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 ex pressed in Escherichia coli, Sequencing revealed that the deduced amino aci d 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 consti tute 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 amylosuc rase was purified by affinity chromatography between fusion protein glutath ione S-transferase-amylosucrase and glutathione-Sepharose 4B, The pure enzy me linearly elongated some branched chains of glycogen, to an average degre e of polymerization of 75.