Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 beta-N-acetylhexosaminidase

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F20%3A00533177" target="_blank" >RIV/61388971:_____/20:00533177 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0141813020334747" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0141813020334747</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ijbiomac.2020.05.273" target="_blank" >10.1016/j.ijbiomac.2020.05.273</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 beta-N-acetylhexosaminidase

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Transglycosidase activity of glycosynthase-type mutants of a fungal GH20 beta-N-acetylhexosaminidase

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

International Journal of Biological Macromolecules

ISSN

0141-8130

e-ISSN

—

Svazek periodika

161

Číslo periodika v rámci svazku

OCT15

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

10

Strana od-do

1206-1215

Kód UT WoS článku

000569753300005

EID výsledku v databázi Scopus

2-s2.0-85086941152