A novel enzymatic tool for transferring GalNAc moiety onto challenging acceptors
Language English Country Netherlands Media print-electronic
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
31740417
DOI
10.1016/j.bbapap.2019.140319
PII: S1570-9639(19)30204-3
Knihovny.cz E-resources
- Keywords
- Glycosylation, Penicillium oxalicum, Pichia pastoris, Substrate specificity, β-N-acetylhexosaminidase,
- MeSH
- beta-N-Acetylhexosaminidases genetics metabolism MeSH
- Glycosylation MeSH
- Kinetics MeSH
- Hydrogen-Ion Concentration MeSH
- Penicillium enzymology MeSH
- Pichia metabolism MeSH
- Recombinant Proteins biosynthesis isolation & purification metabolism MeSH
- Substrate Specificity MeSH
- Temperature MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- beta-N-Acetylhexosaminidases MeSH
- Recombinant Proteins 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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