A Thermophilic Alkalophilic alpha-Amylase from Bacillus sp AAH-31 Shows a Novel Domain Organization among Glycoside Hydrolase Family 13 Enzymes

作者：Saburi Wataru; Morimoto Naoki; Mukai Atsushi; Kim Dae Hoon; Takehana Toshihiko; Koike Seiji; Matsui Hirokazu; Mori Haruhide^*

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

DOI：10.1271/bbb.130284

• alpha-amylase
• neopullulanase
• glycoside hydrolase family 13
• carbohydrate-binding module family 20
• starch-binding domain

alpha-Amylases (EC 3.2.1.1) hydrolyze internal alpha-1,4-glucosidic linkages of starch and related glucans. Bacillus sp. AAH-31 produces an alkalophilic thermophilic alpha-amylase (AmyL) of higher molecular mass, 91 kDa, than typical bacterial alpha-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 alpha-amylases. In contrast to known neopullulanase-like alpha-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 degrees C and from pH 6.4 to 11.9. The k(cat) values for soluble starch, gamma-cyclodextrin, and maltotriose were 103 s(-1), 67.6 s(-1), and 5.33 s(-1), respectively, and the K-m 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.

• 出版日期2013-9