{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T04:31:11Z","timestamp":1786509071117,"version":"build-2736575974"},"reference-count":36,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32072166"],"award-info":[{"award-number":["32072166"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["32072166"],"award-info":[{"award-number":["32072166"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100005196","name":"Chinese Academy of Agricultural Sciences","doi-asserted-by":"publisher","award":["ASTIP-IBFC"],"award-info":[{"award-number":["ASTIP-IBFC"]}],"id":[{"id":"10.13039\/501100005196","id-type":"DOI","asserted-by":"publisher"}]},{"award":["ASTIP-IBFC"],"award-info":[{"award-number":["ASTIP-IBFC"]}],"id":[{"id":"https:\/\/ror.org\/0313jb750","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009377","name":"Education Department of Hunan Province","doi-asserted-by":"publisher","award":["25A0192"],"award-info":[{"award-number":["25A0192"]}],"id":[{"id":"10.13039\/100009377","id-type":"DOI","asserted-by":"publisher"}]},{"award":["25A0192"],"award-info":[{"award-number":["25A0192"]}],"id":[{"id":"https:\/\/ror.org\/05ckg3w11","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002767","name":"Department of Science and Technology of Hunan Province","doi-asserted-by":"publisher","award":["2023RC3137"],"award-info":[{"award-number":["2023RC3137"]}],"id":[{"id":"10.13039\/501100002767","id-type":"DOI","asserted-by":"publisher"}]},{"award":["2023RC3137"],"award-info":[{"award-number":["2023RC3137"]}],"id":[{"id":"https:\/\/ror.org\/04qgr7x96","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Metabolites"],"abstract":"<jats:p>Background\/Objectives: \u03b2-Glucosidases from lactic acid bacteria are valuable biocatalysts for carbohydrate conversion, flavour enhancement, and bioactive glycoside biotransformation. Although GH3 family enzymes have been characterized from several Lactobacillus species, no systematic study exists for Lentilactobacillus buchneri\u2014a GRAS strain with plant-polysaccharide-degrading potential. This work aimed to clone, heterologously express, and comprehensively characterize a novel GH3 \u03b2-glucosidase (LbBgl3) from L. buchneri, with a particular focus on its catalytic properties, substrate profile, stability, and the unexpected EDTA-mediated thermostabilization mechanism. Methods: A novel \u03b2-glucosidase gene (LbBgl3) from L. buchneri was successfully expressed in Escherichia coli. Results: Biochemical characterization revealed that LbBgl3 is a cold-adapted, moderately acidophilic enzyme, exhibiting optimal activity at 37 \u00b0C and pH 5.0, with a maximum specific activity of 799.67 U\u00b7mg\u22121. Under optimal conditions, the enzyme displayed kinetic parameters toward pNPG with a Km of 1.697 mM, Vmax of 442.5 \u03bcmol\u00b7mg\u22121\u00b7min\u22121, kcat of 634.32 s\u22121, and kcat\/Km of 373.79 mM\u22121\u00b7s\u22121. LbBgl3 exhibited high salt tolerance, with activity peaking at ~140% at 0.5 M NaCl and retaining ~68% at 2.0 M NaCl, while showing sensitivity to glucose inhibition. Notably, EDTA significantly enhanced both the activity and stability of LbBgl3. At 50 mM, the relative activity increased to 161%, and stability at 25 \u00b0C was prolonged. Molecular docking simulations suggested that EDTA binds near the substrate-binding pocket, forming hydrogen bonds with Ala57, Gln766, Ser768, and Lys770, thereby stabilizing the local conformation and enhancing thermal resistance. Conclusions: This study presents the first characterization of a GH3 \u03b2-glucosidase from L. buchneri and reveals a non-classical stabilizing effect of EDTA, offering valuable insights for enzyme engineering and biocatalytic applications.<\/jats:p>","DOI":"10.3390\/metabo16080565","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T12:47:36Z","timestamp":1786366056000},"page":"565","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Enzymatic Characterization of a Novel GH3 \u03b2-Glucosidase from Lentilactobacillus buchneri: EDTA-Mediated Enhancement of Thermostability"],"prefix":"10.3390","volume":"16","author":[{"given":"Hui","family":"Tang","sequence":"first","affiliation":[{"name":"Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China"},{"name":"School of Food Science and Bioengineering, Changsha University of Science & Technology, Changsha 410114, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinjian","family":"He","sequence":"additional","affiliation":[{"name":"Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China"},{"name":"National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Can","family":"Li","sequence":"additional","affiliation":[{"name":"School of Food Science and Bioengineering, Changsha University of Science & Technology, Changsha 410114, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tongying","family":"Liu","sequence":"additional","affiliation":[{"name":"National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5532-6564","authenticated-orcid":false,"given":"Hao","family":"Wu","sequence":"additional","affiliation":[{"name":"School of Food Science and Bioengineering, Changsha University of Science & Technology, Changsha 410114, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5736-9261","authenticated-orcid":false,"given":"Mansheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China"},{"name":"National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengjun","family":"Shi","sequence":"additional","affiliation":[{"name":"Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha 410205, China"},{"name":"National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya 572000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,10]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"107423","DOI":"10.1016\/j.fbio.2025.107423","article-title":"Probiotic potential of endophytic bacteria in functional foods: Opportunities and challenges","volume":"72","author":"Sheikh","year":"2025","journal-title":"Food Biosci."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"6359","DOI":"10.1002\/jsfa.13370","article-title":"Modulation of gut-microbiota through probiotics and dietary interventions to improve host health","volume":"104","author":"Dasriya","year":"2024","journal-title":"J. Sci. Food Agric."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"10730","DOI":"10.1080\/10408398.2023.2227896","article-title":"Lactic acid bacteria in the functional food industry: Biotechnological properties and potential applications","volume":"64","author":"Abedin","year":"2024","journal-title":"Crit. Rev. Food Sci. Nutr."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"16028","DOI":"10.1021\/acs.jafc.5c02311","article-title":"Lactic acid bacteria as biofactories: Mechanistic insights, engineering strategies, and future horizons for heterologous enzyme expression","volume":"73","author":"Ullah","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1455","DOI":"10.3168\/jds.2012-6179","article-title":"Lactobacillus helveticus as a tool to change proteolysis and functionality in Swiss-type cheeses","volume":"96","author":"Richoux","year":"2013","journal-title":"J. Dairy Sci."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"5509","DOI":"10.1128\/AEM.01177-13","article-title":"Malic enzyme and malolactic enzyme pathways are functionally linked but independently regulated in Lactobacillus casei BL23","volume":"79","author":"Ferrer","year":"2013","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1187","DOI":"10.1007\/s10529-016-2094-3","article-title":"Biotransformation of gypenoside XVII to compound K by a recombinant \u03b2-glucosidase","volume":"38","author":"Zhong","year":"2016","journal-title":"Biotechnol. Lett."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1007\/s13205-015-0328-z","article-title":"Catalytic properties, functional attributes and industrial applications of \u03b2-glucosidases","volume":"6","author":"Singh","year":"2016","journal-title":"3 Biotech"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Srivastava, N., Rathour, R., Jha, S., Pandey, K., Srivastava, M., Thakur, V.K., Sengar, R.S., Gupta, V.K., Mazumder, P.B., and Khan, A.F. (2019). Microbial beta glucosidase enzymes: Recent advances in biomass conversation for biofuels application. Biomolecules, 9.","DOI":"10.3390\/biom9060220"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/j.biochi.2024.05.009","article-title":"Microbial \u03b2-glucosidases: Recent advances and applications","volume":"225","author":"Magwaza","year":"2024","journal-title":"Biochimie"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"D233","DOI":"10.1093\/nar\/gkn663","article-title":"The carbohydrate-active enzymes database (Cazy): An expert resource for glycogenomics","volume":"37","author":"Cantarel","year":"2009","journal-title":"Nucleic Acids Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"103829","DOI":"10.1016\/j.fbio.2024.103829","article-title":"Review of probiotics, gut microorganisms, and their enzymes involved in the conversion of ginsenosides","volume":"58","author":"Li","year":"2024","journal-title":"Food Biosci."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Xie, Y., Wang, Y., Han, Y., Zhang, J., Wang, S., Lu, S., Wang, H., Lu, F., and Jia, L. (2022). Complete genome sequence of a novel Lactobacillus paracasei TK1501 and its application in the biosynthesis of isoflavone aglycones. Foods, 11.","DOI":"10.3390\/foods11182807"},{"key":"ref_14","doi-asserted-by":"crossref","unstructured":"Hu, Y., Zhai, L., Hong, H., Shi, Z., Zhao, J., and Liu, D. (2022). Study on the biochemical characterization and selectivity of three \u03b2-glucosidases from Bifidobacterium adolescentis ATCC15703. Front. Microbiol., 13.","DOI":"10.3389\/fmicb.2022.860014"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"S2878","DOI":"10.1080\/10942912.2017.1382506","article-title":"\u03b2-Glucosidase activity and bioconversion of isoflavone glycosides to aglycones by potential probiotic bacteria","volume":"20","author":"Yuksekdag","year":"2018","journal-title":"Int. J. Food Prop."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Jin, Y., Wang, P., Li, F., Yu, M., Du, J., Zhao, T., Yi, Q., Tang, H., and Yuan, B. (2024). The effects of Lactobacillus plantarum and Lactobacillus buchneri on the fermentation quality, in vitro digestibility, and aerobic stability of Silphium perfoliatum L. Silage. Animals, 14.","DOI":"10.3390\/ani14152279"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3036","DOI":"10.1093\/bioinformatics\/btx350","article-title":"DeepSite: Protein-binding site predictor using 3D-convolutional neural networks","volume":"33","author":"Doerr","year":"2017","journal-title":"Bioinformatics"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Lopata, A., J\u00f3j\u00e1rt, B., Sur\u00e1nyi, \u00c9.V., Tak\u00e1cs, E., Bez\u00far, L., Leveles, I., Bendes, \u00c1.\u00c1., Viskolcz, B., V\u00e9rtessy, B.G., and T\u00f3th, J. (2019). Beyond chelation: EDTA Tightly binds Taq DNA polymerase, MutT and dUTPase and directly inhibits dNTPase activity. Biomolecules, 9.","DOI":"10.3390\/biom9100621"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2739","DOI":"10.1016\/j.bbagen.2012.11.014","article-title":"Identification of the acid\/base catalyst of a glycoside hydrolase family 3 (GH3) \u03b2-glucosidase from Aspergillus niger ASKU28","volume":"1830","author":"Thongpoo","year":"2013","journal-title":"Biochim. Biophys. Acta-Gen. Subj."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Liu, Y.H., Zhang, N., Ma, J., Zhou, Y.Q., Wei, Q., Tian, C.j., Fang, Y., Zhong, R.Z., Chen, G., and Zhang, S.T. (2023). Advances in cold-adapted enzymes derived from microorganisms. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1152847"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1953","DOI":"10.22207\/JPAM.13.4.07","article-title":"In silico approach to elucidate factors associated with GH1 \u03b2-glucosidase thermostability","volume":"13","author":"Ahmed","year":"2019","journal-title":"J. Pure Appl. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"133449","DOI":"10.1016\/j.ijbiomac.2024.133449","article-title":"Evolutionary history and activity towards oligosaccharides and polysaccharides of GH3 glycosidases from an Antarctic marine bacterium","volume":"275","author":"Marchetti","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"e7106","DOI":"10.7717\/peerj.7106","article-title":"Cloning, purification, and characterization of GH3 \u03b2-glucosidase, MtBgl85, from Microbulbifer thermotolerans DAU221","volume":"7","author":"Pyeon","year":"2019","journal-title":"PeerJ"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4794690","DOI":"10.1155\/2018\/4794690","article-title":"A novel thermostable GH3 \u03b2-glucosidase from Talaromyce leycettanus with broad substrate specificity and significant soybean isoflavone glycosides-hydrolyzing capability","volume":"2018","author":"Li","year":"2018","journal-title":"BioMed Res. Int."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1186\/s13568-017-0360-y","article-title":"Enhancement of active compound, genipin, from Gardeniae Fructus using immobilized glycosyl hydrolase family 3 \u03b2-glucosidase from Lactobacillus antri","volume":"7","author":"Kim","year":"2017","journal-title":"AMB Express"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"103491","DOI":"10.1016\/j.bcab.2025.103491","article-title":"\u03b2-Glucosidase from Lacticaseibacillus casei TISTR 1463: Biochemical characterization and application on biotransformation of pigmented leaf extract from Oryza sativa L. (Luem Pua glutinous rice)","volume":"64","author":"Jaijoi","year":"2025","journal-title":"Biocatal. Agric. Biotechnol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Siddiqi, M.Z., Srinivasan, S., Park, H.Y., and Im, W.-T. (2020). Exploration and characterization of novel glycoside hydrolases from the whole genome of Lactobacillus ginsenosidimutans and enriched production of minor ginsenoside Rg3(s) by a recombinant enzymatic process. Biomolecules, 10.","DOI":"10.3390\/biom10020288"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1016\/j.enzmictec.2014.09.001","article-title":"Purification and functional characterization of the first stilbene glucoside-specific \u03b2-glucosidase isolated from Lactobacillus kimchi","volume":"67","author":"Ko","year":"2014","journal-title":"Enzym. Microb. Technol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Xie, Y., Yan, X., Li, C., Wang, S., and Jia, L. (2024). Characterization and insight mechanism of an acid-adapted \u03b2-Glucosidase from Lactobacillus paracasei and its application in bioconversion of glycosides. Front. Bioeng. Biotechnol., 12.","DOI":"10.3389\/fbioe.2024.1334695"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Muhammad, A.u.R., Gumustop, I., Genel, I., Kurt, I.C., and Ortakci, F. (2025). Comparative genomics of Lentilactobacillus buchneri reveals strain-level hyperdiversity and broad-spectrum CRISPR immunity against human and livestock gut phages. PLoS ONE, 20.","DOI":"10.1371\/journal.pone.0325832"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"245","DOI":"10.1007\/BF02783568","article-title":"Effect of pH on growth and fatty acid composition of Lactobacillus b\u00fcchneri and Lactobacillus fermentum","volume":"59","author":"Johnsson","year":"1996","journal-title":"Appl. Biochem. Biotechnol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"27062","DOI":"10.1038\/srep27062","article-title":"Functional diversity of family 3 \u03b2-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1","volume":"6","author":"Xia","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"7341","DOI":"10.1021\/jf103889m","article-title":"Influence of ethylenediaminetetraacetic acid (EDTA) on the structural stability of endoglucanase from Aspergillus aculeatus","volume":"59","author":"Naika","year":"2011","journal-title":"J. Agric. Food Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1007\/s11756-023-01542-z","article-title":"Ethylenediaminetetraacetic acid enhances structural stability and thermotolerance of recombinant cyclomaltodextrinase from Geobacillus thermopakistaniensis at higher temperatures","volume":"79","author":"Aroob","year":"2024","journal-title":"Biologia"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"141852","DOI":"10.1016\/j.molstruc.2025.141852","article-title":"Exploring enzyme thermostability enhancement through molecular modification: What remains to be known?","volume":"1337","author":"Kang","year":"2025","journal-title":"J. Mol. Struct."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1016\/j.jmgm.2016.01.001","article-title":"Contribution of main chain and side chain atoms and their locations to the stability of thermophilic proteins","volume":"64","author":"Tompa","year":"2016","journal-title":"J. Mol. Graph. Model."}],"container-title":["Metabolites"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-1989\/16\/8\/565\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T04:13:19Z","timestamp":1786507999000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-1989\/16\/8\/565"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,10]]},"references-count":36,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["metabo16080565"],"URL":"https:\/\/doi.org\/10.3390\/metabo16080565","relation":{},"ISSN":["2218-1989"],"issn-type":[{"value":"2218-1989","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,10]]}}}