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Volume 271, Number 39, Issue of September 27, 1996 pp. 24075-24083
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Amino Acid Sequence and Molecular Structure of an Alkaline Amylopullulanase from Bacillus That Hydrolyzes -1,4 and -1,6 Linkages in Polysaccharides at Different Active Sites

(Received for publication, February 1, 1996, and in revised form, May 6, 1996)

Yuji Hatada , Kazuaki Igarashi , Katsuya Ozaki , Katsutoshi Ara , Jun Hitomi , Tohru Kobayashi , Shuji Kawai , Tomoyoshi Watabe ^§ and Susumu Ito

From the  Tochigi Research Laboratories of the Kao Corporation, 2606 Akabane, Ichikai, Haga, Tochigi 321-34, Japan and the ^§ Electron Optics Instruments Training & Application Center of JEOL Datum Ltd., 1156 Nakagami, Akishima, Tokyo 196, Japan

An amylopullulanase from alkalophilic Bacillus sp. KSM-1378 hydrolyzes both -1,6 linkages in pullulan and -1,4 linkages in other polysaccharides, with maximum activity in each case at an alkaline pH, to generate oligosaccharides (Ara, K., Saeki, K., Igarashi, K., Takaiwa, M., Uemura, T., Hagihara, H., Kawai, S., and Ito, S. (1995) Biochim. Biophys. Acta 1243, 315-324). Here, we report the molecular cloning and sequencing of the gene for and the structure of this enzyme and show that its dual hydrolytic activities are associated with two independent active sites. The structural gene contained a single, long open reading frame of 5,814 base pairs, corresponding to 1,938 amino acids that included a signal peptide of 32 amino acids. The molecular mass of the extracellular mature enzyme (Glu³³ through Leu¹⁹³⁸) was calculated to be 211,450 Da, a value close to the 210 kDa determined for the amylopullulanase produced by Bacillus sp. KSM-1378. The amylase and the pullulanase domains were located in the amino-terminal half and in the carboxyl-terminal half of the enzyme, respectively, being separated by a tandem repeat of a sequence of 35 amino acids. Four regions, designated I, II, III, and IV, were highly conserved in each catalytic domain, and they included a putative catalytic triad Asp⁵⁵⁰-Glu⁵⁷⁹-Asp⁶⁴⁵ for the amylase activity and Asp¹⁴⁶⁴-Glu¹⁴⁹³-Asp¹⁵⁸¹ for the pullulanase activity. The purified enzyme was rotary shadowed at a low angle and observed by transmission electron microscopy; it appeared to be a ``castanet-like'' or ``bent dumbbell-like'' molecule with a diameter of approximately 25 nm.

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