Engineering transglycosidase activity into a GH51 α-L-arabinofuranosidase

Directed evolution was applied to the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus to confer better transglycosylation ability, particularly for the synthesis of benzyl alpha-L-arabinofuranosyl-(1,2)-alpha-D-xylopyranoside, starting from p-nitrophenyl alpha-L-arabinofuranoside (donor) and benzyl alpha-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.