A novel enzymatic tool for transferring GalNAc moiety onto challenging acceptors
Jazyk angličtina Země Nizozemsko Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
PubMed
31740417
DOI
10.1016/j.bbapap.2019.140319
PII: S1570-9639(19)30204-3
Knihovny.cz E-zdroje
- Klíčová slova
- Glycosylation, Penicillium oxalicum, Pichia pastoris, Substrate specificity, β-N-acetylhexosaminidase,
- MeSH
- beta-N-acetylhexosaminidasy genetika metabolismus MeSH
- glykosylace MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- Penicillium enzymologie MeSH
- Pichia metabolismus MeSH
- rekombinantní proteiny biosyntéza izolace a purifikace metabolismus MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- beta-N-acetylhexosaminidasy MeSH
- rekombinantní proteiny MeSH
The β-N-acetylhexosaminidase from Penicillium oxalicum (PoHex; EC 3.2.1.52) is a fungal glycosidase with an outstandingly high GalNAcase/GlcNAcase activity ratio. It has a remarkable synthetic capability and can process carbohydrates functionalized at various positions. However, the production in the native fungal host is lengthy, unselective and purification from the fungal medium is complicated and low yielding. We present here a novel production method of this enzyme in the eukaryotic host of Pichia pastoris, followed by elegant one-step purification to homogeneity. The resulting recombinant enzyme has improved biochemical and catalytic properties compared to the fungal wild type. Its good production yield (11 mg/400 mL cultivation medium) greatly expands the scope of synthetic applications. We further demonstrate the synthetic utility and broad acceptor specificity of recombinant PoHex in the glycosylation of a series of challenging acceptors with varying structural architectures, namely secondary and tertiary hydroxyl, aldoxime and a poly-hydroxylated compound.
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