How Site-Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme
The result's identifiers
Result code in 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>
Alternative codes found
RIV/61388971:_____/20:00533157 RIV/68407700:21460/20:00348819 RIV/00216208:11310/20:10414379
Result on the web
<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>
Alternative languages
Result language
angličtina
Original language name
How Site-Directed Mutagenesis Boosted Selectivity of a Promiscuous Enzyme
Original language description
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.
Czech name
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Czech description
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Classification
Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10406 - Analytical chemistry
Result continuities
Project
Result was created during the realization of more than one project. More information in the Projects tab.
Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace
Others
Publication year
2020
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
Advanced Synthesis & Catalysis
ISSN
1615-4150
e-ISSN
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Volume of the periodical
362
Issue of the periodical within the volume
19
Country of publishing house
DE - GERMANY
Number of pages
13
Pages from-to
4138-4150
UT code for WoS article
000563150000001
EID of the result in the Scopus database
2-s2.0-85089864231