کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
33340 | 44971 | 2013 | 9 صفحه PDF | دانلود رایگان |
• Directed evolution has been used to increase the transglycosylation ability of TxAbf.
• Mutants display contrasted biochemical and hydrolysis/transglycosylation profiles.
• STD-NMR pinpointed altered interactions in the catalytic site of mutants.
• F26, W178 and N344 stand out as targets for engineering to improve glycosynthesis.
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.
Journal: New Biotechnology - Volume 30, Issue 5, 25 June 2013, Pages 536–544