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Mutation Hotspot for Changing the Substrate Specificity of beta-N-Acetylhexosaminidase: A Library of GlcNAcases
- 1.0563832 - MBÚ 2023 RIV CH eng J - Journal Article
Nekvasilová, Pavlína - Kulik, Natalia - Kotík, Michael - Petrásková, Lucie - Slámová, Kristýna - Křen, Vladimír - Bojarová, Pavla
Mutation Hotspot for Changing the Substrate Specificity of beta-N-Acetylhexosaminidase: A Library of GlcNAcases.
International Journal of Molecular Sciences. Roč. 23, č. 20 (2022), č. článku 12456. E-ISSN 1422-0067
R&D Projects: GA ČR(CZ) GA22-00262S; GA MŠMT(CZ) LTC20072; GA MŠMT(CZ) LTC19035
Institutional support: RVO:61388971
Keywords : beta-N-acetylhexosaminidase * Talaromyces flavus * Pichia pastoris * site-saturation mutagenesis * site-directed mutagenesis * substrate specificity
OECD category: Biochemistry and molecular biology
Impact factor: 5.6, year: 2022
Method of publishing: Open access
https://www.mdpi.com/1422-0067/23/20/12456
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.
Permanent Link: https://hdl.handle.net/11104/0335625
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