Journal
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
Volume 161, Issue -, Pages 1206-1215Publisher
ELSEVIER
DOI: 10.1016/j.ijbiomac.2020.05.273
Keywords
beta-N-Acetylhexosaminidase; Enzyme engineering; Transglycosidase
Funding
- Czech Science Foundation [2000477S]
- (COST Action) from the Ministry of Education, Youth, and Sports of the Czech Republic [LTC19035, CA17118]
- project ACCORD - Operational Programme Research and Development - ERDF [313021X329]
Ask authors/readers for more resources
beta-N-Acetylhexosaminidases (CAZy GH20, EC 3.2.1.52) are exo-glycosidases specific for cleaving N-acetylglucosamine and N-acetylgalactosamine moieties of various substrates. The beta-N-acetylhexosaminidase from the filamentous fungus Talaromyces flavus (TfHex), a model enzyme in this study, has a broad substrate flexibility and outstanding synthetic ability. We have designed and characterized seven glycosynthase-type variants of TfHex mutated at the catalytic aspartate residue that stabilizes the oxazoline reaction intermediate. Most of the obtained enzyme variants lost the majority of their original hydrolytic activity towards the standard substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (pNP-beta-GlcNAc); moreover, the mutants were not active with the proposed glycosynthase donor 2-acetamido-2-deoxy-D-glucopyranosyl-alpha-fluoride (GlcNAc-alpha-F) either as would be expected in a glycosynthase. Importantly, the mutant enzymes instead retained a strong transglycosylation activity towards the standard substrate pNP-beta-GlcNAc. In summary, five out of seven prepared TfHex variants bearing mutation at the catalytic Asp370 residue acted as efficient transglycosidases, which makes them excellent tools for the synthesis of chitooligosaccharides, with the advantage of processing an inexpensive, stable and commercially available pNP-beta-GlcNAc. (C) 2020 Elsevier B.V. All rights reserved.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available