期刊
CHEMISTRY-A EUROPEAN JOURNAL
卷 27, 期 40, 页码 10323-10334出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/chem.202100110
关键词
enzyme catalysis; hydrolases; multiple sequences alignment; oligosaccharide synthesis; protein engineering; transglycosylation
资金
- Novo Nordisk Foundation [NNF17OC0025660, NNF10CC1016517]
- China Scholarship Council (CSC)
- Villum Foundation
- Region Midi-Pyrenees
- European Regional Development Fund
- SICOVAL
- IBiSa-France
- CNRS
- INRAE
- DTU
- Swedish Foundation [IRC150068, RBP14-0046]
- Swedish Research Council [2019-05605]
- FORMAS [9422016-117]
- Vinnova [2019-05605] Funding Source: Vinnova
- Swedish Foundation for Strategic Research (SSF) [RBP14-0046] Funding Source: Swedish Foundation for Strategic Research (SSF)
- Swedish Research Council [2019-05605] Funding Source: Swedish Research Council
This method describes a straightforward strategy involving rapid in silico analysis of protein sequences to identify single-mutant candidates for improving transglycosylation yields. Requiring minimal prior knowledge of the target enzyme, the method is generic and can validate mutations in one enzyme for transposition to others, even distantly related enzymes.
Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6-12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various D/L-, alpha/beta-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.
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