Covalent connectivity of glycogen in brewer's spent yeast cell walls revealed by enzymatic approaches and dynamic nuclear polarization NMR


Yeast cell walls undergo modifications during the brewing process, leading to a remodelling of their architecture. One significant change is the increased insolubility of the cell wall glycogen pool, likely due to the formation of covalent bonds between glycogen and cell wall polysaccharides. To verify this hypothesis, we extracted the brewer's spent yeast with 4 M KOH, obtaining an insoluble glucan fraction (AE.4 M) primarily composed of (α1 → 4)- and (1 → 3)-linked Glc residues. Dynamic nuclear polarization solid-state NMR of AE.4 M revealed distinct glucan resonances that helped to differentiate between α- and β glucosyl (1 → 4)-linked residues, and confirm covalent linkages between (β1 → 3)-glucans and glycogen through a (β1 → 4)-linkage. The hydrolysis with different endo-glucanases (zymolyase, cellulase, and lichenase) was used to obtain solubilized high molecular weight glycogen fractions. NMR analysis showed that covalent links between glycogen and (β1 → 6)-glucans through (α1 → 6) glycosidic linkage, with branching at the C6 position involving (β1 → 3), and (β1 → 6)-glucans. HPAEC-PAD analysis of the enzymatically released oligosaccharides confirmed covalent linkages of (β1 → 3), (β1 → 6)-, and (β1 → 4)-glucan motifs with (α1 → 4)-glucans. This combination of multiple enzymatic approaches and NMR methods shed light into the role of yeast cell wall glycogen as a structural core covalently linked to other cell wall components during the brewing process.


Rita Bastos, Ildefonso Marín-Montesinos*, Sónia S. Ferreira, Frédéric Mentink-Vigier, Mariana Sardo, Luís Mafra, Manuel A. Coimbra, Elisabete Coelho*

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The authors acknowledge to FCT/MEC for the financial support of the project “Yeast4FoodMed” (POCI-01-045-FEDER-030936 and PTDC/BAA-AGR/30936/2017), LAQV/REQUIMTE (UIDB/50006/2020, UIDP/50006/2020), and CICECO (UIDB/50011/2020, UIDP/50011/2020 & LA/P/0006/2020) through national funds and, where applicable, co-financed by the FEDER - Fundo Europeu de Desenvolvimento Regional, within the PT2020 Partnership Agreement. Rita Bastos was supported by an individual FCT grant (PD/BD/ 114579/2016), Elisabete Coelho thanks the research contract (CDLCTTRI-88-ARH/2018 — REF. 049-88-ARH/2018) funded by national funds (OE), through FCT, in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/ 2016, of August 29, changed by Law 57/2017, of July 19. Ildefonso Marín-Montesinos is supported by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (Grant Agreement 865974). Mariana Sardo also acknowledges FCT for a researcher position (CEECIND/00056/2020). Thanks are also due to the Portuguese NMR Network Partnership Agreement. The authors also acknowledge Professor Artur M. S. Silva, Department of Chemistry of the University of Aveiro for helpful discussions about liquid-state NMR spectra interpretation. Thanks are also due to Super Bock Group SA (Porto, Portugal) for supplying the brewer's spent yeast samples. The National High Magnetic Field Laboratory (NHMFL) is funded by the National Science Foundation Division of Materials Research (DMR-1644779 and DMR-2128556) and the State of Florida. A portion of this work was supported by the NIH P41 GM122698, and from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement no. 101008500.

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