Carbohydrate Research 401 (2015) 64–72
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 a-L-arabinofuranosidase Faten Arab-Jaziri a,b,c,d, Bastien Bissaro a,b,c, Charles Tellier e, Michel Dion e, Régis Fauré a,b,c, Michael J. O’Donohue a,b,c,⇑ a
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 d Agence de l’environnement et de la Maîtrise de l’Energie, 20 Avenue du Grésillé—BP 90406, 49004 Angers Cedex 01, France e UFIP, UMR CNRS 6286, Faculté des Sciences et Techniques, Université de Nantes, 2 rue de la Houssinière, F-44322 Nantes, France b c
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 28 October 2014 Accepted 30 October 2014 Available online 8 November 2014 Keywords: Glycoside hydrolase Transglycosylation Pentose/furanose Xylo-oligosaccharide Inhibition
a b s t r a c t Random mutagenesis was performed on the a-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 (
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Enhancing the chemoenzymatic synthesis of arabinosylated xylo-oligosaccharides by GH51 a-L-arabinofuranosidase Faten Arab-Jaziri a,b,c,d, Bastien Bissaro a,b,c, Charles Tellier e, Michel Dion e, Régis Fauré a,b,c, Michael J. O’Donohue a,b,c,⇑ a
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 d Agence de l’environnement et de la Maîtrise de l’Energie, 20 Avenue du Grésillé—BP 90406, 49004 Angers Cedex 01, France e UFIP, UMR CNRS 6286, Faculté des Sciences et Techniques, Université de Nantes, 2 rue de la Houssinière, F-44322 Nantes, France b c
a r t i c l e
i n f o
Article history: Received 26 September 2014 Received in revised form 28 October 2014 Accepted 30 October 2014 Available online 8 November 2014 Keywords: Glycoside hydrolase Transglycosylation Pentose/furanose Xylo-oligosaccharide Inhibition
a b s t r a c t Random mutagenesis was performed on the a-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 (
