Synthesis of fucosyllactose using a-L-fucosidases GH29 from infant gut microbial metagenome
Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FU...
| Autores: | , , , , , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2024 |
| País: | España |
| Institución: | INCLIVA |
| Repositorio: | r-INCLIVA. Repositorio Institucional de Producción Científica de INCLIVA |
| OAI Identifier: | oai:incliva.fundanetsuite.com:p18506 |
| Acceso en línea: | https://incliva.portalinvestigacion.com/publicaciones/18506 |
| Access Level: | acceso abierto |
| Palabra clave: | alpha-L-fucosidase GH29 Human milk oligosaccharides 2 '-fucosyllactose 3 '-fucosyllactose Protein engineering |
| Sumario: | Fucosyl-oligosaccharides (FUS) provide many health benefits to breastfed infants, but they are almost completely absent from bovine milk, which is the basis of infant formula. Therefore, there is a growing interest in the development of enzymatic transfucosylation strategies for the production of FUS. In this work, the alpha-L-fucosidases Fuc2358 and Fuc5372, previously isolated from the intestinal bacterial metagenome of breastfed infants, were used to synthesize fucosyllactose (FL) by transfucosylation reactions using p-nitrophenyl-alpha-L-fucopyranoside (pNP-Fuc) as donor and lactose as acceptor. Fuc2358 efficiently synthesized the major fucosylated human milk oligosaccharide (HMO) 2 '-fucosyllactose (2 ' FL) with a 35% yield. Fuc2358 also produced the non-HMO FL isomer 3 '-fucosyllactose (3 ' FL) and traces of non-reducing 1-fucosyllactose (1FL). Fuc5372 showed a lower transfucosylation activity compared to Fuc2358, producing several FL isomers, including 2 ' FL, 3 ' FL, and 1FL, with a higher proportion of 3 ' FL. Site-directed mutagenesis using rational design was performed to increase FUS yields in both alpha-L-fucosidases, based on structural models and sequence identity analysis. Mutants Fuc2358-F184H, Fuc2358-K286R, and Fuc5372-R230K showed a significantly higher ratio between 2 ' FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes after 4 h of transfucosylation. The results with the Fuc2358-F184W and Fuc5372-W151F mutants showed that the residues F184 of Fuc2358 and W151 of Fuc5372 could have an effect on transfucosylation regioselectivity. Interestingly, phenylalanine increases the selectivity for alpha-1,2 linkages and tryptophan for alpha-1,3 linkages. These results give insight into the functionality of the active site amino acids in the transfucosylation activity of the GH29 alpha-L-fucosidases Fuc2358 and Fuc5372. |
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