{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T23:01:45Z","timestamp":1777762905426,"version":"3.51.4"},"reference-count":59,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2025,1,29]],"date-time":"2025-01-29T00:00:00Z","timestamp":1738108800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100003086","name":"Eusko Jaurlaritza","doi-asserted-by":"publisher","award":["ELKARTEK bmg22"],"award-info":[{"award-number":["ELKARTEK bmg22"]}],"id":[{"id":"10.13039\/501100003086","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011033","name":"Agencia Estatal de Investigaci\u00f3n","doi-asserted-by":"publisher","award":["PID2020-117552RB-I00"],"award-info":[{"award-number":["PID2020-117552RB-I00"]}],"id":[{"id":"10.13039\/501100011033","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100003989","name":"Ikerbasque, Basque Foundation for Science","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100003989","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["chemistry-europe.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["ChemSusChem"],"published-print":{"date-parts":[[2025,5,19]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Efficient methods for isolating N\u2010glycans are essential to understanding the functions and characteristics of the entire N\u2010glycome. Enzymatic release using PNGaseF is the most effective approach for releasing mammalian N\u2010glycans for analytical purposes. However, the use of PNGaseF for preparative N\u2010glycan isolation is precluded due to the enzyme\u2032s cost and limited stability. In this work, we develop a PNGaseF heterogeneous biocatalyst for the preparative isolation of N\u2010glycans from natural sources. By controlling the immobilization conditions, 100\u201351\u2009% of offered PNGaseF is immobilized on aldehyde\u2010functionalized agarose porous microbeads through distinct protein orientations, achieving different performances. The enzyme orientation through the N\u2010terminus provides the best activity\/operational stability balance, being 20\u2009% more efficient than that randomly oriented. This active and stable heterogeneous biocatalyst eases its application in a packed bed reactor (PBR) for continuous release of free N\u2010glycans from a model glycoprotein. This PBR processes 1\u2005g of ovalbumin from chicken egg white to isolate 95\u2009% of its N\u2010glycans upon operating the PBR for 7\u2005days. Finally, by tuning the flow rate, we can control the profile of N\u2010glycans isolated due to different enzyme kinetics for the deglycosylation reactions. In\u2010line methodologies to isolate N\u2010glycans open new paths for more sustainable protocols to prepare relevant glycans.<\/jats:p>","DOI":"10.1002\/cssc.202402346","type":"journal-article","created":{"date-parts":[[2025,1,16]],"date-time":"2025-01-16T09:12:16Z","timestamp":1737018736000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Preparative Isolation of N\u2010Glycans from Natural Sources Mediated by a Deglycosylating Heterogeneous Biocatalyst in Flow"],"prefix":"10.1002","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2085-4412","authenticated-orcid":false,"given":"Sonia","family":"Serna","sequence":"first","affiliation":[{"name":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE) Basque Research and Technology Alliance (BRTA)  Paseo de Miram\u00f3n 194 20014 Donostia-San Sebasti\u00e1n Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Natalia","family":"Comino","sequence":"additional","affiliation":[{"name":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE) Basque Research and Technology Alliance (BRTA)  Paseo de Miram\u00f3n 194 20014 Donostia-San Sebasti\u00e1n Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Niels C.","family":"Reichardt","sequence":"additional","affiliation":[{"name":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE) Basque Research and Technology Alliance (BRTA)  Paseo de Miram\u00f3n 194 20014 Donostia-San Sebasti\u00e1n Spain"},{"name":"Biomedical Research Networking Center in Bioengineering, Biomaterials, and Nanomedicine (CIBER-BBN)  20014 Donostia-San Sebasti\u00e1n Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0031-1880","authenticated-orcid":false,"given":"Fernando","family":"L\u00f3pez\u2010Gallego","sequence":"additional","affiliation":[{"name":"Center for Cooperative Research in Biomaterials (CIC biomaGUNE) Basque Research and Technology Alliance (BRTA)  Paseo de Miram\u00f3n 194 20014 Donostia-San Sebasti\u00e1n Spain"},{"name":"Ikerbasque Basque Foundation for Science  48009 Bilbao Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2025,1,29]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41580-020-00294-x"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1136\/jitc-2022-005891"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41557-018-0188-3"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1021\/cb501023u"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0969-2126(94)00108-1"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2018.024"},{"key":"e_1_2_8_7_1","first-page":"1","volume":"10","author":"Pan S.","year":"2011","journal-title":"Mol. Cell. Proteomics"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.3389\/fchem.2020.00508"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/0076-6879(82)83019-X"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00053a037"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1042\/bj20030673"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2014.05.029"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10719-018-9827-6"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10719-020-09909-z"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3861"},{"key":"e_1_2_8_16_1","first-page":"58","volume":"30","author":"Fischler D. A.","year":"2019","journal-title":"J. Biomol. Tech."},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1006\/prep.2001.1555"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.aca.2023.340972"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/rcm.1979"},{"key":"e_1_2_8_20_1","doi-asserted-by":"publisher","DOI":"10.1039\/D2CS00083K"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1002\/aic.690380212"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2004.10.006"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1039\/c3cs35464d"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijbiomac.2021.09.006"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cbpa.2019.11.008"},{"key":"e_1_2_8_26_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF01049451"},{"key":"e_1_2_8_27_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac901326u"},{"key":"e_1_2_8_28_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac201665e"},{"key":"e_1_2_8_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2013.10.087"},{"key":"e_1_2_8_30_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2009.02.036"},{"key":"e_1_2_8_31_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.talanta.2022.123652"},{"key":"e_1_2_8_32_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpba.2019.112995"},{"key":"e_1_2_8_33_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.270.49.29493"},{"key":"e_1_2_8_34_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12033-022-00464-6"},{"key":"e_1_2_8_35_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chroma.2020.460934"},{"key":"e_1_2_8_36_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jchromb.2016.02.006"},{"key":"e_1_2_8_37_1","doi-asserted-by":"publisher","DOI":"10.1039\/D3RA04591A"},{"key":"e_1_2_8_38_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.enzmictec.2005.10.014"},{"key":"e_1_2_8_39_1","doi-asserted-by":"publisher","DOI":"10.1002\/smll.201002089"},{"key":"e_1_2_8_40_1","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts343"},{"key":"e_1_2_8_41_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.enzmictec.2009.09.001"},{"key":"e_1_2_8_42_1","doi-asserted-by":"publisher","DOI":"10.1021\/cs400594e"},{"key":"e_1_2_8_43_1","doi-asserted-by":"publisher","DOI":"10.2174\/138527281917150806125708"},{"key":"e_1_2_8_44_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jasms.2008.11.012"},{"key":"e_1_2_8_45_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41929-021-00728-5"},{"key":"e_1_2_8_46_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1044-0305(00)00122-7"},{"key":"e_1_2_8_47_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.11765"},{"key":"e_1_2_8_48_1","doi-asserted-by":"publisher","DOI":"10.3382\/ps\/pey402"},{"key":"e_1_2_8_49_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00731873"},{"key":"e_1_2_8_50_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.carres.2019.04.011"},{"key":"e_1_2_8_51_1","doi-asserted-by":"publisher","DOI":"10.1021\/ac203394r"},{"key":"e_1_2_8_52_1","unstructured":"PNGase F from different manufacturers Promega :https:\/\/www.promega.es\/products\/mass-spectrometry\/glycosidases\/pngase-f\/?catNum=V4831; NEB:https:\/\/www.neb.com\/en\/protocols\/2014\/07\/31\/pngase-f-protocol; Merck:https:\/\/www.sigmaaldrich.com\/ES\/en\/product\/sigma\/p7367."},{"key":"e_1_2_8_53_1","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.T600063-MCP200"},{"key":"e_1_2_8_54_1","doi-asserted-by":"publisher","DOI":"10.7171\/jbt.17-2804-002"},{"key":"e_1_2_8_55_1","doi-asserted-by":"publisher","DOI":"10.1016\/0165-022X(89)90081-X"},{"key":"e_1_2_8_56_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.eng.2023.02.016"},{"key":"e_1_2_8_57_1","doi-asserted-by":"publisher","DOI":"10.1021\/pr049825o"},{"key":"e_1_2_8_58_1","doi-asserted-by":"publisher","DOI":"10.1002\/chem.202301494"},{"key":"e_1_2_8_59_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-0215-7_5"}],"container-title":["ChemSusChem"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/chemistry-europe.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cssc.202402346","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,14]],"date-time":"2025-10-14T04:30:36Z","timestamp":1760416236000},"score":1,"resource":{"primary":{"URL":"https:\/\/chemistry-europe.onlinelibrary.wiley.com\/doi\/10.1002\/cssc.202402346"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,29]]},"references-count":59,"journal-issue":{"issue":"10","published-print":{"date-parts":[[2025,5,19]]}},"alternative-id":["10.1002\/cssc.202402346"],"URL":"https:\/\/doi.org\/10.1002\/cssc.202402346","archive":["Portico"],"relation":{},"ISSN":["1864-5631","1864-564X"],"issn-type":[{"value":"1864-5631","type":"print"},{"value":"1864-564X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,1,29]]},"assertion":[{"value":"2024-10-31","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-01-29","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e202402346"}}