Sequence analysis of the gene encoding amylosucrase from Neisseria polysaccharea and characterization of the recombinant enzyme.
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.
De Montalk
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Centre de Bioingénierie Gilbert Durand, UMR CNRS 5504, LA INRA DGBA, INSA, Complexe Scientifique de Rangueil, 31 077 Toulouse Cedex, France.
Remaud-Simeon
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Willemot
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Planchot
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Monsan
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers; D004926:Escherichia coli; D019143:Evolution, Molecular; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009343:Neisseria / Q000201:enzymology* / Q000235:genetics*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C019700:amylosucrase