title: Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 α-L-arabinofuranosidase.
abstract: Random mutagenesis was performed on the α-l-arabinofuranosidase of Thermobacillus xylanilyticus in order to enhance its ability to perform transarabinofuranosylation using natural xylo-oligosaccharides as acceptors. To achieve this goal, a two-step, high-throughput digital imaging protocol involving a colorimetric substrate was used to screen a library of 30,000 mutants. In the first step this screen selected for hydrolytically-impaired mutants, and in the second step the screen identified mutants whose global activity was improved in the presence of a xylo-oligosaccharide mixture. Thereby, 199 mutants displaying lowered hydrolytic activity and modified properties were detected. In the presence of these xylo-oligosaccharides, most of the 199 (i.e., 70%) enzymes were less inhibited and some (18) mutants displayed an unambiguous alleviation of inhibition (<25% loss of activity). More precise monitoring of reactions catalyzed by the most promising mutants revealed a significant improvement of the synthesis yields of transglycosylation products (up to 18% compared to 9% for the parental enzyme) when xylobiose was present in the reaction. Genetic analysis of improved mutants revealed that many of the amino acid substitutions that correlate with the modified phenotype are located in the vicinity of the active site, particularly in subsite -1. Consequently, we hypothesize that these mutations modify the active site topology or the molecular interaction network of the l-arabinofuranoside donor substrate, thus impairing the hydrolysis and concomitantly favoring transglycosylation onto natural acceptors.
authors.affiliation: Université de Toulouse, INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France; Agence de l'environnement et de la Maîtrise de l'Energie, 20 Avenue du Grésillé-BP 90406, 49004 Angers Cedex 01, France.
authors.forename: Faten
authors.initials: F
authors.lastname: Arab-Jaziri
authors.affiliation: Université de Toulouse, INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Bastien
authors.initials: B
authors.lastname: Bissaro
authors.affiliation: UFIP, UMR CNRS 6286, Faculté des Sciences et Techniques, Université de Nantes, 2 rue de la Houssinière, F-44322 Nantes, France.
authors.forename: Charles
authors.initials: C
authors.lastname: Tellier
authors.affiliation: UFIP, UMR CNRS 6286, Faculté des Sciences et Techniques, Université de Nantes, 2 rue de la Houssinière, F-44322 Nantes, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Dion
authors.affiliation: Université de Toulouse, INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France.
authors.forename: Régis
authors.initials: R
authors.lastname: Fauré
authors.affiliation: Université de Toulouse, INSA,UPS,INP; LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France; INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France; CNRS, UMR5504, F-31400 Toulouse, France. Electronic address: michael.odonohue@insa-toulouse.fr.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: O'Donohue
mesh_terms: D001089:Arabinose / Q000737:chemistry*; D056449:Bacillales / Q000201:enzymology; D020134:Catalytic Domain; D060326:Chemistry Techniques, Synthetic; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: D009844:Oligosaccharides; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase
keywords: Glycoside hydrolase; Inhibition; Pentose/furanose; Transglycosylation; Xylo-oligosaccharide