{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T18:01:41Z","timestamp":1790359301325,"version":"4.1.0"},"reference-count":36,"publisher":"Public Library of Science (PLoS)","issue":"9","license":[{"start":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T00:00:00Z","timestamp":1790294400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005621","name":"Kasetsart University Research and Development Institute","doi-asserted-by":"publisher","award":["FF(KU)16.67"],"award-info":[{"award-number":["FF(KU)16.67"]}],"id":[{"id":"10.13039\/501100005621","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Lactosucrose is a functional trisaccharide with prebiotic properties and growing potential in food and agricultural applications; however, its industrial production is limited by low conversion efficiency and high downstream processing costs. In this study, a whole-cell biocatalyst was developed by displaying \u03b2-galactosidase on\n                    <jats:italic>Lactococcus<\/jats:italic>\n                    sp. KTH0-1S using a nisin-inducible expression system, the USP45 signal peptide, and a LysM anchoring domain. The engineered strain exhibited a maximum whole-cell \u03b2-galactosidase activity of 146.0 Miller units after 4 h of induction. The whole-cell enzyme efficiently catalyzed lactosucrose synthesis from lactose and sucrose, with optimal activity observed at pH 6.0 and 50 \u00b0C. Substrate molar ratio significantly influenced product formation, and the highest lactosucrose concentration (15,940 mg\/L) was achieved at a lactose:sucrose ratio of 1:3. Reusability studies revealed high initial catalytic activity but limited stability upon repeated reaction cycles, likely due to the non-covalent nature of LysM-mediated surface anchoring. Molecular docking analysis supported the experimental observations by identifying a high-affinity substrate-binding pocket capable of accommodating both donor and acceptor substrates, providing a structural rationale for enhanced transgalactosylation under sucrose-enriched conditions. Overall, this study demonstrates the feasibility of using a whole-cell biocatalyst based on\n                    <jats:italic>Lactococcus<\/jats:italic>\n                    sp. for efficient lactosucrose production and highlights its potential as a cost-effective and food-compatible platform for the synthesis of functional oligosaccharides.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0359091","type":"journal-article","created":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T17:45:11Z","timestamp":1790358311000},"page":"e0359091","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["\u03b2-Galactosidase expression on Lactococcus sp. KTH0-1S as a whole-cell biocatalyst for efficient lactosucrose production"],"prefix":"10.1371","volume":"21","author":[{"given":"Nichapat","family":"Ariyakajorn","sequence":"first","affiliation":[{"name":"Kasetsart University","place":["Bangkok, Thailand"],"department":["Department of Biotechnology, Faculty of Agro-Industry"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Napat","family":"Sunsern","sequence":"additional","affiliation":[{"name":"Kasetsart University","place":["Bangkok, Thailand"],"department":["Department of Biotechnology, Faculty of Agro-Industry"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Phitsanu","family":"Pinmanee","sequence":"additional","affiliation":[{"name":"National Center of Genetic Engineering and Biotechnology (BIOTEC)","place":["Pathum Thani, Thailand"],"department":["Biocatalyst Technology Research Team"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Boontiwa","family":"Ninchan","sequence":"additional","affiliation":[{"name":"Kasetsart University","place":["Bangkok, Thailand"],"department":["Department of Biotechnology, Faculty of Agro-Industry"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pimsiriya","family":"Srila","sequence":"additional","affiliation":[{"name":"Kasetsart University","place":["Bangkok, Thailand"],"department":["Department of Biotechnology, Faculty of Agro-Industry"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Konlarat","family":"Phirom-on","sequence":"additional","affiliation":[{"name":"BOKU University","place":["Vienna, Austria"],"department":["Food Biotechnology Laboratory, Department of Biotechnology and Food Sciences"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sivatch","family":"Imrattanarak","sequence":"additional","affiliation":[{"name":"Kasetsart University","place":["Bangkok, Thailand"],"department":["Department of Biotechnology, Faculty of Agro-Industry"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wirat","family":"Vanichsriratana","sequence":"additional","affiliation":[{"name":"Kasetsart University","place":["Bangkok, Thailand"],"department":["Department of Biotechnology, Faculty of Agro-Industry"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0161-1025","authenticated-orcid":true,"given":"Nisit","family":"Watthanasakphuban","sequence":"additional","affiliation":[{"name":"Kasetsart University","place":["Bangkok, Thailand"],"department":["Department of Biotechnology, Faculty of Agro-Industry"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,9,25]]},"reference":[{"issue":"1","key":"pone.0359091.ref001","first-page":"1","article-title":"Dietary prebiotics: Current status and new definition","volume":"7","author":"GR Gibson","year":"2010","journal-title":"Food Science & Technology Bulletin: Functional Foods"},{"issue":"8","key":"pone.0359091.ref002","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1038\/nrgastro.2017.75","article-title":"Expert consensus document: The international scientific association for probiotics and prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics","volume":"14","author":"GR Gibson","year":"2017","journal-title":"Nat Rev Gastroenterol Hepatol"},{"issue":"1","key":"pone.0359091.ref003","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1093\/jn\/136.1.70","article-title":"Dietary fructooligosaccharides affect intestinal barrier function in healthy men","volume":"136","author":"SJM Ten Bruggencate","year":"2006","journal-title":"J Nutr"},{"issue":"1","key":"pone.0359091.ref004","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1007\/s10068-014-0021-8","article-title":"The prebiotic lactosucrose modulates gut metabolites and microbiota in intestinal inflammatory rats","volume":"23","author":"X Zhou","year":"2013","journal-title":"Food Sci Biotechnol"},{"issue":"16","key":"pone.0359091.ref005","doi-asserted-by":"crossref","first-page":"7073","DOI":"10.1007\/s00253-013-5079-3","article-title":"Current studies on physiological functions and biological production of lactosucrose","volume":"97","author":"W Mu","year":"2013","journal-title":"Appl Microbiol Biotechnol"},{"issue":"9","key":"pone.0359091.ref006","doi-asserted-by":"crossref","first-page":"3927","DOI":"10.1021\/jf9002494","article-title":"Effective enzymatic synthesis of lactosucrose and its analogues by beta-D-galactosidase from Bacillus circulans","volume":"57","author":"W Li","year":"2009","journal-title":"J Agric Food Chem"},{"issue":"2","key":"pone.0359091.ref007","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1016\/j.copbio.2010.12.004","article-title":"Impact of microbial transformation of food on health - 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