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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
-amylase family predicted a
(
/
)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
-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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