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Journal of Bacteriology, January 1999, p. 375-381, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Sequence Analysis of the Gene Encoding Amylosucrase from Neisseria polysaccharea and Characterization of the Recombinant Enzyme

G. Potocki De Montalk,1 M. Remaud-Simeon,1 R. M. Willemot,1 V. Planchot,2 and P. Monsan1,*

Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, LA INRA DGBA, INSA, Complexe Scientifique de Rangueil, 31 077 Toulouse Cedex,1 and INRA URPOI 44 316 Nantes Cedex 3,2 France

Received 24 June 1998/Accepted 21 October 1998

The Neisseria polysaccharea gene encoding amylosucrase was subcloned and expressed in Escherichia coli. Sequencing revealed that the deduced amino acid sequence differs significantly from that previously published. Comparison of the sequence with that of enzymes of the alpha -amylase family predicted a (beta /alpha )8-barrel domain. Six of the eight highly conserved regions in amylolytic enzymes are present in amylosucrase. Among them, four constitute the active site in alpha -amylases. These sites were also conserved in the sequence of glucosyltransferases and dextransucrases. Nevertheless, the evolutionary tree does not show strong homology between them. The amylosucrase was purified by affinity chromatography between fusion protein glutathione S-transferase-amylosucrase and glutathione-Sepharose 4B. The pure enzyme linearly elongated some branched chains of glycogen, to an average degree of polymerization of 75.

* Corresponding author. Mailing address: Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, LA INRA DGBA, INSA, Complexe Scientifique de Rangueil, 31 077 Toulouse Cedex, France. Phone: 33 5 61 55 94 15. Fax: 33 5 61 55 94 00. E-mail: monsan{at}insa_tlse.fr.

Journal of Bacteriology, January 1999, p. 375-381, Vol. 181, No. 2
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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