{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T15:05:12Z","timestamp":1785337512857,"version":"3.55.0"},"reference-count":45,"publisher":"American Chemical Society (ACS)","issue":"29","license":[{"start":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T00:00:00Z","timestamp":1784505600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T00:00:00Z","timestamp":1784505600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T00:00:00Z","timestamp":1784505600000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/na","name":"Tianshan Talent Training Program","doi-asserted-by":"publisher","award":["2023TSYCLJ0043"],"award-info":[{"award-number":["2023TSYCLJ0043"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,7,29]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Mannan oligosaccharides (MOS) are valuable prebiotics, and enzymatic hydrolysis by \u03b2-mannanase is the preferred production route. Bacillus-derived GH26 \u03b2-mannanase (gmuG) holds industrial promise but suffers from low catalytic efficiency and weak pH stability. Here, we rationally engineered flexible loops near the active site via molecular dynamics, NMsim (normal mode-based geometric simulation), sequence conservation analysis, and MAESTRO prediction, and obtained the optimal double mutant M1 (E301K\u2013T271V). Relative to the wild type, M1 showed 1.54-fold higher specific activity and nearly 2-fold improved pH stability at pH 8, with a nearly doubled kcat while retaining substrate affinity. Mechanistically, M1 enlarged the active site, shortened the attack distance of the catalytic residue, increased \u03b1-helix content, and rigidified neighboring loops. Fermentation optimization in Escherichia coli gave a maximum yield of 924.34 U\/mL. This work offers an effective strategy for \u03b2-mannanase engineering and a high-performance biocatalyst for industrial MOS production.<\/jats:p>","DOI":"10.1021\/acs.jafc.6c03707","type":"journal-article","created":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T10:15:45Z","timestamp":1784542545000},"page":"22975-22987","source":"Crossref","is-referenced-by-count":0,"title":["Rational Design\nof Flexible Loops Adjacent to the\nActive Site Enhances the Catalytic Efficiency of\n                    <i>Bacillus<\/i>\n                    \u03b2-Mannanase"],"prefix":"10.1021","volume":"74","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4883-0308","authenticated-orcid":true,"given":"Yuan","family":"Li","sequence":"first","affiliation":[{"name":"Xinjiang University , , ,","place":["Urumqi, China, 830017"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuwei","family":"Ma","sequence":"additional","affiliation":[{"name":"Xinjiang University , , ,","place":["Urumqi, China, 830017"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tongli","family":"Li","sequence":"additional","affiliation":[{"name":"Xinjiang University , , ,","place":["Urumqi, China, 830017"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Wang","sequence":"additional","affiliation":[{"name":"Xinjiang University , , ,","place":["Urumqi, China, 830017"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aixia","family":"Ma","sequence":"additional","affiliation":[{"name":"Xinjiang University , , ,","place":["Urumqi, China, 830017"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Loredana","family":"Capobianco","sequence":"additional","affiliation":[{"name":"University of Salento , , ,","place":["Lecce, Italy, 73100"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kongfang","family":"Yu","sequence":"additional","affiliation":[{"name":"Xinjiang University , , ,","place":["Urumqi, China, 830017"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Vincenza","family":"Dolce","sequence":"additional","affiliation":[{"name":"University of Calabria , , Arcavacata di Rende , ,","place":["Cosenza, Italy, 87036"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jinyao","family":"Li","sequence":"additional","affiliation":[{"name":"Xinjiang University , , ,","place":["Urumqi, China, 830017"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,7,20]]},"reference":[{"key":"2026072910064760200_cit1","doi-asserted-by":"publisher","first-page":"232","DOI":"10.1016\/j.foodchem.2017.10.071","article-title":"Preparation and Prebiotic\nPotential of Pectin Oligosaccharides Obtained from Citrus Peel Pectin","volume":"244","author":"Zhang","year":"2018","journal-title":"Food Chem."},{"key":"2026072910064760200_cit2","doi-asserted-by":"publisher","first-page":"109893","DOI":"10.1016\/j.enzmictec.2021.109893","article-title":"Production\nand in Vitro Evaluation of Prebiotic Manno-Oligosaccharides\nPrepared with a Recombinant Aspergillus Niger Endo-Mannanase,\nMan26A","volume":"150","author":"Magengelele","year":"2021","journal-title":"Enzyme Microb. Technol."},{"key":"2026072910064760200_cit3","doi-asserted-by":"publisher","first-page":"110197","DOI":"10.1016\/j.enzmictec.2023.110197","article-title":"Characterization of\na Novel GH26 \u03b2-Mannanase from Paenibacillus Polymyxa and Its Application in the Production of Mannooligosaccharides","volume":"165","author":"Gao","year":"2023","journal-title":"Enzyme Microb. Technol."},{"key":"2026072910064760200_cit4","doi-asserted-by":"publisher","first-page":"368","DOI":"10.1016\/j.carbpol.2017.10.083","article-title":"Protective Effects\nof Natural and Partially Degraded Konjac Glucomannan on Bifidobacteria against Antibiotic Damage","volume":"181","author":"Mao","year":"2018","journal-title":"Carbohydr. Polym."},{"key":"2026072910064760200_cit5","doi-asserted-by":"publisher","first-page":"42","DOI":"10.1016\/j.foodchem.2017.03.121","article-title":"Physicochemical\nProperties and Cellular Protection against Oxidation of Degraded Konjac\nGlucomannan Prepared by \u03b3-Irradiation","volume":"231","author":"Jian","year":"2017","journal-title":"Food Chem."},{"issue":"8","key":"2026072910064760200_cit6","doi-asserted-by":"publisher","first-page":"692","DOI":"10.2174\/0929866529666220615161603","article-title":"Production and Functional Characterization\nof a Novel Mannanase from Alteromonadaceae Bacterium\nBs31","volume":"29","author":"Ding","year":"2022","journal-title":"Protein Pept. Lett."},{"issue":"28","key":"2026072910064760200_cit7","doi-asserted-by":"publisher","first-page":"10221","DOI":"10.1080\/10408398.2023.2222165","article-title":"Functional\n\u03b2-Mannooligosaccharides:\nSources, Enzymatic Production and Application as Prebiotics","volume":"64","author":"Rana","year":"2024","journal-title":"Crit. Rev. Food Sci. Nutr."},{"issue":"1","key":"2026072910064760200_cit8","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/j.biotechadv.2016.11.001","article-title":"Production, Properties,\nand Applications of Endo-\u03b2-Mannanases","volume":"35","author":"Srivastava","year":"2017","journal-title":"Biotechnol. Adv."},{"issue":"6","key":"2026072910064760200_cit9","doi-asserted-by":"publisher","first-page":"169","DOI":"10.1007\/s11274-024-03985-1","article-title":"Research and\nApplication Progress of Microbial \u03b2-Mannanases:\nA Mini-Review","volume":"40","author":"Wang","year":"2024","journal-title":"World J. Microbiol. Biotechnol."},{"issue":"8","key":"2026072910064760200_cit10","doi-asserted-by":"publisher","first-page":"1166","DOI":"10.1016\/j.procbio.2013.06.014","article-title":"Improving the Acidic Stability of\na \u03b2-Mannanase from Bacillus Subtilis by Site-Directed\nMutagenesis","volume":"48","author":"Xu","year":"2013","journal-title":"Process Biochem."},{"key":"2026072910064760200_cit11","doi-asserted-by":"publisher","first-page":"50","DOI":"10.1016\/j.enzmictec.2018.07.005","article-title":"Rational Design\nof a Thermophilic \u03b2-Mannanase fromBacillus Subtilis TJ-102 to\nImprove Its Thermostability","volume":"118","author":"Wang","year":"2018","journal-title":"Enzyme Microb.\nTechnol."},{"key":"2026072910064760200_cit12","doi-asserted-by":"publisher","first-page":"70","DOI":"10.1016\/j.enzmictec.2019.02.003","article-title":"Cloning and Expression\nof a \u03b2-Mannanase Gene from Bacillus Sp. MK-2 and Its Directed\nEvolution by Random Mutagenesis","volume":"124","author":"Zhang","year":"2019","journal-title":"Enzyme Microb.\nTechnol."},{"issue":"23","key":"2026072910064760200_cit13","doi-asserted-by":"publisher","first-page":"14453","DOI":"10.1021\/acs.jafc.5c01791","article-title":"Enhancing Catalytic Activity of a Baeyer-Villiger Monooxygenase from\nOceanicola Granulosus: Simultaneous Engineering of the Distal Site\nand Active Site","volume":"73","author":"Du","year":"2025","journal-title":"J. Agric. Food Chem."},{"issue":"16","key":"2026072910064760200_cit14","doi-asserted-by":"publisher","first-page":"9973","DOI":"10.1021\/acs.jafc.5c01807","article-title":"Modification of Flexible\nRegions for Enhanced Thermal Stability of Alkaline Amylase","volume":"73","author":"Liu","year":"2025","journal-title":"J. Agric. Food Chem."},{"issue":"34","key":"2026072910064760200_cit15","doi-asserted-by":"publisher","first-page":"12785","DOI":"10.1021\/acs.jafc.3c03871","article-title":"Simultaneously\nEnhanced Thermostability\nand Catalytic Activity of Xylanase from Streptomyces Rameus L2001\nby Rigidifying Flexible Regions in Loop Regions of the N-Terminus","volume":"71","author":"Wu","year":"2023","journal-title":"J. Agric. Food Chem."},{"issue":"11","key":"2026072910064760200_cit16","doi-asserted-by":"publisher","first-page":"5805","DOI":"10.1021\/acs.jafc.3c07957","article-title":"Critical Roles of Acidic Residues\nin Loop Regions of the Structural Surface for the Salt Tolerance of\na GH39 \u03b2-d-Xylosidase","volume":"72","author":"Cao","year":"2024","journal-title":"J. Agric. Food\nChem."},{"key":"2026072910064760200_cit17","doi-asserted-by":"publisher","first-page":"127373","DOI":"10.1016\/j.ijbiomac.2023.127373","article-title":"Improvement of the Fibrinolytic Activity,\nAcid Resistance and Thermostability of Nattokinase by Surface Charge\nEngineering","volume":"253","author":"Li","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"issue":"25","key":"2026072910064760200_cit18","doi-asserted-by":"publisher","first-page":"14241","DOI":"10.1021\/acs.jafc.4c02335","article-title":"Enhanced Thermostability\nof Nattokinase by Computation-Based Rational Redesign of Flexible\nRegions","volume":"72","author":"Chen","year":"2024","journal-title":"J. Agric. Food Chem."},{"issue":"1","key":"2026072910064760200_cit19","doi-asserted-by":"publisher","first-page":"51","DOI":"10.1186\/s12934-025-02681-5","article-title":"Enhanced Thermostability\nof Nattokinase by Rational Design of Disulfide Bond","volume":"24","author":"Yu","year":"2025","journal-title":"Microb. 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Technol."},{"issue":"7873","key":"2026072910064760200_cit22","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly Accurate Protein\nStructure Prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"issue":"2","key":"2026072910064760200_cit23","doi-asserted-by":"publisher","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK: A Program\nto Check the Stereochemical Quality of Protein Structures","volume":"26","author":"Laskowski","year":"1993","journal-title":"J. Appl. Crystallogr."},{"issue":"28","key":"2026072910064760200_cit24","doi-asserted-by":"publisher","first-page":"17788","DOI":"10.1021\/acs.jafc.5c03873","article-title":"Amino Acid Residue\n131 Plays a Key Role in the Catalytic\nPerformance of Nattokinase","volume":"73","author":"Li","year":"2025","journal-title":"J. Agric. Food Chem."},{"key":"2026072910064760200_cit25","doi-asserted-by":"publisher","first-page":"116","DOI":"10.1186\/s12859-015-0548-6","article-title":"MAESTRO\u2013Multi Agent Stability Prediction upon\nPoint Mutations","volume":"16","author":"Laimer","year":"2015","journal-title":"BMC Bioinf."},{"issue":"10","key":"2026072910064760200_cit26","doi-asserted-by":"publisher","first-page":"3128","DOI":"10.1021\/acs.jcim.3c00336","article-title":"Binding Site Detection\nRemastered:\nEnabling Fast, Robust, and Reliable Binding Site Detection and Descriptor\nCalculation with DoGSite3","volume":"63","author":"Graef","year":"2023","journal-title":"J. Chem. Inf. Model."},{"issue":"16","key":"2026072910064760200_cit27","doi-asserted-by":"publisher","first-page":"2785","DOI":"10.1002\/jcc.21256","article-title":"AutoDock4 and AutoDockTools4: Automated\nDocking with Selective Receptor Flexibility","volume":"30","author":"Morris","year":"2009","journal-title":"J. Comput. Chem."},{"issue":"10","key":"2026072910064760200_cit28","doi-asserted-by":"publisher","first-page":"5293","DOI":"10.1021\/acs.jcim.1c00743","article-title":"Assembly of Biomolecular\nGigastructures and Visualization with the Vulkan Graphics API","volume":"61","author":"Ozvoldik","year":"2021","journal-title":"J. Chem. Inf. Model."},{"key":"2026072910064760200_cit29","doi-asserted-by":"publisher","first-page":"109358","DOI":"10.1016\/j.bioorg.2025.109358","article-title":"Synergistic\nEngineering of Nattokinase Terminal and Calcium-Binding Regions for\nEnhanced Catalytic Performance and Oral Stability","volume":"168","author":"Li","year":"2026","journal-title":"Bioorg. Chem."},{"issue":"20","key":"2026072910064760200_cit30","doi-asserted-by":"publisher","first-page":"2981","DOI":"10.1093\/bioinformatics\/btu426","article-title":"YASARA View - Molecular Graphics for All Devices -\nfrom Smartphones to Workstations","volume":"30","author":"Krieger","year":"2014","journal-title":"Bioinformatics"},{"issue":"8","key":"2026072910064760200_cit31","doi-asserted-by":"publisher","first-page":"3696","DOI":"10.1021\/acs.jctc.5b00255","article-title":"ff14SB: Improving the Accuracy of\nProtein Side Chain and Backbone Parameters from ff99SB","volume":"11","author":"Maier","year":"2015","journal-title":"J. Chem. Theory Comput."},{"issue":"11","key":"2026072910064760200_cit32","doi-asserted-by":"publisher","first-page":"5596","DOI":"10.1021\/acs.jctc.6b00701","article-title":"Multiple Staggered\nMesh Ewald: Boosting the Accuracy\nof the Smooth Particle Mesh Ewald Method","volume":"12","author":"Wang","year":"2016","journal-title":"J.\nChem. Theory Comput."},{"issue":"13","key":"2026072910064760200_cit33","doi-asserted-by":"publisher","first-page":"996","DOI":"10.1002\/jcc.23899","article-title":"New Ways to Boost Molecular\nDynamics Simulations","volume":"36","author":"Krieger","year":"2015","journal-title":"J. Comput. Chem."},{"key":"2026072910064760200_cit34","doi-asserted-by":"publisher","first-page":"1119232","DOI":"10.3389\/fmicb.2023.1119232","article-title":"Thermostability Modification of \u03b2-Mannanase from\nAspergillus Niger via Flexibility Modification Engineering","volume":"14","author":"Tan","year":"2023","journal-title":"Front. Microbiol."},{"key":"2026072910064760200_cit35","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1099\/00221287-146-1-65","article-title":"Proteome analysis of Bacillus subtilis extracellular\nproteins: a two-dimensional protein electrophoretic study","volume":"146","author":"Hirose","year":"2000","journal-title":"Microbiology"},{"issue":"2","key":"2026072910064760200_cit36","doi-asserted-by":"publisher","first-page":"186","DOI":"10.1007\/s00284-013-0460-0","article-title":"Enhanced Activity\nof Rhizomucor Miehei Lipase by Deglycosylation\nof Its Propeptide in Pichia Pastoris","volume":"68","author":"Liu","year":"2014","journal-title":"Curr.\nMicrobiol."},{"issue":"7","key":"2026072910064760200_cit37","doi-asserted-by":"publisher","first-page":"4152","DOI":"10.1021\/acs.jafc.4c09651","article-title":"Mining and Modification\nof Key Functional Regions of the Nattokinase Propeptide","volume":"73","author":"Ma","year":"2025","journal-title":"J. Agric. Food Chem."},{"issue":"W1","key":"2026072910064760200_cit38","doi-asserted-by":"publisher","first-page":"W310","DOI":"10.1093\/nar\/gks478","article-title":"NMSim Web\nServer: Integrated Approach\nfor Normal Mode-Based Geometric Simulations of Biologically Relevant\nConformational Transitions in Proteins","volume":"40","author":"Kru\u0308ger","year":"2012","journal-title":"Nucleic\nAcids Res."},{"issue":"26","key":"2026072910064760200_cit39","doi-asserted-by":"publisher","first-page":"16454","DOI":"10.1021\/acs.jafc.5c02217","article-title":"Structure-Guided\nTunnel\nEngineering to Reveal the Molecular Basis of Sugar Chain Extension\nof Inulosucrase","volume":"73","author":"Ni","year":"2025","journal-title":"J. Agric. Food Chem."},{"issue":"23","key":"2026072910064760200_cit40","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202307779","article-title":"Rationally\nEngineering pH Adaptation of Acid-Induced Arginine Decarboxylase from Escherichia Coli to Alkaline Environments to Efficiently\nBiosynthesize Putrescine","volume":"11","author":"Wang","year":"2024","journal-title":"Adv. Sci."},{"key":"2026072910064760200_cit41","doi-asserted-by":"publisher","first-page":"1505584","DOI":"10.3389\/fnut.2024.1505584","article-title":"Modification of the\nActive Centre of Nattokinase to Enhance Its Thermostability Using\na Strategy Based on Molecular Dynamics Simulation, Steered Dynamics\nSimulation, and Conservative Prediction","volume":"11","author":"Li","year":"2024","journal-title":"Front.\nNutr."},{"issue":"5","key":"2026072910064760200_cit42","doi-asserted-by":"publisher","first-page":"4623","DOI":"10.1021\/acs.jafc.5c15833","article-title":"Design\nGlycosyltransferases with High Glycosyl Transfer Efficiency to Efficiently\nProduce Isoquercetin from Quercetin","volume":"74","author":"Wang","year":"2026","journal-title":"J. Agric.\nFood Chem."},{"key":"2026072910064760200_cit43","doi-asserted-by":"publisher","first-page":"712","DOI":"10.1016\/j.ijbiomac.2017.04.061","article-title":"Potassium and Sodium\nIons Enhance the Activity and Thermostability of 1,4-\u03b1-Glucan\nBranching Enzyme from Geobacillus Thermoglucosidasius in the Presence\nof Glycerol","volume":"102","author":"Ban","year":"2017","journal-title":"Int. J. Biol. Macromol."},{"key":"2026072910064760200_cit44","doi-asserted-by":"publisher","first-page":"130153","DOI":"10.1016\/j.biortech.2023.130153","article-title":"Engineering\nSerine Hydroxymethyltransferases\nfor Efficient Synthesis of L-Serine in Escherichia Coli","volume":"393","author":"Teng","year":"2024","journal-title":"Bioresour. Technol."},{"issue":"49","key":"2026072910064760200_cit45","doi-asserted-by":"publisher","first-page":"31477","DOI":"10.1021\/acs.jafc.5c08476","article-title":"Interfacial\nEngineering Circumvents\nActivity-Stability Trade-Off in L-Threonine Aldolase for High-Yield\nL-Threo-MTPS","volume":"73","author":"Ruan","year":"2025","journal-title":"J. Agric. Food Chem."}],"container-title":["Journal of Agricultural\nand Food Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jafc.6c03707","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/29\/22975\/66043041\/acs.jafc.6c03707.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jafcau\/article-pdf\/74\/29\/22975\/66043041\/acs.jafc.6c03707.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T14:21:44Z","timestamp":1785334904000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jafcau\/article\/74\/29\/22975\/5216479\/Rational-Design-of-Flexible-Loops-Adjacent-to-the"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,7,20]]},"references-count":45,"journal-issue":{"issue":"29","published-online":{"date-parts":[[2026,7,20]]},"published-print":{"date-parts":[[2026,7,29]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jafc.6c03707","relation":{},"ISSN":["0021-8561","1520-5118"],"issn-type":[{"value":"0021-8561","type":"print"},{"value":"1520-5118","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,7,20]]}}}