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Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 β-N-acetylhexosaminidase
J. Kapešová, L. Petrásková, N. Kulik, Z. Straková, P. Bojarová, K. Markošová, M. Rebroš, V. Křen, K. Slámová
Jazyk angličtina Země Nizozemsko
Typ dokumentu časopisecké články
Odkazy
PubMed
32522540
DOI
10.1016/j.ijbiomac.2020.05.273
Knihovny.cz E-zdroje
- MeSH
- aktivace enzymů MeSH
- beta-N-acetylhexosaminidasy genetika metabolismus MeSH
- houby enzymologie genetika MeSH
- hydrolýza MeSH
- katalýza MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- mutace * MeSH
- proteinové inženýrství MeSH
- substrátová specifita MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
β-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 β-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-β-D-glucopyranoside (pNP-β-GlcNAc); moreover, the mutants were not active with the proposed glycosynthase donor 2-acetamido-2-deoxy-d-glucopyranosyl-α-fluoride (GlcNAc-α-F) either as would be expected in a glycosynthase. Importantly, the mutant enzymes instead retained a strong transglycosylation activity towards the standard substrate pNP-β-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-β-GlcNAc.
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- $a Kapešová, Jana $u Laboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, Prague 4 CZ 14220, Czech Republic
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- $a β-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 β-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-β-D-glucopyranoside (pNP-β-GlcNAc); moreover, the mutants were not active with the proposed glycosynthase donor 2-acetamido-2-deoxy-d-glucopyranosyl-α-fluoride (GlcNAc-α-F) either as would be expected in a glycosynthase. Importantly, the mutant enzymes instead retained a strong transglycosylation activity towards the standard substrate pNP-β-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-β-GlcNAc.
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- $a Markošová, Kristína $u Institute of Biotechnology, Slovak University of Technology, Radlinského 9, Bratislava, SK 81237, Slovakia
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