名 Morimoto, Naoki
著者名 Mukai, Atsushi
著者名 Kim, Dae Hoon
著者名 Takehana, Toshihiko
著者名 Koike, Seiji
e-Rad_Researcher 90109504 著者名 Matsui, Hirokazu
e-Rad_Researcher 80241363 著者名 Mori, Haruhide
言語	英語
キーワード	α-amylase
	neopullulanase
	glycoside hydrolase family 13
	carbohydrate-binding module family 20
	starch-binding domain
発行日	2013-09
出版者	Taylor & Francis
収録物名	Bioscience, Biotechnology, and Biochemistry
巻(号)	77(9)
ページ	1867-1873
抄録	α-Amylases (EC 3.2.1.1) 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.
資源タイプ	学術雑誌論文
出版タイプ	Accepted Manuscript
権利情報	This is an Accepted Manuscript of an article published by Taylor & Francis in Bioscience biotechnology and biochemistry on 2013 Sep, available online: http://wwww.tandfonline.com/10.1271/bbb.130284
PISSN	0916-8451
EISSN	1347-6947
NCID	AA10824164
関連情報	PMID 24018662
関連情報 (isVersionOf)	出版社版DOI https://doi.org/10.1271/bbb.130284