title: Engineering transglycosidase activity into a GH51 α-l-arabinofuranosidase.
abstract: Directed evolution was applied to the α-l-arabinofuranosidase from Thermobacillus xylanilyticus to confer better transglycosylation ability, particularly for the synthesis of benzyl α-l-arabinofuranosyl-(1,2)-α-d-xylopyranoside, starting from p-nitrophenyl α-l-arabinofuranoside (donor) and benzyl α-d-xylopyranoside (acceptor). The aim was to obtain mutants displaying both lower hydrolytic and greater transglycosylation activities to favour the stable production of the target disaccharide. The implementation of a simple chromogenic screen ultimately provided three mutant enzymes whose properties correspond to those sought after. These all displayed lowered hydrolytic activity and conserved or slightly improved transfer activity, while one of them also displayed lowered secondary hydrolysis of the transglycosylation product. DNA sequence analysis of the mutants revealed between three and seven point mutations and biochemical analysis combined with STD-NMR experiments indicated that distinct molecular mechanisms were active among the three mutants.
authors.affiliation: Université de Toulouse, INSA, UPS, INP, 135 Avenue de Rangueil, F-31077 Toulouse, France.
authors.forename: Faten
authors.initials: F
authors.lastname: Arab-Jaziri
authors.forename: Bastien
authors.initials: B
authors.lastname: Bissaro
authors.forename: Michel
authors.initials: M
authors.lastname: Dion
authors.forename: Olivier
authors.initials: O
authors.lastname: Saurel
authors.forename: David
authors.initials: D
authors.lastname: Harrison
authors.forename: Fernando
authors.initials: F
authors.lastname: Ferreira
authors.forename: Alain
authors.initials: A
authors.lastname: Milon
authors.forename: Charles
authors.initials: C
authors.lastname: Tellier
authors.forename: Régis
authors.initials: R
authors.lastname: Fauré
authors.forename: Michael J
authors.initials: MJ
authors.lastname: O'Donohue
mesh_terms: D056449:Bacillales / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004187:Disaccharides / Q000138:chemical synthesis* / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation
chemical_list: D001426:Bacterial Proteins; D004187:Disaccharides; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase