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Applied and Environmental Microbiology, October 1999, p. 4652-4658, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Characteristics of Two Forms of alpha -Amylases and Structural Implication

Kohji Ohdan,1 Takashi Kuriki,1,* Hiroki Kaneko,2 Jiro Shimada,2 Toshikazu Takada,2 Zui Fujimoto,3 Hiroshi Mizuno,3,4 and Shigetaka Okada1

Biochemical Research Laboratory, Ezaki Glico Co., Ltd., Utajima 4-6-5, Nishiyodogawa-ku, Osaka 555-8502,1 Fundamental Research Laboratories, NEC Corporation, Miyukigaoka, Tsukuba, Ibaraki 305-0841,2 Department of Biotechnology, National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602,3 and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8572,4 Japan

Received 24 May 1999/Accepted 16 July 1999

Complete (Ba-L) and truncated (Ba-S) forms of alpha -amylases from Bacillus subtilis X-23 were purified, and the amino- and carboxyl-terminal amino acid sequences of Ba-L and Ba-S were determined. The amino acid sequence deduced from the nucleotide sequence of the alpha -amylase gene indicated that Ba-S was produced from Ba-L by truncation of the 186 amino acid residues at the carboxyl-terminal region. The results of genomic Southern analysis and Western analysis suggested that the two enzymes originated from the same alpha -amylase gene and that truncation of Ba-L to Ba-S occurred during the cultivation of B. subtilis X-23 cells. Although the primary structure of Ba-S was approximately 28% shorter than that of Ba-L, the two enzyme forms had the same enzymatic characteristics (molar catalytic activity, amylolytic pattern, transglycosylation ability, effect of pH on stability and activity, optimum temperature, and raw starch-binding ability), except that the thermal stability of Ba-S was higher than that of Ba-L. An analysis of the secondary structure as well as the predicted three-dimensional structure of Ba-S showed that Ba-S retained all of the necessary domains (domains A, B, and C) which were most likely to be required for functionality as alpha -amylase.

* Corresponding author. Mailing address: Biochemical Research Laboratory, Ezaki Glico Co., Ltd., Utajima 4-6-5, Nishiyodogawa-ku, Osaka 555-8502, Japan. Phone: 81-6-6477-8425. Fax: 81-6-6477-8362. E-mail: kuriki-takashi{at}glico.co.jp.

Applied and Environmental Microbiology, October 1999, p. 4652-4658, Vol. 65, No. 10
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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