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Applied and Environmental Microbiology, September 1999, p. 4163-4170, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Expression of the Isoamylase Gene of Flavobacterium odoratum KU in Escherichia coli and Identification of Essential Residues of the Enzyme by Site-Directed Mutagenesis

Jun-ichi Abe,* Chiaki Ushijima, and Susumu Hizukuri

Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1-21-24, Kagoshima 890, Japan

Received 3 February 1999/Accepted 15 July 1999

The isoamylase gene from Flavobacterium odoratum KU was cloned into and expressed in Escherichia coli JM109. The promoter of the gene was successful in E. coli, and the enzyme produced was excreted into the culture medium, depending on the amount of the enzyme expressed. The enzyme found in the culture medium showed almost the same Mr, heat-inactivating constant, and N-terminal sequence as those of the enzyme accumulated in the periplasmic space. This result indicated that the enzyme accumulated in an active form at the periplasm was transported out of the cell. The primary sequence of the enzyme, which was deduced from its nucleotide sequence, showed that the mature enzyme consisted of 741 amino acid residues. By changing five possible residues to Ala independently, it was found that Asp-374, Glu-422, and Asp-497 were essential. The sequences around those residues were highly conserved in isoamylases of different origins and the glycogen operon protein X, GlgX. The comparison of the distance between these essential residues with those of various amylases suggested that the bacterial and plant isoamylase but not GlgX had a longer fourth loop than the other amylases. This longer fourth loop had a possible role in accommodating the long branched chains of native glycogens and starches.

* Corresponding author. Mailing address: Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto-1-21-24, Kagoshima 890, Japan. Phone and fax: 81-99-285-8642. E-mail: j_abe{at}chem.agri.kagoshima-u.ac.jp.

Applied and Environmental Microbiology, September 1999, p. 4163-4170, Vol. 65, No. 9
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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