How Site-Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388963%3A_____%2F20%3A00533157" target="_blank" >RIV/61388963:_____/20:00533157 - isvavai.cz</a>

Nalezeny alternativní kódy

RIV/61388971:_____/20:00533157 RIV/68407700:21460/20:00348819 RIV/00216208:11310/20:10414379

Výsledek na webu

<a href="https://onlinelibrary.wiley.com/doi/abs/10.1002/adsc.202000604" target="_blank" >https://onlinelibrary.wiley.com/doi/abs/10.1002/adsc.202000604</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/adsc.202000604" target="_blank" >10.1002/adsc.202000604</a>

Jazyk výsledku

angličtina

Název v původním jazyce

How Site-Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme

Popis výsledku v původním jazyce

beta-N-Acetylhexosaminidases (GH20, EC 3.2.1.52) areexo-glycosidases with a dual activity for cleaving bothN-acetylglucosamine (GlcNAc) andN-acetylgalactosamine (GalNAc) units from glycostructures. This substrate promiscuity is a hurdle in the selective synthesis ofN-acetylhexosamine oligosaccharides combining both GlcNAc and GalNAc units since there are hardly any GalNAc transferring enzymes available for synthetic applications. We present here site-directed mutagenesis of a synthetically potent promiscuous beta-N-acetylhexosaminidase fromTalaromyces flavus(TfHex), which, as a wild type, exhibits a GalNAcase/GlcNAcase ratio of 1.2. On the basis of molecular modeling, we identified crucial amino acid residues responsible for its GalNAcase/GlcNAcase selectivity. Six site-directed mutants were prepared, heterologously expressed inPichia pastoris, purified, and kinetically characterized. As a result, novel engineered enzymes with an up to 7-times higher selectivity for either GalNAc or GlcNAc substrates were obtained, preserving the favorable properties of the wild typeTfHex, mainly its transglycosylation potential and tolerance to functional groups in the substrate molecule. The substrate selectivity and transglycosylation yield were further corroborated by reaction engineering. The new selective and synthetically capable enzymes were applied in the preparation of tailoredN-acetylhexosamines.

Název v anglickém jazyce

How Site-Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme

Popis výsledku anglicky

beta-N-Acetylhexosaminidases (GH20, EC 3.2.1.52) areexo-glycosidases with a dual activity for cleaving bothN-acetylglucosamine (GlcNAc) andN-acetylgalactosamine (GalNAc) units from glycostructures. This substrate promiscuity is a hurdle in the selective synthesis ofN-acetylhexosamine oligosaccharides combining both GlcNAc and GalNAc units since there are hardly any GalNAc transferring enzymes available for synthetic applications. We present here site-directed mutagenesis of a synthetically potent promiscuous beta-N-acetylhexosaminidase fromTalaromyces flavus(TfHex), which, as a wild type, exhibits a GalNAcase/GlcNAcase ratio of 1.2. On the basis of molecular modeling, we identified crucial amino acid residues responsible for its GalNAcase/GlcNAcase selectivity. Six site-directed mutants were prepared, heterologously expressed inPichia pastoris, purified, and kinetically characterized. As a result, novel engineered enzymes with an up to 7-times higher selectivity for either GalNAc or GlcNAc substrates were obtained, preserving the favorable properties of the wild typeTfHex, mainly its transglycosylation potential and tolerance to functional groups in the substrate molecule. The substrate selectivity and transglycosylation yield were further corroborated by reaction engineering. The new selective and synthetically capable enzymes were applied in the preparation of tailoredN-acetylhexosamines.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10406 - Analytical chemistry

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

Advanced Synthesis & Catalysis

ISSN

1615-4150

e-ISSN

—

Svazek periodika

362

Číslo periodika v rámci svazku

19

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

13

Strana od-do

4138-4150

Kód UT WoS článku

000563150000001

EID výsledku v databázi Scopus

2-s2.0-85089864231