Mutation Hotspot for Changing the Substrate Specificity of beta-N-Acetylhexosaminidase: A Library of GlcNAcases
The result's identifiers
Result code in IS VaVaI
<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F22%3A00563832" target="_blank" >RIV/61388971:_____/22:00563832 - isvavai.cz</a>
Result on the web
<a href="https://www.mdpi.com/1422-0067/23/20/12456" target="_blank" >https://www.mdpi.com/1422-0067/23/20/12456</a>
DOI - Digital Object Identifier
<a href="http://dx.doi.org/10.3390/ijms232012456" target="_blank" >10.3390/ijms232012456</a>
Alternative languages
Result language
angličtina
Original language name
Mutation Hotspot for Changing the Substrate Specificity of beta-N-Acetylhexosaminidase: A Library of GlcNAcases
Original language description
beta-N-Acetylhexosaminidase from Talaromyces flavus (TfHex, EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal beta-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.
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
10608 - Biochemistry and molecular biology
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
2022
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů
Data specific for result type
Name of the periodical
International Journal of Molecular Sciences
ISSN
1422-0067
e-ISSN
1422-0067
Volume of the periodical
23
Issue of the periodical within the volume
20
Country of publishing house
CH - SWITZERLAND
Number of pages
17
Pages from-to
12456
UT code for WoS article
000872833300001
EID of the result in the Scopus database
2-s2.0-85140984333