title: Increased amylosucrase activity and specificity, and identification of regions important for activity, specificity and stability through molecular evolution.
abstract: Amylosucrase is a transglycosidase which belongs to family 13 of the glycoside hydrolases and transglycosidases, and catalyses the formation of amylose from sucrose. Its potential use as an industrial tool for the synthesis or modification of polysaccharides is hampered by its low catalytic efficiency on sucrose alone, its low stability and the catalysis of side reactions resulting in sucrose isomer formation. Therefore, combinatorial engineering of the enzyme through random mutagenesis, gene shuffling and selective screening (directed evolution) was applied, in order to generate more efficient variants of the enzyme. This resulted in isolation of the most active amylosucrase (Asn387Asp) characterized to date, with a 60% increase in activity and a highly efficient polymerase (Glu227Gly) that produces a longer polymer than the wild-type enzyme. Furthermore, judged from the screening results, several variants are expected to be improved concerning activity and/or thermostability. Most of the amino acid substitutions observed in the totality of these improved variants are clustered around specific regions. The secondary sucrose-binding site and beta strand 7, connected to the important Asp393 residue, are found to be important for amylosucrase activity, whereas a specific loop in the B-domain is involved in amylosucrase specificity and stability.
authors.affiliation: Laboratoire Biotechnologie-Bioprocédés, UMR CNRS 5504, UMR INRA 792, Toulouse, France.
authors.forename: Bart A
authors.initials: BA
authors.lastname: van der Veen
authors.forename: Lars K
authors.initials: LK
authors.lastname: Skov
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Potocki-Véronèse
authors.forename: Michael
authors.initials: M
authors.lastname: Gajhede
authors.forename: Pierre
authors.initials: P
authors.lastname: Monsan
authors.forename: Magali
authors.initials: M
authors.lastname: Remaud-Simeon
mesh_terms: D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D019143:Evolution, Molecular; D014644:Genetic Variation; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D005964:Glucosyltransferases; C019700:amylosucrase